U.S. patent application number 11/355904 was filed with the patent office on 2007-03-01 for mass tags for quantitative analyses.
This patent application is currently assigned to Applera Corporation. Invention is credited to Subhakar Dey, Kuo-Liang Hsi, Helena Huang, Joe Y. Lam, Darryl J. C. Pappin, Sasi Pillai, Subhasish Purkayastha, Krishna G. Upadhya, Xiongwei Yan, Pau-Miau Yuan, Sylvia W. Yuen.
Application Number | 20070048752 11/355904 |
Document ID | / |
Family ID | 38459505 |
Filed Date | 2007-03-01 |
United States Patent
Application |
20070048752 |
Kind Code |
A1 |
Yan; Xiongwei ; et
al. |
March 1, 2007 |
Mass tags for quantitative analyses
Abstract
This invention pertains to methods, mixtures, kits and/or
compositions for the determination of analytes by mass analysis
using unique labeling reagents or sets of unique labeling reagents.
The labeling reagents can be isomeric or isobaric and can be used
to produce mixtures suitable for multiplex analysis of the labeled
analytes.
Inventors: |
Yan; Xiongwei; (Dublin,
CA) ; Yuan; Pau-Miau; (San Jose, CA) ; Yuen;
Sylvia W.; (San Mateo, CA) ; Hsi; Kuo-Liang;
(Fremont, CA) ; Lam; Joe Y.; (Castro Valley,
CA) ; Upadhya; Krishna G.; (Union City, CA) ;
Dey; Subhakar; (North Billerica, MA) ; Pappin; Darryl
J. C.; (Boxborough, MA) ; Pillai; Sasi;
(Littleton, MA) ; Huang; Helena; (Boxborough,
MA) ; Purkayastha; Subhasish; (Acton, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Applera Corporation
Framingham
MA
|
Family ID: |
38459505 |
Appl. No.: |
11/355904 |
Filed: |
February 15, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11179060 |
Jul 11, 2005 |
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11355904 |
Feb 15, 2006 |
|
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60679183 |
May 9, 2005 |
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60587138 |
Jul 12, 2004 |
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Current U.S.
Class: |
435/6.12 ;
424/1.11; 514/19.3; 514/2.4; 514/21.91; 514/3.7; 544/276;
544/373 |
Current CPC
Class: |
C07D 239/54 20130101;
C07D 239/553 20130101; C07K 7/08 20130101; G01N 2458/15 20130101;
Y10T 436/143333 20150115; C07K 5/0806 20130101; G01N 33/6848
20130101; C07K 7/06 20130101; Y10T 436/24 20150115; C07K 5/1013
20130101; C07B 2200/05 20130101; C07D 295/185 20130101 |
Class at
Publication: |
435/006 ;
514/019; 424/001.11; 544/276; 544/373 |
International
Class: |
A61K 51/00 20060101
A61K051/00; C12Q 1/68 20060101 C12Q001/68; C07D 473/12 20070101
C07D473/12; C07D 403/02 20070101 C07D403/02 |
Claims
1. A kit comprising at least two different compounds represented by
the following formula: ##STR38## or a salt form and/or hydrate form
thereof, wherein independently for each different compound: RG is a
nucleophilic group or an electrophilic group, or a reaction product
of an analyte with a nucleophilic group.sub.=or an electrophilic
group; r and t are both 0 or one of r and t is 1 and the other is
0; S' is a cleavable linker coupled to a solid support or an
affinity ligand; X and Y are each a bond, wherein X couples an atom
or an optional substituent of each of RP and LK to thereby link RP
to LK, and Y couples an atom or an optional substituent of LK to
RG; RP and LK are each optionally and independently substituted,
wherein RP and LK are each independently a heteroaryl or
heterocycloalkyl, or a linear or branched aliphatic or
heteroaliphatic group substituted or interrupted with a heteroaryl
or heterocycloalkyl group; or LK is a linking moiety and RP is a
tertiary amine, a 4-9 membered nitrogenous heteroaryl or
heterocycloalkyl bonded at a ring nitrogen to X, a 5-6 membered
arylmethylene, a 5-6 membered heteroarylmethylene, or a 5-6
membered heterocycloalkyl; and RP has a unique gross mass for each
compound, and LK has a unique gross mass for each compound that
compensates for the difference in unique gross mass between the RP
for each compound such that the aggregate gross mass of the RP and
LK for each compound is the same, provided that; RP and LK are not
both selected from the group consisting of amino acids,
nucleotides, oligonucleotides, peptides, and proteins; and when t
is 0, the group RP is not an optionally substituted 5, 6 or 7
membered heterocycloalkyl comprising a ring nitrogen atom that is
N-alkylated with a substituted or unsubstituted moiety of the
formula --C(J).sub.2-LK'-- such that LK' is --C(O)--, --C(S)--,
--C(NH)--, or --C(NRz)-, wherein Rz is an alkyl group comprising
one to eight carbon atoms which may optionally contain a heteroatom
or optionally substituted aryl group wherein the carbon atoms of
the alkyl and aryl groups independently comprise linked hydrogen,
deuterium and/or fluorine atoms and each J is the same or different
and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz).sub.2, fluorine,
chlorine, bromine or iodine.
2. The kit of claim 1, wherein all compounds of the kit are
isobaric.
3. The kit of claim 2, wherein the compounds are isobaric
isomers.
4. The kit of claim 2, wherein the compounds are isobaric
isotopologues.
5. The kit of claim 1, where each compound of the kit comprises a
unique isotopically coded reporter.
6. The kit of claim 1, wherein r and t are both 0.
7. (canceled)
8. (canceled)
9. (canceled)
10. The kit of claim 6, wherein at least one compound is
represented by structural formula V: RP.sup.5--X-LK.sup.5--Y--RG V
wherein: RP.sup.5 is a reporter group and LK.sup.5 is a linking
moiety represented by structural formula E: ##STR39## wherein each
n, independently, is an integer from 1 to 3; and each R,
independently, is H, D, an alkyl, a heteroalkyl, an aryl, a
heteroaryl, or a halo group.
11. The kit of claim 10, wherein at least one compound is
represented by structural formula D: ##STR40##
12. The kit of claim 6, wherein at least one compound is
represented by structural formula I: RP.sup.1--X-LK.sup.1--Y--RG I
wherein: RP.sup.1 is a reporter group represented by structural
formula A: ##STR41## wherein, Ring A is aromatic; each Z is
independently CH, CR.sup.2, or N, provided that no more than two Z
groups are N; n is 1 or 2; each R.sup.2 is independently selected
from hydrogen, deuterium, --OH, halogen, --CN, --NO.sub.2, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,
heteroalkyl, heterocycloalkyl, --R.sup.3, or -T-R.sup.3; each
R.sup.3 is independently hydrogen, deuterium, alkyl, alkenyl,
alkynyl, aryl, arylalkyl, heteroalkyl, heterocycloalkyl,
heteroaryl, or heteroaralkyl; T is --O--, --NR.sup.4--, --S--,
--C(O)--, --S(O)--, --SO.sub.2--, --NR.sup.4C(O)--,
--C(O)NR.sup.4--, --NR.sup.4SO.sub.2--, --SO.sup.2NR.sup.4--,
--C(O)O--, --OC(O)--, --NR.sup.4C(O)O--, or --OC(O)NR.sup.4--; each
R.sup.4 is independently hydrogen, deuterium, alkyl, heteroalkyl,
aryl, or aralkyl; LK.sup.1 is a linking moiety; X is a bond between
an atom of the reporter and LK.sup.1; and Y is a bond between an
atom of the linker and an atom of RG, wherein at least one of
RP.sup.1 and LK.sup.1 is isotopically enriched with one or more
heavy atom isotopes.
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. The kit of claim 6, wherein at least one compound is
represented by Structural Formula III: RP.sup.3--X-LK.sup.3--Y--RG
III wherein: RP.sup.3 is a reporter group represented by structural
formula C: ##STR42## wherein, each of R.sup.x and R.sup.y is
independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, or heteroalkyl, wherein optional substituents for
R.sup.x and R.sup.y are independently selected from hydrogen,
deuterium, --OH, halogen, --CN, --NO.sub.2, --R.sup.3, -T-R.sup.3,
ribose, deoxyribose or phosphate, or R.sup.x and R.sup.y are taken
together to form a Ring C': ##STR43## wherein, ring C' is
heteroaryl or heterocycloalkyl, wherein the substituents for Ring
C' are independently hydrogen, deuterium, --OH, halogen, --CN,
--NO.sub.2, alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, heteroalkyl, --R.sup.3, -T-R.sup.3, ribose,
deoxyribose or phosphate; each R.sup.3 is independently hydrogen,
deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl,
heterocycloalkyl, heteroaryl, or heteroaralkyl; T is --O--,
--NR.sup.4--, --S--, --C(O)--, --S(O)--, --SO.sub.2--,
--NR.sup.4C(O)--, --C(O)NR.sup.4--, --NR.sup.4SO.sub.2--,
--SO.sub.2NR.sup.4--, --C(O)O--, --OC(O)--, --NR.sup.4C(O)O--, or
--OC(O)NR.sup.4--; each R.sup.4 is independently hydrogen,
deuterium, alkyl, heteroalkyl, aryl, or aralkyl; LK.sup.3 is a
linking moiety, provided that when R.sup.x and R.sup.y are taken
together to form Ring C', then the ring nitrogen that links R.sup.x
and R.sup.y is linked to a group other than a substituted or
unsubstituted moiety of the formula --C(J).sub.2-LK'-- such that
LK' is --C(O)--, --C(S)--, --C(NH)--, or --C(NRz)-, wherein Rz is
is an alkyl group comprising one to eight carbon atoms which may
optionally contain a heteroatom or optionally substituted aryl
group wherein the carbon atoms of the alkyl and aryl groups
independently comprise linked hydrogen, deuterium and/or fluorine
atoms and J is the same or different and is H, deuterium (D), Rz,
ORz, SRz, NHRz, N(Rz).sub.2, fluorine, chlorine, bromine or iodine;
X is a bond between an atom of the reporter and LK.sup.3; and Y is
a bond between an atom of the linker and an atom of RG, wherein at
least one of RP.sup.3 and LK.sup.3 is isotopically enriched with
one or more heavy atom isotopes.
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. The kit of claim 6, wherein RP comprises an optionally
substituted piperazinyl and LK is an aryl or cycloalkyl, or a
linear or branched aliphatic or heteroaliphatic group substituted
or interrupted with an aryl or cycloalkyl.
35. A kit comprising a compound represented by the Structural
Formula VI: RP.sup.6--X-LK.sup.6--Y--RG VI or a salt form and/or
hydrate form thereof, wherein RP.sup.6 and LK.sup.6 are each
independently a heteroaryl or heterocycloalkyl, or a linear or
branched aliphatic or heteroaliphatic group substituted or
interrupted with a heteroaryl or heterocycloalkyl, wherein at least
one of RP.sup.6 or LK.sup.6 comprises an optionally substituted
nucleobase, or a linear or branched aliphatic or heteroaliphatic
group substituted or interrupted with an optionally substituted
nucleobase; optional substituents for RP.sup.6 and LK.sup.6 are
independently selected from hydrogen, deuterium, --OH, halogen,
--CN, --NO.sub.2, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
arylalkyl, heteroarylalkyl, heteroalkyl, heterocycloalkyl,
--R.sup.3, -T-R.sup.3, ribose, deoxyribose, or phosphate; each
R.sup.3 is independently hydrogen, deuterium, alkyl, alkenyl,
alkynyl, aryl, arylalkyl, heteroalkyl, heterocycloalkyl,
heteroaryl, or heteroaralkyl; T is --O--, --NR.sup.4--, --S--,
--C(O)--, --S(O)--, --SO.sub.2--, --NR.sup.4C(O)--,
--C(O)NR.sup.4--, --NR.sup.4SO.sub.2--, --SO.sub.2NR.sup.4--,
--C(O)O--, --OC(O)--, --NR.sup.4C(O)O--, or --OC(O)NR.sup.4--; each
R.sup.4 is independently hydrogen, deuterium, alkyl, heteroalkyl,
aryl, or aralkyl; X is a bond between an atom of the reporter and
LK.sup.6; and Y is a bond between an atom of the linker and an atom
of RG, wherein at least one of RP.sup.6 and LK.sup.6 is
isotopically enriched with one or more heavy atom isotopes.
36. The kit of claim 35, wherein only LK.sup.6 is a nucleobase.
37. The kit of claim 6, wherein at least one compound is
represented by Structural Formula IV: RP.sup.4--X-LK.sup.4--Y--RG
IV wherein RP.sup.4 and LK.sup.4 are each independently a
heteroaryl or heterocycloalkyl, or a linear or branched aliphatic
or heteroaliphatic group substituted or interrupted with a
heteroaryl or heterocycloalkyl; optional substituents for RP.sup.4
and LK.sup.4 are independently selected from hydrogen, deuterium,
--OH, halogen, --CN, --NO.sub.2, alkyl, alkenyl, alkynyl, aryl,
heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl,
heterocycloalkyl, --R.sup.3, -T-R.sup.3, ribose, deoxyribose, or
phosphate; each R.sup.3 is independently hydrogen, deuterium,
alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl,
heterocycloalkyl, heteroaryl, or heteroaralkyl; T is --O--,
--NR.sup.4--, --S--, --C(O)--, --S(O)--, --SO.sub.2--,
--NR.sup.4C(O)--, --C(O)NR.sup.4--, --NR.sup.4SO.sub.2--,
--SO.sub.2NR.sup.4--, --C(O)O--, --OC(O)--, --NR.sup.4C(O)O--, or
--OC(O)NR.sup.4--; each R.sup.4 is independently hydrogen,
deuterium, alkyl, heteroalkyl, aryl, or aralkyl; X is a bond
between an atom of the reporter and LK.sup.4; and Y is a bond
between an atom of the linker and an atom of RG, wherein at least
one of RP.sup.4 and LK.sup.4 is isotopically enriched with one or
more heavy atom isotopes; provided that if RP.sup.4 is a
heterocycloalkyl, the heterocycloalkyl is not a 5, 6 or 7 membered
heterocycloalkyl comprising a ring nitrogen atom that is
N-alkylated with a substituted or unsubstituted moiety of the
formula --C(J).sub.2-LK'-- such that LK' is --C(O)--, --C(S)--,
--C(NH)--, or --C(NRz)-, wherein Rz is an alkyl group comprising
one to eight carbon atoms which may optionally contain a heteroatom
or optionally substituted aryl group wherein the carbon atoms of
the alkyl and aryl groups independently comprise linked hydrogen,
deuterium and/or fluorine atoms and J is the same or different and
is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz).sub.2, fluorine,
chlorine, bromine or iodine.
38. The kit of claim 37, wherein, the heteroaryl or
heterocycloalkyl groups in RP.sup.4 and LK.sup.4 are each
independently selected from optionally substituted imidazolyl,
furyl, pyrrolyl, thienyl, oxazolyl, thiazolyl, isoxazolyl,
isothiazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyrimidyl,
pyrazinyl, pyridazinyl, quinolyl, isoquinolinyl, indazolyl,
benzoxazolyl, benzisooxazolyl, benzofuryl, benzothiazolyl,
indolizinyl, imidazopyridinyl, pyrazolyl, triazolyl, isothiazolyl,
oxazolyl, tetrazolyl, benzimidazolyl, benzothiazolyl,
benzoisothiazolyl, benzothiadiazolyl, benzoxadiazolyl, indolyl,
tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl,
purinyl, pyrrolo[2,3]pyrimidyl, pyrazolo[3,4]pyrimidyl,
benzo(b)thienyl, morpholinyl, piperidinyl, piperazinyl,
pyrrolidinyl, and thiomorpholinyl.
39. The kit of claim 38, wherein for at least one compound, at
least one of RP.sup.4 or LK.sup.4 comprises an optionally
substituted piperazinyl, or a linear or branched aliphatic or
heteroaliphatic group substituted or interrupted with
piperazinyl.
40. The kit of claim 39, wherein for at least one compound,
RP.sup.4 comprises an optionally substituted piperazinyl.
41. The kit of claim 38, wherein at least one of RP.sup.4 or
LK.sup.4 comprises an optionally substituted nucleobase, or a
linear or branched aliphatic or heteroaliphatic group substituted
or interrupted with an optionally substituted nucleobase.
42. The kit of claim 41, wherein LK.sup.4 comprises an optionally
substituted nucleobase, or a linear or branched aliphatic or
heteroaliphatic group substituted or interrupted with an optionally
substituted nucleobase.
43. The kit of claim 42, wherein for at least one compound,
LK.sup.4 comprises an optionally substituted nucleobase.
44. The kit of claim 43, wherein the nucleobase is an optionally
substituted 9H-purin-6-amine (adenine), 2-amino-1H-purin-6(9H)-one
(guanine), 4-aminopyrimidin-2(1H)-one (cytosine),
5-methylpyrimidine-2,4(1H,3H)-dione (thymine) or
pyrimidine-2,4(1H,3H)-dione (uracil).
45. The kit of claim 44, wherein RP.sup.4 comprises an optionally
substituted piperazinyl.
46. The kit of claim 45, wherein at least one compound is
represented by a structural formula selected from: ##STR44##
wherein, R.sup.5 is --C(J).sub.2-C(O)--, --C(J).sub.2-C(S)--,
--C(J).sub.2-C(NH)--, or --C(J).sub.2-C(NR)--, wherein R.sup.z is
an alkyl group comprising one to eight carbon atoms that may
optionally contain a heteroatom or optionally substituted aryl
group wherein the carbon atoms of the alkyl and aryl groups
independently comprise linked hydrogen, deuterium and/or fluorine
atoms; and each J is the same or different and is H, deuterium (D),
Rz, ORz, SRz, NHRz, N(Rz).sub.2, fluorine, chlorine, bromine or
iodine R.sup.6 and R7 are each independently alkyl, alkenyl,
alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl,
heterocycloalkyl, --R.sup.3, -T-R.sup.3, ribose, deoxyribose, or
phosphate; wherein each R.sup.3 is independently hydrogen,
deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl,
heterocycloalkyl, heteroaryl, or heteroaralkyl; R.sup.8 and R.sup.9
are each independently H, deuterium (D), fluorine, chlorine,
bromine, iodine, or a halogenated alkyl.
47. The kit of claim 46, wherein at least one compound is:
##STR45## ##STR46## an isotopologue thereof.
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. (canceled)
54. (canceled)
55. (canceled)
56. (canceled)
57. (canceled)
58. A mixture comprising a plurality of labeled analytes
represented by the following formula: ##STR47## or a salt form
and/or hydrate form thereof, wherein independently for each labeled
analyte: r and t are both 0 or one of r and t is I and the other is
0; S' is a cleavable linker coupled to a solid support or an
affinity ligand; X and Y are each a bond, wherein X couples an atom
or an optional substituent of each of RP and LK to thereby link RP
to LK, and Y couples an atom or an optional substituent of LK to
-Analyte; RP and LK are each optionally and independently
substituted, wherein RP and LK are are each independently a
heteroaryl or heterocycloalkyl, or a linear or branched aliphatic
or heteroaliphatic group substituted or interrupted with a
heteroaryl or heterocycloalkyl; or LK is a linking moiety and RP is
a tertiary amine, a 4-9 membered nitrogenous heteroaryl or
heterocycloalkyl bonded at a ring nitrogen to X, a 5-6 membered
arylmethylene, a 5-6 membered heteroarylmethylene, or a 5-6
membered heterocycloalkyl; and RP has a unique gross mass for each
labeled analyte, and LK has a unique gross mass for each labeled
analyte that compensates for the difference in unique gross mass
between the RP for each labeled analyte such that the aggregate
gross mass of the RP and LK for each labeled analyte is the same,
provided that; RP and LK are not both selected from the group
consisting of naturally occurring amino acids, nucleotides,
oligonucleotides, peptides, and proteins; and when t is 0, the
group RP is not an optionally substituted 5, 6 or 7 membered
heterocycloalkyl comprising a ring nitrogen atom that is
N-alkylated with a substituted or unsubstituted moiety of the
formula --C(J).sub.2-LK'-- such that LK' is --C(O)--, --C(S)--,
--C(NH)--, or --C(NRz)-, wherein Rz is an alkyl group comprising
one to eight carbon atoms which may optionally contain a heteroatom
or optionally substituted aryl group wherein the carbon atoms of
the alkyl and aryl groups independently comprise linked hydrogen,
deuterium and/or fluorine atoms and each J is the same or different
and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz).sub.2, fluorine,
chlorine, bromine or iodine.
59. (canceled)
60. (canceled)
61. An isotopically enriched compound represented by the following
formula: RP--X-LK--Y--RG or a salt form and/or hydrate form
thereof, wherein: RG is a nucleophilic group or an electrophilic
group, or a reaction product of an analyte with a nucleophilic
group or an electrophilic group; X and Y are each a bond, wherein X
couples an atom or an optional substituent of each of RP and LK to
thereby link RP to LK, and Y couples an atom or an optional
substituent of LK to RG; RP and LK are each optionally and
independently substituted, wherein RP and LK are are each
independently a heteroaryl or heterocycloalkyl, or a linear or
branched aliphatic or heteroaliphatic group substituted or
interrupted with a heteroaryl or heterocycloalkyl; or LK is a
linking moiety and RP is a tertiary amine, a 4-9 membered
nitrogenous heteroaryl or heterocycloalkyl bonded at a ring
nitrogen to X, a 5-6 membered arylmethylene, a 5-6 membered
heteroarylmethylene, or a 5-6 membered heterocycloalkyl; and at
least two atoms of the compound are isotopically enriched with a
heavy atom isotope, provided that; RP and LK are not both selected
from the group consisting of naturally occurring amino acids,
nucleotides, oligonucleotides, peptides, and proteins; and when t
is 0, the group RP is not an optionally substituted 5, 6 or 7
membered heterocycloalkyl comprising a ring nitrogen atom that is
N-alkylated with a substituted or unsubstituted moiety of the
formula --C(J).sub.2-LK'-- such that LK' is --C(O)--, --C(S)--,
--C(NH)--, or --C(NRz)-, wherein Rz is an alkyl group comprising
one to eight carbon atoms which may optionally contain a heteroatom
or optionally substituted aryl group wherein the carbon atoms of
the alkyl and aryl groups independently comprise linked hydrogen,
deuterium and/or fluorine atoms and each J is the same or different
and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz).sub.2, fluorine,
chlorine, bromine or iodine.
62. (canceled)
63. (canceled)
64. (canceled)
65. A method comprising: a) reacting two or more samples, each
sample comprising one or more analytes, with a different labeling
reagent to thereby produce two or more differently labeled samples
each comprising one or more labeled analytes, wherein the labeling
reagents are represented by the formula: ##STR48## or a salt form
and/or hydrate form thereof, wherein independently for each
labeling reagent: RG is a nucleophilic group or an electrophilic
group; r and t are both 0 or one of r and t is 1 and the other is
0; S' is a cleavable linker coupled to a solid support or an
affinity ligand; X and Y are each a bond, wherein X couples an atom
or an optional substituent of each of RP and LK to thereby link RP
to LK, and Y couples an atom or an optional substituent of LK to
RG; RP and LK are each optionally and independently substituted,
wherein RP and LK are are each independently a heteroaryl or
heterocycloalkyl, or a linear or branched aliphatic or
heteroaliphatic group substituted or interrupted with a heteroaryl
or heterocycloalkyl; or LK is a linking moiety and RP is a tertiary
amine, a 4-9 membered nitrogenous heteroaryl or heterocycloalkyl
bonded at a ring nitrogen to X, a 5-6 membered arylmethylene, a 5-6
membered heteroarylmethylene, or a 5-6 membered heterocycloalkyl;
and RP has a unique gross mass for each labeled analyte, and LK has
a unique gross mass for each labeled analyte that compensates for
the difference in unique gross mass between the RP for each labeled
analyte such that the aggregate gross mass of the RP and LK for
each labeled analyte is the same; and b) mixing two or more of the
labeled samples, or a portion thereof, and optionally one or more
calibration standards to thereby produce the mixture, provided
that; RP and LK are not both selected from the group consisting of
naturally occurring amino acids, nucleotides, oligonucleotides,
peptides, and proteins; and when t is 0, the group RP is not an
optionally substituted 5, 6 or 7 membered heterocycloalkyl
comprising a ring nitrogen atom that is N-alkylated with a
substituted or unsubstituted moiety of the formula
--C(J).sub.2-LK'-- such that LK' is --C(O)--, --C(S)--, --C(NH)--,
or --C(NRz)-, wherein Rz is an alkyl group comprising one to eight
carbon atoms which may optionally contain a heteroatom or
optionally substituted aryl group wherein the carbon atoms of the
alkyl and aryl groups independently comprise linked hydrogen,
deuterium and/or fluorine atoms and each J is the same or different
and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz).sub.2, fluorine,
chlorine, bromine or iodine.
66. (canceled)
67. (canceled)
68. (canceled)
69. (canceled)
70. (canceled)
71. (canceled)
72. (canceled)
73. (canceled)
74. (canceled)
75. (canceled)
76. (canceled)
77. (canceled)
78. (canceled)
79. (canceled)
80. (canceled)
81. (canceled)
82. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 11/179,060, filed on Jul. 11, 2005, which
claims the benefit of U.S. Application No. 60/679,183, filed on May
9, 2005, and U.S. Application No. 60/587,138, filed on Jul. 12,
2004. The entire teachings of the above applications are
incorporated herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0002] The skilled artisan will understand that the drawings,
described below, are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0003] FIGS. 1A-1H show the structural formulae of a set of eight
isobaric mass tags each of which have the same molecular weight but
which will fragment to yield a signature ion having a different
molecular weight when subjected to dissociative energy levels.
[0004] FIG. 2A is a QTRAP.TM. 2000 MS analysis of SEQ ID No.: 1
which was alkylated with mass tag (32).
[0005] FIG. 2B is a QTRAP.TM. 2000 MS analysis of SEQ ID No.: 2
which was alkylated with mass tag (32).
[0006] FIG. 3A is a QTRAP.TM. 2000 MS/MS analysis of SEQ ID No.: 1
which was alkylated with mass tag (32).
[0007] FIG. 3B is a QTRAP.TM. 2000 MS/MS analysis of SEQ ID No.: 2
which was alkylated with mass tag (32).
[0008] FIG. 4A is a MS analysis of SEQ ID No.: 1, which was
alkylated with mass tag (32), using a 4700 Proteomic Analyzer.
[0009] FIG. 4B is a MS analysis of SEQ ID No.: 3, which was
alkylated with mass tag (32), using a 4700 Proteomic Analyzer.
[0010] FIG. 5A is a MS/MS analysis of SEQ ID No.: 1, which was
alkylated with mass tag (32), using a 4700 Proteomic Analyzer.
[0011] FIG. 5B is a MS/MS analysis of SEQ ID No.: 3, which was
alkylated with mass tag (32), using a 4700 Proteomic Analyzer.
[0012] FIG. 6A shows a MRM experiment performed on a QTRAP.TM. 2000
of two samples, in which one sample has been alkylated with mass
tag (32) and the other which has been alkylated with mass tag (33),
wherein the ratio of the sample label with mass tag (32) to the
sample labeled with mass tag (33) is 1:0.05.
[0013] FIG. 6B shows a MRM experiment performed on a QTRAP.TM. 2000
of two samples, in which one sample has been alkylated with mass
tag (32) and the other which has been alkylated with mass tag (33),
wherein the ratio of the sample label with mass tag (32) to the
sample labeled with mass tag (33) is 1:1.
[0014] FIG. 6C shows a MRM experiment performed on a QTRAP.TM. 2000
of two samples, in which one sample has been alkylated with mass
tag (32) and the other which has been alkylated with mass tag (33),
wherein the ratio of the sample label with mass tag (32) to the
sample labeled with mass tag (33) is 1:10.
[0015] FIG. 7A-1 and 7A-2 are mass spectra in the MS/MS mode of the
sample in FIG. 6A using a 4700 Proteomic Analyzer.
[0016] FIG. 7B-1 and 7B-2 are mass spectra in the MS/MS mode of the
sample in FIG. 6B using a 4700 Proteomic Analyzer.
[0017] FIG. 7C-1 and 7C-2 are mass spectra in the MS/MS mode of the
sample in FIG. 6C using a 4700 Proteomic Analyzer.
[0018] FIG. 8 illustrates exemplary formulas of leaving groups (LG)
for the alcohol or thiol group of an active ester wherein each G is
independently O or S, typically O.
[0019] FIGS. 9A-9B illustrate moieties i-xiv, which can be
comprised by the LK group in some embodiments.
[0020] FIG. 10 illustrates Protocol I and II for amine acylation to
generate a reactive group on a mass tag.
[0021] FIG. 11 illustrates the synthesis of Mass Tag (2).
[0022] FIG. 12 illustrates the synthesis of Mass Tag (3).
[0023] FIG. 13 illustrates Mass Tags (4) and (5).
[0024] FIG. 14 illustrates the syntheses of Mass Tags (6), (7) and
(8).
[0025] FIG. 15 illustrates the syntheses of Mass Tags (9), (10) and
(11).
[0026] FIG. 16 illustrates the synthesis of Mass Tag (12).
[0027] FIG. 17 illustrates Mass Tags (14) and (15).
[0028] FIG. 18 illustrates a general protocol for syntheses of Mass
Tags (16), (17), (18), (19) and (20).
[0029] FIG. 19 illustrates the syntheses of Mass Tags (21), (22),
(23) and (24).
[0030] FIG. 20 illustrates the synthesis of Mass Tag (25).
[0031] FIG. 21 illustrates the synthesis of Mass Tag (26).
[0032] FIG. 22 illustrates the synthesis of Mass Tag (27).
[0033] FIG. 23 illustrates the synthesis of Mass Tag (28).
[0034] FIG. 24 illustrates the synthesis of
FmocGly-Ser(Bzl-.sup.13C.sub.6) (29)
[0035] FIG. 25 illustrates the syntheses of resin bound Mass Tags
(30), (31) and (32).
[0036] FIG. 26 illustrates the synthesis of a labeling reagent/mass
tag (XX) ((37a)) comprising a thymine nucleobase.
[0037] FIG. 27A illustrates a known procedure for the synthesis of
6-methyl uracil from which a labeling reagent (mass tag) comprising
the 6-methyl uracil nucleobase ((37b)) can be prepared.
[0038] FIG. 27B illustrates various commercially available
isotopically substituted versions of ethyl acetoacetate that can be
used in the preparation of isotopically enriched versions of
6-methyl uracil.
[0039] FIG. 27C illustrates various commercially available
isotopically substituted versions of urea that can be used in the
preparation of isotopically enriched versions of 6-methyl
uracil.
[0040] FIGS. 28A and 28B illustrate various isotopically enriched
versions of 6-methyl uracil that can be prepared using the
compounds illustrated in FIGS. 27B and 27C in combination with the
procedure illustrated in FIG. 27A. Atoms labeled with * are heavy
atom isotopes.
[0041] FIGS. 29A-E illustrates various isotopically encoded
labeling reagents that can be prepared using the procedures and
commercially available compounds illustrated in FIGS. 26, 27A, 27B,
27C, and isotopically substituted 6-methyl uracils illustrated in
FIGS. 28A and 28B.
[0042] FIG. 30 illustrates the chemical structures of Mass Tag
labeled Glu-Fib peptides (38) and (39).
[0043] FIG. 31 illustrates the chemical structure of the Mass Tag
labeled Glu-Fib peptide (40).
[0044] FIG. 32 illustrates the synthesis of Mass Tag labeled
Glu-Fib peptide (41).
[0045] FIG. 33 illustrates the synthesis of Mass Tag labeled
Glu-Fib peptide (42).
[0046] FIG. 34 illustrates the synthesis of Mass Tag labeled
Glu-Fib peptide (43).
[0047] FIG. 35 illustrates the syntheses of Mass Tags (44) and
(45).
[0048] FIG. 36 illustrates the synthesis of Mass Tag labeled
Glu-Fib peptide (46).
[0049] FIG. 37 illustrates the chemical structures of Mass Tag
labeled Glu-Fib peptide (47) and Mass Tag (48)
[0050] FIG. 38 illustrates the synthesis of Mass Tag labeled
Glu-Fib peptide (49).
1. Introduction
[0051] This invention pertains to methods, mixtures, kits and/or
compositions for the determination of an analyte or analytes by
mass analysis. An analyte can be any molecule of interest.
Non-limiting examples of analytes include, but are not limited to,
proteins, peptides, oligonucleotides, carbohydrates, lipids,
steroids, amino acids and small molecules of less than 1500
daltons.
[0052] Labeling reagents and labeled analytes can be represented by
a compound of the general formula: ##STR1## or a salt form and/or
hydrate form thereof, wherein RG can be a reactive group that
reacts with an analyte or the reaction product of the reactive
group and the analyte. A labeled analyte therefore can have the
general formula: ##STR2## The compound can be tethered to a solid
support or moieties for linking it to a solid support via S'. The
variables RG, RP, X, LK, S', r, t, and Y are described in more
detail below.
[0053] Sets of isomeric or isobaric labeling reagents can be used
to label the analytes of two or more different samples wherein the
labeling reagent can be different for each different sample and
wherein the labeling reagent can comprise a unique reporter, "RP",
that can be associated with the sample from which the labeled
analyte originated. Hence, information, such as the presence and/or
amount of the reporter, can be correlated with the presence and/or
amount (often expressed as a concentration and/or quantity) of the
analyte in a sample even from the analysis of a complex mixture of
labeled analytes derived by mixing the reaction products obtained
from the labeling of different samples. Analysis of such complex
sample mixtures can be performed in a manner that allows for the
determination of one or a plurality of analytes from the same or
from multiple samples in a multiplex manner. Thus, the methods,
mixtures, kits and/or compositions of this invention are
particularly well suited for the multiplex analysis of complex
sample mixtures. For example, they can be used in proteomic
analysis and/or genomic analysis as well as for correlation studies
related to genomic and/or proteomic analysis.
2. Definitions
[0054] For the purposes of interpreting of this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice versa.
In the event that any definition set forth below conflicts with any
other document, including any incorporated herein by reference for
all purposes, the definition set forth below shall control:
[0055] As used herein, "analyte" refers to any molecule of interest
that may be determined. Non-limiting examples of analytes can
include, but are not limited to, proteins, peptides, nucleotides,
oligonucleotides (both DNA or RNA), carbohydrates, lipids,
steroids, amino acids and/or other small molecules with a molecular
weight of less than 1500 daltons. The source of the analyte, or the
sample comprising the analyte, is not a limitation as it can come
from any source. The analyte or analytes can be natural or
synthetic. Non-limiting examples of sources for the analyte, or the
sample comprising the analyte, include but are not limited to cells
or tissues, or cultures (or subcultures) thereof. Non-limiting
examples of analyte sources include, but are not limited to, crude
or processed cell lysates (including whole cell lysates), body
fluids, tissue extracts or cell extracts. Still other non-limiting
examples of sources for the analyte include but are not limited to
fractions from a separations process such as a chromatographic
separation or an electrophoretic separation. Body fluids include,
but are not limited to, blood, urine, feces, spinal fluid, cerebral
fluid, amniotic fluid, lymph fluid or a fluid from a glandular
secretion. By processed cell lysate we mean that the cell lysate is
treated, in addition to the treatments needed to lyse the cell, to
thereby perform additional processing of the collected material.
For example, the sample can be a cell lysate comprising one or more
analytes that are peptides formed by treatment of the total protein
component of a crude cell lysate with a proteolytic enzyme to
thereby digest precursor protein or proteins. For the avoidance of
doubt, the term analyte can include the original analyte and
compounds derived therefrom, unless from the context a clearly
contrary meaning is intended. For example, in some embodiments, the
term analyte can apply to a protein as well as to the peptides
derived therefrom by digestion of said protein.
[0056] As used herein, "fragmentation" refers to the breaking of a
covalent bond.
[0057] As used herein, "fragment" refers to a product of
fragmentation (noun) or the operation of causing fragmentation
(verb).
[0058] It is well accepted that the mass of an atom or molecule can
be approximated, often to the nearest whole number atomic mass unit
or the nearest tenth or hundredth of an atomic mass unit. As used
herein, "gross mass" refers to the absolute mass as well as to the
approximate mass within a range where the use of isotopes of
different atom types are so close in mass that they are the
functional equivalent for the purpose of balancing the mass of the
reporter and/or linker moieties (so that the gross mass of the
reporter/linker combination is the same within a set or kit of
isobaric or isomeric labeling reagents) whether or not the very
small difference in mass of the different isotopes types used can
be detected.
[0059] For example, the common isotopes of oxygen have a gross mass
of 16.0 (actual mass 15.9949) and 18.0 (actual mass 17.9992), the
common isotopes of carbon have a gross mass of 12.0 (actual mass
12.00000) and 13.0 (actual mass 13.00336) and the common isotopes
of nitrogen have a gross mass of 14.0 (actual mass 14.0031) and
15.0 (actual mass 15.0001). Whilst these values are approximate,
one of skill in the art will appreciate that if one uses the
.sup.18O isotope in one reporter of a set, the additional 2 mass
units (over the isotope of oxygen having a gross mass of 16.0) can,
for example, be compensated for in a different reporter of the set
comprising .sup.16O by incorporating, elsewhere in the reporter,
two carbon .sup.13C atoms, instead of two .sup.12C atoms, two
.sup.15N atoms, instead of two .sup.14N atoms or even one .sup.13C
atom and one .sup.15N atom, instead of a .sup.12C and a .sup.14N,
to compensate for the .sup.18O. In this way the two different
reporters of the set are the functional mass equivalent (i.e. have
the same gross mass) since the very small actual differences in
mass between the use of two .sup.13C atoms (instead of two .sup.12C
atoms), two .sup.15N atoms (instead of two .sup.14N atoms), one
.sup.13C and one .sup.15N (instead of a .sup.12C and .sup.14N) or
one .sup.18O atom (instead of one .sup.16O atom), to thereby
achieve an increase in mass of two Daltons, in all of the labels of
the set or kit, is not an impediment to the nature of the
analysis.
[0060] This can be illustrated with reference to FIGS. 1A-1H. In
FIG. 1A, the reporter/linker combination (FIG. 1A, not including
the reactive iodo group; chemical formula:
C.sub.11.sup.13C.sub.5H.sub.20N.sup.15N.sub.2O.sub.6) has two
.sup.15N atoms and five .sup.13C atom and a total theoretical mass
of 357.2213. By comparison, the reporter/linker isobar shown in
FIG. 1C (chemical formula
C.sub.10.sup.13C.sub.6H.sub.20N.sub.2.sup.15NO.sub.6) has one
.sup.15N atom and six .sup.13C atom and a total theoretical mass of
357.2279. The compounds in FIGS. 1A and C are isobars that are
structurally and chemically indistinguishable, except for heavy
atom isotope content, although there is a slight absolute mass
difference (mass 357.2213 vs. mass 357.2279, respectively).
However, the gross mass of the compounds in FIGS. 1A and 1C is
357.2 for the purposes of this invention since this is not an
impediment to the analysis whether or not the mass spectrometer is
sensitive enough to measure the small difference between the
absolute mass of the isobars in FIGS. 1A and 1C.
[0061] From FIGS. 1A-1H, it is clear that the distribution of the
same heavy atom isotopes within a structure is not the only
consideration for the creation of sets of isomeric and/or isobaric
labeling reagents. It is possible to mix heavy atom isotope types
to achieve isomers or isobars of a desired gross mass. In this way,
both the selection (combination) of heavy atom isotopes as well as
their distribution is available for consideration in the production
of the isomeric and/or isobaric labeling reagents useful for
embodiments of this invention.
[0062] As used herein, "isotopically enriched" refers to a compound
(e.g. labeling reagent) that has been enriched synthetically with
one or more heavy atom isotopes (e.g. stable isotopes such as
deuterium, .sup.13C, .sup.15N, .sup.18O, .sup.37Cl or .sup.81Br).
Because isotopic enrichment is not 100% effective, there can be
impurities of the compound that are of lesser states of enrichment
and these will have a lower mass. Likewise, because of
over-enrichment (undesired enrichment) and because of natural
isotopic abundance, there can be impurities of greater mass. In
some embodiments, each incorporated heavy atom isotope can be
present in at least 80 percent isotopic purity. In some
embodiments, each incorporated heavy atom isotope can be present in
at least 93 percent isotopic purity. In some embodiments, each
incorporated heavy atom isotope can be present in at least 96
percent isotopic purity.
[0063] As used herein, compounds that are "isotopologues" have the
same chemical composition but differ in isotopic composition
(number of isotopic substitutions), e.g., the methane isotopologues
CH.sub.4, CH.sub.3D, and CH.sub.2D.sub.2.
[0064] As used herein, compounds that are "isobaric isotopologues"
are those that have the same chemical composition and differ in
isotopic composition but have the same gross mass as measured by a
mass spectrometer (e.g., for the methane isobaric isotopologues
.sup.14CH.sub.4, .sup.13CH.sub.3D, and CH.sub.2D.sub.2, each has a
gross mass of 18 atomic mass units).
[0065] Some embodiments are an isotopically enriched compound that
can have at least two atoms that are isotopically enriched. In
various embodiments, the isotopically enriched compound can have 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, or more atoms that are isotopically enriched.
The chemical structure of the compound can be represented by any of
the preceding formulas wherein the variables are as defined
generally and in classes and subclasses described herein.
[0066] As used herein, "labeling reagent" refers to a moiety
suitable to mark an analyte for determination. The term label is
synonymous with the terms tag and mark and other equivalent terms
and phrases. For example, a labeled analyte can also be referred to
as a tagged analyte or a marked analyte. Accordingly the terms
"label", "tag", "mark" and derivatives of these terms, are
interchangeable and refer to a moiety suitable to mark, or that has
marked, an analyte for determination.
[0067] As used herein a "mass tag," as used herein, refers to a
labeling reagent that can be used to label or mark an analyte by
adding a group having a particular gross mass to the analyte. A set
of mass tags includes two or more mass tags, each of which adds a
group having the same mass to an analyte that is labeled. However,
each of the mass tags in the set of mass tags will fragment when
dissociative energy is applied to a signature ion having a
different mass from the signature ions of other mass tags in the
set. Mass tag and labeling reagent are equivalent terms for the
purposes of this description. Thus, a set of mass tags is the
equivalent of a set of labeling reagents.
[0068] As used herein, "support", "solid support" or "solid
carrier" refers to any solid phase material upon which a labeling
reagent or analyte can be immobilized. Immobilization can, for
example, be used to label analytes or be used to prepare a labeling
reagent, whether or not the labeling occurs on the support. Solid
support encompasses terms such as "resin", "synthesis support",
"solid phase", "surface" "membrane" and/or "support". A solid
support can be composed of organic polymers such as polystyrene,
polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy,
and polyacrylamide, as well as co-polymers and grafts thereof. A
solid support can also be inorganic, such as glass, silica,
controlled-pore-glass (CPG), or reverse-phase silica. The
configuration of a solid support can be in the form of beads,
spheres, particles, granules, a gel, a membrane or a surface.
Surfaces can be planar, substantially planar, or non-planar. Solid
supports can be porous or non-porous, and can have swelling or
non-swelling characteristics. A solid support can be configured in
the form of a well, depression or other container, vessel, feature
or location. A plurality of solid supports can be configured in an
array at various locations, addressable for robotic delivery of
reagents, or by detection methods and/or instruments.
[0069] As used herein, a "library" is a plurality of different
compounds (e.g., labeling reagents, mass tags, labeled analytes, or
the like), typically 5, 10, 25, 50, 100, 250 or more different
compounds. A library is typically configured for ease of
sequential, random, and/or parallel access to one, a plurality,
and/or all of the different compounds therein. For example, the
plurality of different compounds a library can be in the same
flask, or can be immobilized on one or more solid supports, or the
like. Typically, a library can have at least two different
compounds immobilized at different locations, e.g., on physically
distinct supports (e.g., beads, spheres, particles, granules, or
the like) or at addressable locations on the same support (e.g., as
a random or regular array on a solid support). The different
compounds in a library can be contacted with other compounds (e.g.,
one or more analytes can be reacted with a library of labeling
reagents) or can be analyzed (e.g., a library of labeled analytes
can be analyzed), or the like. For example, in some embodiments, a
library can comprise a plurality of different labeling reagents,
wherein each different labeling reagent can be immobilized at a
known address in a regular array on a solid support. The library
can be used to label a plurality of separate analyte samples with
particular labeling reagents by separately spotting (contacting)
the analyte samples to each different immobilized labeling reagent,
thereby producing a plurality of labeled analytes. In another
example, a library of labeling reagents can have a different
labeling reagent immobilized on each of a plurality of solid
particles. The library can be employed by contacting each solid
particle with a different analyte sample, whereby a plurality of
labeled analytes are immobilized to the solid particles.
[0070] As used herein, an "affinity ligand" refers to a molecule
that is a member of a molecular recognition system.
[0071] As used herein, a "molecular recognition system" refers to a
system of at least two molecules or complexes which have a high
capacity of molecular recognition for each other and a high
capacity to specifically bind to each other. In a some embodiments,
the binding is specific, and the affinity ligand is part of a
binding pair.
[0072] Unless specified as a covalent bond, the term "bind" or
"bound" includes both covalent and non-covalent associations.
[0073] "Specific binding," as used herein, refers to when an
affinity ligand of a molecular recognition system binds one or more
other molecule or complex, with specificity sufficient to
differentiate between the molecule or complex and other components
or contaminants of a sample. Molecular recognition systems for use
in the invention are conventional and are not described here in
detail. Techniques for preparing and utilizing such systems are
well known in the art and are exemplified in the publication of
Tijssen, P., "Laboratory Techniques in Biochemistry and Molecular
Biology Practice and Theories of Enzyme Immunoassays" (1988), eds.
Burdon and Knippenberg, New York:Elsevier, the entire teachings of
which are incorporated herein. Examples of molecular recognition
systems include, for example, an antigen/antibody, an
antigen/antibody fragment, an avidin/biotin, a streptavidin/biotin,
a protein A/I.sub.g or a lectin/carbohydrate.
[0074] As used herein, "natural isotopic abundance" refers to the
level (or distribution) of one or more isotopes found in a compound
based upon the natural prevalence of an isotope or isotopes in
nature. For example, a natural compound obtained from living plant
matter can typically contain about 1.08% .sup.13C relative to
.sup.12C
[0075] As used herein, "amino acid" refers to a group represented
by --NH--CHR.sup.#--C(O)--, wherein R.sup.# is hydrogen, deuterium,
an aliphatic group, a substituted aliphatic group, an aromatic
group or a substituted aromatic group. A "naturally-occurring amino
acid" is found in nature. Examples include glycine, alanine,
valine, leucine, isoleucine, aspartic acid, glutamic acid, serine,
threonine, glutamine, asparagine, arginine, lysine, ornithine,
proline, hydroxyproline, phenylalanine, tyrosine, tryptophan,
cysteine, methionine and histidine. In some embodiments, R.sup.#
can be a side-chain of a naturally-occurring amino acid. Examples
of naturally occurring amino acid side-chains include methyl
(alanine), isopropyl (valine), sec-butyl (isoleucine),
--CH.sub.2CH(--CH.sub.3).sub.2 (leucine), benzyl (phenylalanine),
p-hydroxybenzyl (tyrosine), --CH.sub.2--OH (serine), --CHOHCH.sub.3
(threonine), --CH.sub.2-3-indoyl (tryptophan), --CH.sub.2COOH
(aspartic acid), --CH.sub.2CH.sub.2COOH (glutamic acid),
--CH.sub.2C(O)NH.sub.2 (asparagine), --CH.sub.2CH.sub.2C(O)NH.sub.2
(glutamine), --CH.sub.2SH, (cysteine), --CH.sub.2CH.sub.2SCH.sub.3
(methionine), --(CH.sub.2).sub.4NH.sub.2 (lysine),
--(CH.sub.2).sub.3NH.sub.2 (ornithine),
-{(CH).sub.2}.sub.4NHC(.dbd.NH)NH.sub.2 (arginine) and
--CH.sub.2-3-imidazoyl (histidine).
[0076] The side-chains of other naturally-occurring amino acids
comprise a heteroatom-containing functional group, e.g., an alcohol
(serine, tyrosine, hydroxyproline and threonine), an amine (lysine,
ornithine, histidine and arginine), a thiol (cysteine) or a
carboxylic acid (aspartic acid and glutamic acid). When the
heteroatom-containing functional group is modified to include a
protecting group, the side-chain is referred to as the "protected
side-chain" of an amino acid. In some embodiments, R.sup.w is a
protected side-chain of an amino acid.
[0077] The selection of a suitable protecting group depends upon
the functional group being protected, the conditions to which the
protecting group is being exposed and to other functional groups
that may be present in the molecule. Suitable protecting groups for
the functional groups discussed above are well known in the art and
many examples are described in Greene and Wuts, "Protective Groups
in Organic Synthesis", John Wiley & Sons (1991). The skilled
artisan can select, using no more than routine experimentation,
suitable protecting groups for use in the disclosed synthesis,
including protecting groups other than those described below, as
well as conditions for applying and removing the protecting
groups.
[0078] As used herein, a "peptide" refers to a polymer comprising
two or more amino acids linked together by amide (peptide)
bonds.
[0079] As used herein, the terms "optionally substituted" and
"substituted or unsubstituted" are equivalent.
[0080] As used herein, a halo group refers to --F, --Cl, --Br, or
--I.
[0081] As used herein, the term "alkyl," refers to a straight
chained or branched C.sub.1-C.sub.20 hydrocarbon or a cyclic
C.sub.3-C.sub.20 hydrocarbon that is completely saturated. When
used herein the term "alkyl" refers to a group that may be
substituted or unsubstituted. In some embodiments, alkyl can be a
straight chained or branched C.sub.1-C.sub.6 hydrocarbon or a
cyclic C.sub.3-C.sub.6 hydrocarbon that is completely
saturated.
[0082] As used herein, the term "alkylene" refers to a straight or
branched alkyl chain or a cyclic alkyl that is optionally
substituted and that has at least two points of attachment to at
least two moieties (e.g., {--CH.sub.2--, methylene},
--{CH.sub.2CH.sub.2--, ethylene}, ##STR3## etc., wherein the
brackets indicate the points of attachement). When used herein the
term "alkylene" refers to a group that may be substituted or
unsubstituted.
[0083] As used herein, the term "alkenyl" refers to straight
chained or branched C.sub.2-C.sub.20 hydrocarbons or cyclic
C.sub.3-C.sub.20 hydrocarbons that have one or more double bonds.
When used herein the term "alkenyl" refers to a group that can be
substituted or unsubstituted. In some embodiments, alkenyl groups
can be straight chained or branched C.sub.2-C.sub.6 hydrocarbon or
cyclic C.sub.3-C.sub.6 hydrocarbons that have one or more double
bonds.
[0084] As used herein, the term "alkenylene" refers to an alkenyl
group that has two points of attachment to at least two moieties.
When used herein the term "alkenylene" refers to a group that may
be substituted or unsubstituted.
[0085] As used herein, the term "alkynyl" refers to straight
chained or branched C.sub.2-C.sub.20 hydrocarbons or cyclic
C.sub.3-C.sub.20 hydrocarbons that have one or more triple bonds.
When used herein the term "alkynyl" refers to a group that can be
substituted or unsubstituted. In some embodiments, alkynyl groups
can be straight chained or branched C.sub.2-C.sub.6 hydrocarbon or
cyclic C.sub.3-C.sub.6 hydrocarbons that have one or more triple
bonds.
[0086] As used herein, the term "alkynylene" refers to an alkynyl
group that has two points of attachment to at least two moieties.
When used herein the term "alkynylene" refers to a group that may
be substituted or unsubstituted.
[0087] As used herein, the term "aliphatic" refers to any of the
straight, branched, or cyclic alkyl, alkenyl, and alkynyl moieties
as defined above. When used herein the term "aliphatic" refers to a
group that may be substituted or unsubsituted.
[0088] As used herein, the term "heteroalkyl" refers to an alkyl
group in which one or more methylene groups in the alkyl chain is
replaced by a heteroatom such as --O--, --S--, and --NR--. R can be
a hydrogen, deuterium, alkyl, aryl, arylalkyl, alkenyl, alkynyl,
heteroaryl, heteroarylalkyl, or heterocycloalkyl. When used herein,
the term "heteroalkyl" refers to a group that can be substituted or
unsubstituted.
[0089] As used herein, the term "heteroalkylene" refers to a group
having the formula -{(alkylene-X').sub.r-alkylene}-, wherein X',
for each occurrence, is --O--, --NR--, or --S--; and r is an
integer from 1 to 10. When used herein, the term "heteroalkylene"
refers to a group that can be substituted or unsubstituted. In some
embodiments, r can be an integer from 1 to 5.
[0090] As used herein, the term "azaalkylene" refers to a
heteroalkylene wherein at least one X' is --NR--. When used herein,
the term "azaalkylene" refers to a group that can be substituted or
unsubstituted.
[0091] The term "aryl," as used herein, either alone or as part of
another moiety (e.g., arylalkyl, etc.), refers to carbocyclic
aromatic groups such as phenyl. Aryl groups also include fused
polycyclic aromatic ring systems in which a carbocyclic aromatic
ring is fused to another carbocyclic aromatic ring (e.g.,
1-naphthyl, 2-naphthyl, 1-anthracyl, 2-anthracyl, etc.) or in which
a carbocylic aromatic ring is fused to one or more carbocyclic
non-aromatic rings (e.g., tetrahydronaphthylene, indan, etc.). As
used herein, the term "aryl" refers to a group that may be
substituted or unsubstituted.
[0092] As used herein, the term "arylene" refers to an aryl group
that has at least two points of attachment to at least two moieties
(e.g., phenylene, etc.). The point of attachment of an arylene
fused to a carbocyclic, non-aromatic ring may be on either the
aromatic, non-aromatic ring. As used herein, the term "arylene"
refers to a group that may be substituted or unsubstituted.
[0093] As used herein, the term "arylalkyl" refers to an aryl group
that is attached to another moiety via an alkylene linker. As used
herein, the term "arylalkyl" refers to a group that may be
substituted or unsubstituted.
[0094] As used herein, the term "arylalkylene" refers to an
arylalkyl group that has at least two points of attachment to at
least two moieties. The second point of attachment can be on either
the aromatic ring or the alkylene. As used herein, the term
"arylalkylene" refers to a group that may be substituted or
unsubstituted. When an arylalkylene is substituted, the
substituents may be on either or both of the aromatic ring or the
alkylene portion of the arylalkylene.
[0095] As used herein, the term "heteroaryl," refers to an aromatic
heterocycle which comprises 1, 2, 3 or 4 heteroatoms independently
selected from nitrogen, sulfur and oxygen. As used herein, the term
"heteroaryl" refers to a group that may be substituted or
unsubstituted. A heteroaryl may be fused to one or two rings, such
as a cycloalkyl, a heterocycloalkyl, an aryl, or a heteroaryl. The
point of attachment of a heteroaryl to a molecule may be on the
heteroaryl, cycloalkyl, heterocycloalkyl or aryl ring, and the
heteroaryl group may be attached through carbon or a heteroatom.
Heteroaryl groups may be substituted or unsubstituted. Examples of
heteroaryl groups include imidazolyl, furyl, pyrrolyl, thienyl,
oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, thiadiazolyl,
oxadiazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl,
quinolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzisooxazolyl,
benzofuryl, benzothiazolyl, indolizinyl, imidazopyridinyl,
pyrazolyl, triazolyl, isothiazolyl, oxazolyl, tetrazolyl,
benzimidazolyl, benzothiazolyl, benzoisothiazolyl,
benzothiadiazolyl, benzoxadiazolyl, indolyl, tetrahydroindolyl,
azaindolyl, imidazopyridyl, quinazolinyl, purinyl,
pyrrolo[2,3]pyrimidyl, pyrazolo[3,4]pyrimidyl or benzo(b)thienyl,
each of which is optionally substituted.
[0096] As used herein, the term "heteroarylene" refers to a
heteroaryl group that has at least two points of attachment to at
least two moieties. As used herein, the term "heteroarylene" refers
to a group that may be substituted or unsubstituted.
[0097] As used herein, the term "azaarylene" refers to a
heteroarylene in which one of the heteroatoms is a nitrogen.
Azaarylenes may also comprise 1, 2, or 3 non-nitrogen heteroatoms
such as S and O. As used herein, the term "azaarylene" refers to a
group that may be substituted or unsubstituted.
[0098] As used herein, the term "heteroarylalkyl" refers to a
heteroaryl group that is attached to another moiety via an alkylene
linker. As used herein, the term "heteroarylalkyl" refers to a
group that may be substituted or unsubstituted.
[0099] As used herein, the term "heteroarylalkylene" refers to a
heteroarylalkyl group that has at least two points of attachment to
at least two moieties. The second points of attachment can be on
either the hetroaromatic ring or the alkylene. As used herein, the
term "heteroarylalkylene" referst to a group that may be
substituted or unsubstituted. When a heteroarylalkylene is
substituted, the substituents may be on either or both of the
heteroaromatic ring or the alkylene portion of the
heteroarylalkylene.
[0100] As used herein, the term "heterocycloalkyl" refers to a
non-aromatic ring which comprise one or more oxygen, nitrogen or
sulfur (e.g., morpholine, piperidine, piperazine, pyrrolidine, and
thiomorpholine). As used herein, the term "heterocycloalkyl" refers
to a group that may be substituted or unsubstituted.
[0101] As used herein, the term "heterocycloalkylene" refers to a
heterocycloalkyl that has at least two points of attachment to at
least two moieties. As used herein, the term "heterocycloalkylene"
refers to a group that may be substituted or unsubstituted.
[0102] As used herein, the term "azacycloalkylene" refers to a
heterocycloalkylene in which one heteroatom is a nitrogen.
Azacycloalkylenes may also comprise 1, 2, or 3 non-nitrogen
heteroatoms such as S and O. As used herein, the term
"azacycloalkylene" refers to a group that may be substituted or
unsubstituted.
[0103] Suitable substituents for an alkyl, alkylene, alkenylene,
alkynylene, heteroalkyl, heteroalkylene, azaalkylene,
heterocycloalkyl, heterocycloalkylene, azacycloalkylene, aryl,
arylene, arylalkyl, arylalkylene, heteroaryl, heteroarylene,
azaarylene, heteroarylalkyl, and heteroarylalkylene groups include
any substituent that is stable under the reaction conditions used
to label analytes with the mass tags of the invention. Examples of
substituents for an alkyl, an alkylene, alkenylene, alkynylene,
heteroalkyl, heteroalkylene, azaalkylene, heterocycloalkyl,
heterocycloalkylene, azacycloalkylene, aryl, arylene, arylalkyl,
arylalkylene, heteroaryl, heteroarylene, azaarylene,
heteroarylalkyl, and heteroarylalkylene include deuterium, an aryl
(e.g., phenyl) group, an arylalkyl (e.g., benzyl) group, a nitro
group, a cyano group, a halo (e.g., fluorine, chlorine, bromine and
iodine) group, a alkyl (e.g., methyl, ethyl, isopropyl, cyclohexyl,
etc.) group, a haloalkyl (e.g., trifluoromethyl) group, an alkoxy
(e.g., methoxy, ethoxy, etc.) group, a hydroxy group,
--NR.sup.wR.sup.w, --NR.sup.wC(O)R.sup.o, --C(O)NR.sup.wR.sup.w,
--C(O)R.sup.w, --C(O)OR.sup.w, wherein each R.sup.w is
independently, hydrogen, deuterium, an alkyl, an aryl, or an
arylalkyl; and R.sup.o for each occurrence is, independently, an
alkyl, an aryl, or an arylalkyl. In addition, substituents for an
aryl, an arylene, a heteroaryl or a heteroarylene can be a group
that includes an affinity ligand or a group that includes a solid
support.
[0104] In addition, alkyl, alkylene, heteroalkyl, heteroalkylene,
azaalkylene, a heterocycloalkyl, a heterocycloalkylene
azacycloalkylene groups, and any saturated portion of a alkenyl,
alkenylene, alkynyl, alkynylene, arylalkyl, arylalkylene,
heteroarylalkyl, and heteroarylalkylene groups, may also be
substituted with .dbd.O, .dbd.S, .dbd.N--R.sup.w.
[0105] When a heterocycloalkyl, heterocycloalkylene, heteroaryl,
heteroarylene, heteroarylalkyl, or heteroarylalkylene group
contains a nitrogen atom, it may be substituted or unsubstituted.
When a nitrogen atom in the aromatic ring of a heteroaryl group has
a substituent the nitrogen may be a quaternary nitrogen.
[0106] Suitable substituents for an aliphatic group, non-aromatic
heterocyclic group, benzylic group, an aryl group ring carbon and a
heteroaryl ring carbon are those which do not substantially
interfere with the labeling reaction of the reactive group of the
disclosed compounds. Examples of suitable substituents can include
deuterium, --OH, halogen (--F, --Cl, --Br, --I), --CN, --NO.sub.2,
--OR.sup.a, --C(O)R.sup.a, --OC(O)R.sup.a, --C(O)OR.sup.a,
--SR.sup.a, --C(S)R.sup.a, --OC(S)R.sup.a, --C(S)OR.sup.a,
--C(O)SR.sup.a, --C(S)SR.sup.a, --S(O)R.sup.a, --SO.sub.2R.sup.a,
--SO.sub.3R.sup.a, --PO.sub.2R.sup.aR.sup.b,
--PO.sub.3R.sup.aR.sup.b, --OPO.sub.3R.sup.aR.sup.b,
--N(R.sup.aR.sup.b), --C(O)N(R.sup.aR.sup.b),
--C(O)NR.sup.aNR.sup.bSO.sub.2R.sup.c,
--C(O)NR.sup.aSO.sub.2R.sup.c, --C(O)NR.sup.aCN,
--SO.sub.2N(R.sup.aR.sup.b), --NR.sup.aSO.sub.2R.sup.c,
--NR.sup.cC(O)R.sup.a, --NR.sup.cC(O)OR.sup.a,
--NR.sup.cC(O)N(R.sup.aR.sup.b), --C(NR.sup.c)--N(R.sup.aR.sup.b),
--NR--C(NR.sup.c)--N(R.sup.aR.sup.b), NR.sup.aN(R.sup.aR.sup.b),
--CR.sup.c.dbd.CR.sup.aR.sup.b, --C.ident.CR.sup.a, .dbd.O, .dbd.S,
.dbd.CR.sup.aR.sup.b, .dbd.NR.sup.a, .dbd.NOR.sup.a,
.dbd.NNR.sup.a, optionally substituted alkyl, optionally
substituted cycloalkyl, optionally substituted aliphatic,
optionally substituted cycloaliphatic, optionally substituted
non-aromatic heterocyclic, optionally substituted benzyl,
optionally substituted aryl, and optionally substituted heteroaryl,
wherein R.sup.a-R.sup.d are each independently --H, deuterium (D),
or an optionally substituted aliphatic, optionally substituted
cycloaliphatic, optionally substituted non-aromatic heterocyclic,
optionally substituted benzyl, optionally substituted aryl, or
optionally substituted heteroaryl, preferably an alkyl, benzylic or
phenyl group. In addition, --N(R.sup.aR.sup.b), taken together, can
be an optionally substituted heterocyclic group.
[0107] A non-aromatic heterocyclic group, benzylic group or aryl
group can also have an aliphatic or substituted aliphatic group as
a substituent. A substituted aliphatic group can also have a
non-aromatic heterocyclic ring, a substituted a non-aromatic
heterocyclic ring, benzyl, substituted benzyl, aryl or substituted
aryl group as a substituent. A substituted aliphatic, non-aromatic
heterocyclic group, substituted aryl, or substituted benzyl group
can have more than one substituent.
[0108] Suitable substituents for heteroaryl ring nitrogen atoms
having three covalent bonds to other heteroaryl ring atoms include
--OH and lower alkoxy (preferably C1-C4 alkoxy). Substituted
heteroaryl ring nitrogen atoms that have three covalent bonds to
other heteroaryl ring atoms are positively charged, which can be
balanced by counteranions such as chloride, bromide, formate,
acetate and the like. Examples of other suitable counteranions are
provided in the section below directed to pharmacologically
acceptable salts.
[0109] Suitable substituents for nitrogen atoms having two covalent
bonds to other atoms (e.g., heteroaryl ring nitrogen atoms having
two covalent bonds to other ring atoms) include, for example,
optionally substituted alkyl, optionally substituted cycloalkyl,
optionally substituted aliphatic, optionally substituted
cycloaliphatic, optionally substituted heterocyclic, optionally
substituted benzyl, optionally substituted aryl, optionally
substituted heteroaryl, --CN, --NO.sub.2, --OR.sup.a,
--C(O)R.sup.a, --OC(O)R.sup.a, --C(O)OR.sup.a, --SR.sup.a,
--S(O)R.sup.a, --SO.sub.2R.sup.a, --SO.sub.3R.sup.a,
--N(R.sup.aR.sup.b), --C(O)N(R.sup.aR.sup.b),
--C(O)NR.sup.aNR.sup.bSO.sub.2R.sup.c,
--C(O)NR.sup.aSO.sub.2R.sup.c, --C(O)NR.sup.aCN,
--SO.sub.2N(R.sup.aR.sup.b), --SO.sub.2N(R.sup.aR.sup.b),
--NR.sup.cC(O)R.sup.a, --NR.sup.cC(O)OR.sup.a,
--NR.sup.cC(O)N(R.sup.aR.sup.b), and the like. More typically, the
substituents for nitrogen atoms having two covalent bonds to other
atoms can be alkyl, substituted alkyl (including haloalkyl),
phenyl, substituted phenyl, --S(O).sub.2-(alkyl),
--S(O).sub.2--NH(alkyl) and --S(O).sub.2--NH(alkyl).sub.2.
[0110] A nitrogen-containing heteroaryl or non-aromatic heterocycle
can be substituted with oxygen to form an N-oxide, e.g., as in a
pyridyl N-oxide, piperidyl N-oxide, and the like.
[0111] As used herein, the term "salt form," includes a salt of a
compound (labeling reagent), or a mixture of salts of a compound.
In addition, zwitterionic forms of a compound are also included in
the term "salt form." Salts of mass tags having an amine, or other
basic group can be obtained, for example, by reacting with a
suitable organic or inorganic acid, such as hydrogen chloride,
hydrogen bromide, acetic acid, perchloric acid and the like.
Compounds with a quaternary ammonium group may also contain a
counteranion such as chloride, bromide, iodide, acetate,
perchlorate and the like. Salts of compounds having a carboxylic
acid, or other acidic functional group, can be prepared by reacting
the compound with a suitable base, for example, a hydroxide base.
Accordingly, salts of acidic functional groups may have a
countercation, such as sodium, potassium, magnesium, calcium,
etc.
[0112] The term "hydrate form" comprises any hydration state of a
compound or a mixture of more than one hydration state of a
compound. For example, a mass tag of the invention can be a
hemihydrate, a monohydrate, a dihydrate, etc.
3. General
Overview
The Reactive Group:
[0113] The variable "RG" of the labeling reagent or reagents used
in the method, mixture, kit and/or composition embodiments can be
either a reactive group, e.g., an electrophilic group or a
nucleophilic group that is capable of reacting with one or more
reactive analytes of a sample, or the reaction product of the
reactive group and the analyte. The reactive group can be
preexisting or it can be prepared in-situ. In some embodiments,
in-situ preparation of the reactive group can proceed in the
absence of the reactive analyte and in some embodiments, it can
proceed in the presence of the reactive analyte. For example, a
carboxylic acid group can be modified in-situ with water-soluble
carbodiimide (e.g. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride; EDC) to thereby prepare an electrophilic group that
can be reacted with a nucleophilic group such as an amine group. In
some embodiments, activation of the carboxylic acid group of a
labeling reagent with EDC can be performed in the presence of an
amine (nucleophilic group) containing analyte. In some embodiments,
the amine (nucleophilic group) containing analyte can also be added
after the initial reaction with EDC is performed. In some
embodiments, the reactive group can be generated in-situ by the
in-situ removal of a protecting group. Consequently, any existing
or newly created reagent or reagents that can effect the
derivatization of analytes by the reaction of nucleophilic groups
and/or electrophilic groups are contemplated by the method,
mixture, kit and/or composition embodiments of this invention.
[0114] Where the reactive group of the labeling reagent is an
electrophilic group, it can react with a suitable nucleophilic
group of the analyte or analytes. Where the reactive group of the
labeling reagent is a nucleophilic group, it can react with a
suitable electrophilic group of the analyte or analytes. Numerous
pairs of suitable nucleophilic groups and electrophilic groups are
known and often used in the chemical and biochemical arts.
Non-limiting examples of reagents comprising suitable nucleophilic
or electrophilic groups that can be coupled to analytes (e.g. such
as proteins, peptides, nucleotides, carbohydrates, lipids, steroids
or other small molecules of less that 1500 daltons) to effect their
derivatization, are described in the Pierce Life Science &
Analytical Research Products Catalog & Handbook (a Perstorp
Biotec Company), Rockford, Ill. 61105, USA. Other suitable reagents
are well known in the art and are commercially available from
numerous other vendors such as Sigma-Aldrich.
[0115] The reactive group of a labeling reagent can be an amine
reactive group. For example the amine reactive group can be an
active ester. Active esters are well known in peptide synthesis and
refer to certain esters that are easily reacted with the N-.alpha.
amine of an amino acid under conditions commonly used in peptide
synthesis. The amine reactive active ester can be an
N-hydroxysuccinimidyl ester, a N-hydroxysulfosuccinimidyl ester, a
pentafluorophenyl ester, a 2-nitrophenyl ester, a 4-nitrophenyl
ester, a 2,4-dinitrophenylester or a 2,4-dihalophenyl ester.
[0116] FIG. 8 illustrates exemplary formulas of leaving groups (LG)
for the alcohol or thiol group of an active ester wherein each G is
independently O or S, but typically O. All of these groups are
alcohol or thiol groups known to form active esters in the field of
peptide chemistry wherein said alcohol or thiol group is displaced
by the reaction of the N-.alpha.-amine of the amino acid with the
carbonyl carbon of the ester. It should be apparent that the active
ester (e.g. N-hydroxysuccinimidyl ester) of any suitable
labelling/tagging reagent described herein could be prepared using
well-known procedures (See: Greg T. Hermanson (1996). "The
Chemistry of Reactive Groups" in "Bioconjugate Techniques" Chapter
2 pages 137-165, Academic Press, (New York); also see: Innovation
And Perspectives In Solid Phase Synthesis, Editor: Roger Epton,
SPCC (UK) Ltd, Birmingham, 1990). Methods for the formation of
active esters of morpholine acetic acid, piperidine acetic acid,
piperazine acetic acid and N-substituted piperazine acetic acids
compounds that are representative examples of labeling reagents of
the general formula: RP--X-LK--Y--RG are described in co-pending
and commonly owned U.S. patent application Ser. No. 10/751,354,
filed on Jan. 27, 2004 the entire teachings of which are
incorporated herein by reference for all purposes.
[0117] In some embodiments, the reactive group of the labeling
reagent can be a mixed anhydride since mixed anhydrides are known
to efficiently react with amine groups to thereby produce amide
bonds.
[0118] The reactive group of a labeling reagent can be a thiol
reactive group. For example, the thiol reactive group can be a
malemide, an alkyl halide, an aryl halide of an .alpha.-halo-acyl
(a.k.a. acyl halide). Halide and halo refer to atoms of fluorine,
chlorine, bromine or iodine. In some embodiments, the RG group is
I--(CH.sub.2)C(O)--.
[0119] The reactive group of a labeling reagent can be a hydroxyl
reactive group. For example, the hydroxyl reactive group can be a
trityl-halide or a silyl-halide reactive moiety. The trityl-halide
reactive moieties can be substituted (e.g. Y-methoxytrityl,
Y-dimethoxytrityl, Y-trimethoxytrityl, etc) or unsubstituted
wherein Y is defined below. The silyl reactive moieties can be
alkyl substituted silyl halides, such as Y-dimethylsilyl,
Y-ditriethylsilyl, Y-dipropylsilyl, Y-diisopropylsilyl, etc.)
wherein Y is defined below.
[0120] The reactive group of the labeling reagent can be a
nucleophilic group. In some embodiments, the RG group is an amine
group, a hydroxyl group, a thiol group or an --NH--NH.sub.2 group,
more typically an amine group, a hydroxyl group, or a thiol
group.
[0121] The reactive group can be a group capable of reacting with a
guanidine group on an analyte. In some embodiments, the RG group is
##STR4##
[0122] The reactive group can be a photoreactive group. In some
embodiments, the RG group is ##STR5##
[0123] In some embodiments, the labeling reagents of the invention
comprise 2 or more RG groups. Thus, a labeling reagent of formula
RP--X-LK--(Y--RG).sub.y is provided wherein y is 1-3. In some
embodiments, y is 2.
The Reporter Moiety:
[0124] The reporter moiety of the labeling reagent or reagents used
in the method, mixture, kit and/or composition embodiments is a
group that has a unique mass (or mass to charge ratio) that can be
determined. Accordingly, each reporter of a set can have a unique
gross mass. Different reporters can comprise one or more heavy atom
isotopes to achieve their unique mass. For example, isotopes of
carbon (.sup.12C, .sup.13C and .sup.14C), nitrogen (.sup.14N and
.sup.15 N), oxygen (.sup.16O and .sup.18O) or hydrogen (hydrogen,
deuterium and tritium) exist and can be used in the preparation of
a diverse group of reporter moieties. Examples of stable heavy atom
isotopes include .sup.13C, .sup.15N, .sup.18O and deuterium. Cost
of the labeling reagent can be reduced and isotopic purity
increased by avoiding .sup.18O in the reagent. These examples of
isotopes are not limiting as other light and heavy atom isotopes
can also be used in the reporter. Basic starting materials suitable
for preparing reporters comprising light and heavy atom isotopes
are available from various commercial sources such as Cambridge
Isotope Laboratories, Andover, Mass. (See: list or "basic starting
materials" at www.isotope.com) and Isotec (a division of
Sigma-Aldrich). Cambridge Isotope Laboratories and Isotec will also
prepare desired compounds under custom synthesis contracts. Id.
[0125] A unique reporter can be associated with a sample of
interest thereby labeling one or multiple analytes of that sample
with a labeling reagent comprising the reporter. In this way
information about the reporter can be associated with information
about one or all of the analytes of the sample. However, the
reporter need not be physically linked to an analyte when the
reporter is determined. Rather, the unique gross mass of the
reporter can, for example, be determined in a second mass analysis
of a tandem mass analyzer, after ions of the labeled analyte are
fragmented to thereby produce daughter fragment ions and detectable
reporters. The determined reporter can be used to identify the
sample from which a determined analyte originated. Further, the
amount of the unique reporter, either relative to the amount of
other reporters or relative to one or more calibration standards
(e.g. an analyte labeled with a specific reporter), can be used to
determine the relative or absolute amount (often expressed as a
concentration and/or quantity) of analyte in the sample or samples.
Therefore information, such as the amount of one or more analytes
in a particular sample, can be associated with the reporter moiety
that is used to label each particular sample. Where the identity of
the analyte or analytes is also determined, that information can be
correlated with information pertaining to the different reporters
to thereby facilitate the determination of the identity and amount
of each labeled analyte in one or a plurality of samples.
[0126] The reporter either comprises a fixed charge or is capable
of becoming ionized. Because the reporter either comprises a fixed
charge or is capable of being ionized, the labeling reagent might
be isolated or used to label the reactive analyte in a salt or
zwitterionic form. Ionization of the reporter facilitates its
determination in a mass spectrometer. Accordingly, the reporter can
be determined as a ion, sometimes referred to as a signature ion.
When ionized, the reporter can comprise one or more net positive or
negative charges. Thus, the reporter can comprise one or more
acidic groups or basic groups since such groups can be easily
ionized in a mass spectrometer. For example, the reporter can
comprise one or more basic nitrogen atoms (positive charge) or one
or more ionizable acidic groups such as a carboxylic acid group,
sulfonic acid group or phosphoric acid group (negative charge). In
some embodiments, the reporter can comprise a substituted or
unsubstituted benzyl ion.
[0127] The reporter can be selected so that it does not
substantially sub-fragment under conditions typical for the
analysis of the analyte. The reporter can be chosen so that it does
not substantially sub-fragment under conditions of dissociative
energy applied to cause fragmentation of both bonds X and Y of at
least a portion of selected ions of a labeled analyte in a mass
spectrometer. By "does not substantially sub-fragment" we mean that
fragments of the reporter are difficult or impossible to detect
above background noise when applied to the successful analysis of
the analyte of interest. The gross mass of a reporter can be
intentionally selected to be different as compared with the mass of
the analyte sought to be determined or any of the expected
fragments of the analyte. For example, where proteins or peptides
are the analytes, the reporter's gross mass can be chosen to be
different as compared with any naturally occurring amino acid or
peptide, or expected fragments thereof. This can facilitate analyte
determination since, depending on the analyte, the lack of any
possible components of the sample having the same coincident mass
can add confidence to the result of any analysis. Examples of mass
ranges where little background can be expected for peptides can be
found in Table 1. TABLE-US-00001 TABLE 1 Possible "Quiet Zones" For
Selection Of Label Fragment Ion m/z M/z start-end 10-14 19-22 24-26
31-38 40-40 46-50 52-52 58-58 61-69 71-71 74-83 89-97 103-109
113-119 121-125 128-128 131-135 137-147 149-154 156-156 160-174
177-182 184-184 188-189 191-191 202-207 210-210 216-222 224-226
[0128] The gross mass of a reporter can be less than 250 Daltons.
Such a small molecule can be easily determined in the second mass
analysis, free from other components of the sample having the same
coincident mass in the first mass analysis. In this context, the
second mass analysis can be performed, typically in a tandem mass
spectrometer, on selected ions that are determined in the first
mass analysis. Because ions of a particular mass to charge ratio
can be specifically selected out of the first mass analysis for
possible fragmentation and further mass analysis, the non-selected
ions from the first mass analysis are not carried forward to the
second mass analysis and therefore do not contaminate the spectrum
of the second mass analysis. Furthermore, the sensitivity of a mass
spectrometer and the linearity of the detector (for purposes of
quantitation) can be quite robust in this low mass range.
Additionally, the present state of mass spectrometer technology can
allow for baseline mass resolution of less than one Dalton in this
mass range. These factors may prove to be useful advancements to
the state of the art.
The Linker Moiety:
[0129] The linker moiety represented by LK, LK.sup.1, LK.sup.2,
LK.sup.3, LK.sup.4, LK.sup.5 and LK.sup.6 of the compounds used
with the method, mixture, kit and/or composition embodiments links
the reporter to the analyte or the reporter to the reactive group
depending on whether or not a reaction with the analyte has
occurred. The linker can be selected to produce a neutral species
when both bonds X and Y are fragmented (i.e. undergoes neutral loss
upon fragmentation of both bonds X and Y). The linker can be a very
small moiety such as a carbonyl or thiocarbonyl group. For example,
the linker can comprise at least one heavy atom isotope and
comprise the formula: ##STR6## wherein each R.sup.1 is the same or
different and is an alkyl group comprising one to eight carbon
atoms which may optionally contain a heteroatom or a substituted or
unsubstituted aryl group wherein the carbon atoms of the alkyl and
aryl groups independently comprise linked hydrogen, deuterium
and/or fluorine atoms. The linker can be a larger moiety such as
amino acid or heteroalkyl. The linker can be a polymer or a
biopolymer (e.g., a peptide). The linker can be designed to
sub-fragment when subjected to dissociative energy levels;
including sub-fragmentation to thereby produce one or more neutral
fragments of the linker. In some embodiments, only neutral
fragments are produced from the linker.
[0130] FIGS. 9A-9B depict moieties i-xiv, which can be comprised by
the LK group in some embodiments. Each bond terminated with the
wavy line indicates the point of attachment to a reporter, support,
reactive group or analyte. The linker moiety can comprise one or
more heavy atom isotopes such that its mass compensates for the
difference in gross mass between the reporters for each labeled
analyte of a mixture or for the reagents of set and/or kit.
Moreover, the aggregate gross mass (i.e. the gross mass taken as a
whole) of the reporter-linker combination can be the same for each
labeled analyte of a mixture or for the reagents of set and/or kit.
More specifically, the linker moiety can compensate for the
difference in gross mass between reporters of labeled analytes from
different samples wherein the unique gross mass of the reporter
correlates with the sample from which the labeled analyte
originated and the aggregate gross mass of the reporter-linker
combination is the same for each labeled analyte of a sample
mixture regardless of the sample from which it originated. In this
way, the gross mass of identical analytes in two or more different
samples can have the same gross mass when labeled and then mixed to
produce a sample mixture.
[0131] For example, the labeled analytes, or labeling reagent
(e.g., mass tags) of a set and/or kit for labeling the analytes,
can be isomers or isobars. Thus, if ions of a particular mass to
charge ratio (taken from the sample mixture) are selected (i.e.
selected ions) in a mass spectrometer from an initial mass analysis
of the sample mixture, identical analytes from the different
samples that make up the sample mixture are represented in the
selected ions in proportion to their respective concentration
and/or quantity in the sample mixture. Accordingly, the linker not
only links the reporter to the analyte, it also can serve to
compensate for the differing masses of the unique reporter moieties
to thereby harmonize the gross mass of the reporter-linker
combination in the labeled analytes of the various samples.
[0132] Because the linker can act as a mass balance for the
reporter in the labeling reagents such that the aggregate gross
mass of the reporter-linker combination is the same for all
reagents of a set or kit, the greater the number of atoms in the
linker, the greater the possible number of different
isomeric/isobaric labeling reagents of a set and/or kit. Stated
differently, generally the greater the number of atoms that a
linker comprises, the greater number of potential reporter-linker
combinations exist since isotopes can be substituted at most any
position in the linker to thereby produce isomers or isobars of the
linker portion wherein the linker portion is used to offset the
differing masses of the reporter portion and thereby create a set
of reporter-linker isomers or isobars. Such diverse sets of
labeling reagents are particularly well suited for multiplex
analysis of analytes in the same and/or different samples.
[0133] The total number of labeling reagents of a set and/or kit
can be two, three, four, five, six, seven, eight, nine, ten or
more. The diversity of the labeling reagents of a set or kit is
limited only by the number of atoms of the reporter and linker
moieties, the heavy atom isotopes available to substitute for the
light isotopes and the various synthetic configurations in which
the isotopes can be synthetically placed. As suggested above
however, numerous isotopically enriched basic starting materials
are readily available from manufacturers such as Cambridge Isotope
Laboratories and Isotec. Such isotopically enriched basic starting
materials can be used in the synthetic processes used to produce
sets of isobaric and isomeric labeling reagents or be used to
produce the isotopically enriched starting materials that can be
used in the synthetic processes used to produce sets of isobaric
and isomeric labeling reagents. Some examples of the preparation of
isobaric labeling reagents suitable for use in a set of labeling
reagents can be found in the Examples section, below.
The Reporter-Linker Combination:
[0134] The labeling reagents described herein comprise reporters
and linkers that are linked through the bond X. As described above,
the reporter-linker combination can be identical in gross mass for
each member of a set and/or kit of labeling reagents. Moreover,
bond X of the reporter-linker combination of the labeling reagents
can be designed to fragment, in at least a portion of the selected
ions, when subjected to dissociative energy levels thereby
releasing the reporter from the analyte. Accordingly, the gross
mass of the reporter (as a m/z ratio) and its intensity can be
observed directly in MS/MS analysis.
[0135] The reporter-linker combination can comprise various
combinations of the same or different heavy atom isotopes amongst
the various labeling reagents of a set or kit. In the scientific
literature this has sometimes been referred to as coding or isotope
coding. For example, Abersold et al. has disclosed the isotope
coded affinity tag (ICAT; see WO 00/11208). In one respect, the
reagents of Abersold et al. differ from the labeling reagents of
this invention in that Abersold does not teach two or more same
mass labeling reagents such as isomeric or isobaric labeling
reagents.
Mass Spectrometers/Mass Spectrometry (MS):
[0136] The methods of this invention can be practiced using tandem
mass spectrometers and other mass spectrometers that have the
ability to select and fragment molecular ions. Tandem mass
spectrometers (and to a lesser degree single-stage mass
spectrometers) have the ability to select and fragment molecular
ions according to their mass-to-charge (m/z) ratio, and then record
the resulting fragment (daughter) ion spectra. More specifically,
daughter fragment ion spectra can be generated by subjecting
selected ions to dissociative energy levels (e.g. collision-induced
dissociation (CID)). For example, ions corresponding to labeled
peptides of a particular m/z ratio can be selected from a first
mass analysis, fragmented and reanalyzed in a second mass analysis.
Representative instruments that can perform such tandem mass
analysis include, but are not limited to, magnetic four-sector,
tandem time-of-flight, triple quadrupole, ion-trap, and hybrid
quadrupole time-of-flight (Q-TOF) mass spectrometers.
[0137] These types of mass spectrometers may be used in conjunction
with a variety of ionization sources, including, but not limited
to, electrospray ionization (ESI) and matrix-assisted laser
desorption ionization (MALDI). Ionization sources can be used to
generate charged species for the first mass analysis where the
analytes do not already possess a fixed charge. Additional mass
spectrometry instruments and fragmentation methods include
post-source decay in MALDI-MS instruments and high-energy CID using
MALDI-TOF (time of flight)-TOF MS. For a recent review of tandem
mass spectrometers please see: R. Aebersold and D. Goodlett, Mass
Spectrometry in Proteomics. Chem. Rev. 101: 269-295 (2001). Also
see U.S. Pat. No. 6,319,476, herein incorporated by reference for
all purposes, for a discussion of TOF-TOF mass analysis
techniques.
Fragmentation by Dissociative Energy Levels:
[0138] It is well accepted that bonds can fragment as a result of
the processes occurring in a mass spectrometer. Moreover, bond
fragmentation can be induced in a mass spectrometer by subjecting
ions to dissociative energy levels. For example, the dissociative
energy levels can be produced in a mass spectrometer by
collision-induced dissociation (CID). Those of ordinary skill in
the art of mass spectrometry will appreciate that other exemplary
techniques for imposing dissociative energy levels that cause
fragmentation include, but are not limited to, photo dissociation,
electron capture and surface induced dissociation.
[0139] The process of fragmenting bonds by collision-induced
dissociation involves increasing the kinetic energy state of
selected ions to a point where bond fragmentation occurs. For
example, kinetic energy can be transferred by collision with an
inert gas (such as nitrogen, helium or argon) in a collision cell.
The amount of kinetic energy that can be transferred to the ions is
proportional to the number of gas molecules that are allowed to
enter the collision cell. When more gas molecules are present, a
greater amount of kinetic energy can be transferred to the selected
ions, and less kinetic energy is transferred when there are fewer
gas molecules present.
[0140] It is therefore clear that the dissociative energy level in
a mass spectrometer can be controlled. It is also well accepted
that certain bonds are more labile than other bonds. The lability
of the bonds in an analyte or the reporter-linker moiety depends
upon the nature of the analyte or the reporter-linker moiety.
Accordingly, the dissociative energy levels can be adjusted so that
the analytes and/or the labels (e.g. the reporter-linker
combinations) can be fragmented in a manner that is determinable.
One of skill in the art will appreciate how to make such routine
adjustments to the components of a mass spectrometer to thereby
achieve the appropriate level of dissociative energy to thereby
fragment at least a portion of ions of labeled analytes into
ionized reporter moieties and daughter fragment ions.
[0141] For example, dissociative energy can be applied to ions that
are selected/isolated from the first mass analysis. In a tandem
mass spectrometer, the extracted ions can be subjected to
dissociative energy levels and then transferred to a second mass
analyzer. The selected ions can have a selected mass to charge
ratio. The mass to charge ratio can be within a range of mass to
charge ratios depending upon the characteristics of the mass
spectrometer. When collision induced dissociation is used, the ions
can be transferred from the first to the second mass analyzer by
passing them through a collision cell where the dissociative energy
can be applied to thereby produce fragment ions. For example the
ions sent to the second mass analyzer for analysis can include all,
some, or a portion, of the remaining (unfragmented) selected ions,
as well as reporter ions (signature ions) and daughter fragment
ions of the labeled analyte.
Analyte Determination by Computer Assisted Database Analysis:
[0142] In some embodiments, analytes can be determined based upon
daughter-ion fragmentation patterns that are analyzed by
computer-assisted comparison with the spectra of known or
"theoretical" analytes. For example, the daughter fragment ion
spectrum of a peptide ion fragmented under conditions of low energy
CID can be considered the sum of many discrete fragmentation
events. The common nomenclature differentiates daughter fragment
ions according to the amide bond that breaks and the peptide
fragment that retains charge following bond fission.
Charge-retention on the N-terminal side of the fissile amide bond
results in the formation of a b-type ion. If the charge remains on
the C-terminal side of the broken amide bond, then the fragment ion
is referred to as a y-type ion. In addition to b- and y-type ions,
the CID mass spectrum may contain other diagnostic fragment ions
(daughter fragment ions). These include ions generated by neutral
loss of ammonia (-17 amu) from glutamine, lysine and arginine or
the loss of water (-18 amu) from hydroxyl-containing amino acids
such as serine and threonine. Certain amino acids have been
observed to fragment more readily under conditions of low-energy
CID than others. This is particularly apparent for peptides
containing proline or aspartic acid residues, and even more so at
aspartyl-proline bonds (Mak, M. et al., Rapid Commun. Mass
Spectrom., 12: 837-842) (1998). Accordingly, the peptide bond of a
Z-pro dimer or Z-asp dimer, wherein Z is any natural amino acid,
pro is proline and asp is aspartic acid, will tend to be more
labile as compared with the peptide bond between all other amino
acid dimer combinations.
[0143] For peptide and protein samples therefore, low-energy CID
spectra contain redundant sequence-specific information in
overlapping b- and y-series ions, internal fragment ions from the
same peptide, and immonium and other neutral-loss ions.
Interpreting such CID spectra to assemble the amino acid sequence
of the parent peptide de novo is challenging and time-consuming.
The most significant advances in identifying peptide sequences have
been the development of computer algorithms that correlate peptide
CID spectra with peptide sequences that already exist in protein
and DNA sequence databases. Such approaches are exemplified by
programs such as SEQUEST (Eng, J. et al. J. Am. Soc. Mass
Spectrom., 5: 976-989 (1994)) and MASCOT (Perkins, D. et al.
Electrophoresis, 20: 3551-3567 (1999)).
[0144] In brief, experimental peptide CID spectra (MS/MS spectra)
are matched or correlated with `theoretical` daughter fragment ion
spectra computationally generated from peptide sequences obtained
from protein or genome sequence databases. The match or correlation
is based upon the similarities between the expected mass and the
observed mass of the daughter fragment ions in MS/MS mode. The
potential match or correlation is scored according to how well the
experimental and `theoretical` fragment patterns coincide. The
constraints on databases searching for a given peptide amino acid
sequence are so discriminating that a single peptide CID spectrum
can be adequate for identifying any given protein in a whole-genome
or expressed sequence tag (EST) database. For other reviews please
see: Yates, J. R. Trends, Genetics, 16: 5-8 (2000) and Yates, J.
R., Electrophoresis 19: 893-900 (1998).
[0145] Accordingly, daughter fragment ion analysis of MS/MS spectra
can be used not only to determine the analyte of a labeled analyte,
it can also be used to determine analytes from which the determined
analyte originated. For example, identification of a peptide in the
MS/MS analysis can be can be used to determine the protein from
which the peptide was cleaved as a consequence of an enzymatic
digestion of the protein. It is envisioned that such analysis can
be applied to other analytes, such as oligonucleotides.
Bonds X and Y:
[0146] X is a bond between an atom of the reporter and an atom of
the linker. Y is a bond between an atom of the linker and an atom
of either the reactive group or, if the labeling reagent has been
reacted with a reactive analyte, the analyte. Bonds X and Y of the
various labeling reagents (i.e. RP--X-LK--Y--RG) that can be used
in the embodiments of this invention can fragment, in at least a
portion of selected ions, when subjected to dissociative energy
levels. Therefore, the dissociative energy level can be adjusted in
a mass spectrometer so that both bonds X and Y fragment in at least
a portion of the selected ions of the labeled analytes (i.e.
RP--X-LK--Y-Analyte). Fragmentation of bond X releases the reporter
from the analyte so that the reporter can be determined
independently from the analyte. Fragmentation of bond Y releases
the reporter-linker combination from the analyte, or the linker
from the analyte, depending on whether or not bond X has already
been fragmented. Bond Y can be more labile than bond X. Bond X can
be more labile than bond Y. Bonds X and Y can be of the same
relative lability.
[0147] In some embodiments, bond X can be more labile than bond Y.
In some embodiments, bond X cleaves and bond Y remains intact. In
still other embodiments, bond X cleaves and bond Y cleaves.
[0148] When the analyte of interest is a protein or peptide, the
relative lability of bonds X and Y can be adjusted with regard to
an amide (peptide) bond. Bond X, bond Y or both bonds X and Y can
be more, equal or less labile as compared with a typical amide
(peptide) bond. For example, under conditions of dissociative
energy, bond X and/or bond Y can be less prone to fragmentation as
compared with the peptide bond of a Z-pro dimer or Z-asp dimer,
wherein Z is any natural amino acid, pro is proline and asp is
aspartic acid. In some embodiments, bonds X and Y will fragment
with approximately the same level of dissociative energy as a
typical amide bond. In some embodiments, bonds X and Y will
fragment at a greater level of dissociative energy as compared with
a typical amide bond.
[0149] In some embodiments, bonds X and Y can also exist such that
fragmentation of bond Y results in the fragmentation of bond X, and
vice versa. In this way, both bonds X and Y can fragment
essentially simultaneously such that no substantial amount of
analyte, or daughter fragment ion thereof, comprises a partial
label in the second mass analysis. By "substantial amount of
analyte" we mean that less than 25%, and preferably less than 10%,
partially labeled analyte can be determined in the MS/MS
spectrum.
[0150] Because there can be a clear demarcation between labeled and
unlabeled fragments of the analyte in the spectra of the second
mass analysis (MS/MS), this feature can simplify the identification
of the analytes from computer assisted analysis of the daughter
fragment ion spectra. Moreover, because the fragment ions of
analytes can, in some embodiments, be either fully labeled or
unlabeled (but not partially labeled) with the reporter/linker
moiety, there can be little or no scatter in the masses of the
daughter fragment ions caused by isotopic distribution across
fractured bonds such as would be the case where isotopes were
present on each side of a single labile bond of a partially labeled
analyte routinely determined in the second mass analysis.
Labeling of Analytes:
[0151] Analytes can be labeled by reacting a functional group of
the analyte with the reactive group (RG) of the labeling reagent.
As discussed previously, the functional group on the analyte can be
one of an electrophilic group or a nucleophilic group and the
functional group (i.e. the RG or reactive group) of the labeling
reagent can be the other of the electrophilic group or a
nucleophilic group. The electrophilic group and nucleophilic group
can react to form a covalent link between the analyte and the
labeling reagent.
[0152] The labeling reaction can take place in solution. In some
embodiments, one of the analyte or the labeling reagent can be
support bound. The labeling reaction can sometimes be performed in
aqueous conditions. Aqueous conditions can be selected for the
labeling of biomolecules such as proteins, peptides, nucleotides
and oligonucleotides. The labeling reaction can sometimes be
performed in organic solvent or a mixture of organic solvents.
Organic solvents can be selected for analytes that are small
molecules. Mixtures of water and organic solvent or organic
solvents can be used across a broad range. For example, a solution
of water and from about 60 percent to about 95 percent organic
solvent or solvents (v/v) can be prepared and used for labeling the
analyte. In some embodiments, a solution of water and from about 65
percent to about 80 percent organic solvent or solvents (v/v) can
be prepared and used for labeling the analyte. Non-limiting
examples of organic solvents include N,N'-dimethylformamide (DMF),
acetonitrile (ACN), and alcohol such as methanol, ethanol, propanol
and/or butanol.
[0153] When performing a labeling reaction, the pH can be
modulated. The pH can be in the range of 4-10. The pH can be
outside this range. Generally, the basicity of non-aqueous
reactions can be modulated by the addition of non-nucleophilic
organic bases. Non-limiting examples of suitable bases include
N-methylmorpholine, triethylamine and N,N-diisopropylethylamine.
Alternatively, the pH of water containing solvents can be modulated
using biological buffers such as
(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid) (HEPES) or
4-morpholineethane-sulfonic acid (MES) or inorganic buffers such as
sodium carbonate and/or sodium bicarbonate. Because at least one of
the reactive groups can be electrophilic, it can be desirable to
select the buffer to not contain any nucleophilic groups. Those of
skill in the are will appreciate other buffers that can be used to
modulate the pH of a labeling reaction, with the application of
ordinary experimentation, so as to facilitate the labeling of an
analyte with a labeling reagent.
Sample Processing:
[0154] In certain embodiments of this invention, a sample can be
processed prior to, as well as after, labeling of the analytes.
Processing can be applied to the whole of a sample, or a fraction
thereof. Processing can be applied to sample mixtures or a fraction
thereof. Processing can be used to de-complexify the sample or be
used to put the sample in a better form for analysis. The
processing can facilitate the labeling of the analytes. The
processing can facilitate the analysis of the sample components
(e.g. labeled analytes). The processing can simplify the handling
of the samples. The processing can facilitate two or more of the
foregoing.
[0155] For example, a sample can be treated with an enzyme. The
enzyme can be a protease (to degrade proteins and peptides), a
nuclease (to degrade oligonucleotides) or some other enzyme. The
enzyme can be chosen to have a very predictable degradation
pattern. Two or more proteases and/or two or more nuclease enzymes
may also be used together, or with other enzymes, to thereby
degrade sample components.
[0156] For example, the proteolytic enzyme trypsin is a serine
protease that cleaves peptide bonds between lysine or arginine and
an unspecific amino acid to thereby produce peptides that comprise
an amine terminus (N-terminus) and lysine or arginine carboxyl
terminal amino acid (C-terminus). In this way the peptides from the
cleavage of the protein are predictable and their presence and/or
quantity, in a sample from a trypsin digest, can be indicative of
the presence and/or quantity of the protein of their origin.
Moreover, the free amine termini of a peptide can be a good
nucleophile that facilitates its labeling. Other exemplary
proteolytic enzymes include papain, pepsin, ArgC, LysC, V8
protease, AspN, pronase, chymotrypsin and carboxypeptidase C.
[0157] For example, a protein (e.g. protein Z) might produce three
peptides (e.g. peptides B, C and D) when digested with a protease
such as trypsin. Accordingly, a sample that has been digested with
a proteolytic enzyme, such as trypsin, and that when analyzed is
confirmed to contain peptides B, C and D, can be said to have
originally comprised the protein Z. The quantity of peptides B, C
and D will also correlate with the quantity of protein Z in the
sample that was digested. In this way, any determination of the
identity and/or quantify of one or more of peptides B, C and D in a
sample (or a fraction thereof), can be used to identify and/or
quantify protein Z in the original sample (or a fraction
thereof).
[0158] Because activity of the enzymes is predictable, the sequence
of peptides that are produced from degradation of a protein of
known sequence can be predicted. With this information,
"theoretical" peptide information can be generated. A determination
of the "theoretical" peptide fragments in computer assisted
analysis of daughter fragment ions (as described above) from mass
spectrometry analysis of an actual sample can therefore be used to
determine one or more peptides or proteins in one or more unknown
samples.
[0159] In some cases, sample processing can include treatment of
precursors to the analyte or analytes to be labeled. For example,
if the analyte or analytes to be labeled are peptides derived from
a digested protein and the labeling reagent is, for this example,
selected to react with amine groups (e.g. N-.alpha.-amine groups
and N-.epsilon.-amine group of lysine) of the peptide or peptide
analytes, the protein (the analyte precursor molecule) of the
sample may be processed in a manner that facilitates the labeling
reaction. In this example, the protein can be reduced with a
reducing agent (e.g. tris[2-carboxyethyl]phosphine (TCEP)) and the
thiol groups then blocked by reaction with a blocking reagent (e.g.
methyl methanethiosulfonate (MMTS)). In this way the thiol groups
of the protein are blocked and therefore do not interfere with the
labeling reaction between the amines of the analytes and labeling
reagent.
[0160] Those of skill in the art will appreciate that treatment of
certain other precursor molecules can be performed using readily
available reagents and protocols that can be adapted with the aid
of routing experimentation. The precise choices or reagents and
conditions can be selected depending on the nature of the analyte
to be labeled and the labeling reagent.
[0161] In some embodiments, sample processing can include the
immobilization of the analytes or analyte precursors to a solid
support, whether labeled with a labeling reagent or not. In some
embodiments, immobilization can facilitate reducing sample
complexity. In some embodiments, immobilization can facilitate
analyte labeling. In some embodiments, immobilization can
facilitate analyte precursor labeling. In some embodiments,
immobilization can facilitate selective labeling of a fraction of
sample components comprising a certain property (e.g. they comprise
or lack cysteine moieties). The immobilization can facilitate two
or more of the foregoing.
Separations:
[0162] In some embodiments, the processing of a sample or sample
mixture of labeled analytes can involve separation. One or more
separations can be performed on the labeled or unlabeled analytes,
labeled or unlabeled analyte precursors, or fractions thereof. One
or more separations can be performed on one or more fractions
obtained from a solid phase capture. Separations can be preformed
on two or more of the foregoing.
[0163] For example, a sample mixture comprising differentially
labeled analytes from different samples can be prepared. By
differentially labeled we mean that each of the labels comprises a
unique property that can be identified (e.g. comprises a unique
reporter moiety that produces a unique "signature ion" in MS/MS
analysis). In order to analyze the sample mixture, components of
the sample mixture can be separated and mass analysis performed on
only a fraction of the sample mixture. In this way, the complexity
of the analysis can be substantially reduced since separated
analytes can be individually analyzed for mass thereby increasing
the sensitivity of the analysis process. Of course the analysis can
be repeated one or more time on one or more additional fractions of
the sample mixture to thereby allow for the analysis of all
fractions of the sample mixture.
[0164] Separation conditions under which identical analytes that
are differentially labeled co-elute at a concentration, or in a
quantity, that is in proportion to their abundance in the sample
mixture can be used to determine the amount of each labeled analyte
in each of the samples that comprise the sample mixture provided
that the amount of each sample added to the sample mixture is
known. Accordingly, in some embodiments, separation of the sample
mixture can simplify the analysis whilst maintaining the
correlation between signals determined in the mass analysis (e.g.
MS/MS analysis) with the amount of the differently labeled analytes
in the sample mixture.
[0165] The separation can be performed by chromatography. For
example, liquid chromatography/mass spectrometry (LC/MS) can be
used to effect such a sample separation and mass analysis.
Moreover, any chromatographic separation process suitable to
separate the analytes of interest can be used. For example, the
chromatographic separation can be normal phase chromatography,
reversed-phase chromatography, ion-exchange chromatography, size
exclusion chromatography or affinity chromatorgraphy.
[0166] The separation can be performed electrophoretically.
Non-limiting examples of electrophoretic separations techniques
that can be used include, but are not limited to, 1D
electrophoretic separation, 2D electrophoretic separation and/or
capillary electrophoretic separation.
[0167] An isobaric labeling reagent or a set of reagents can be
used to label the analytes of a sample. Isobaric labeling reagents
are particularly useful when a separation step is performed because
the isobaric labels of a set of labeling reagents are structurally
and chemically indistinguishable (and can be indistinguishable by
gross mass until fragmentation removes the reporter from the
analyte). Thus, all analytes of identical composition that are
labeled with different isobaric labels can chromatograph in exactly
the same manner (i.e. co-elute). Because they are structurally and
chemically indistinguishable, the eluent from the separation
process can comprise an amount of each isobarically labeled analyte
that is in proportion to the amount of that labeled analyte in the
sample mixture. Furthermore, from the knowledge of how the sample
mixture was prepared (portions of samples, an other optional
components (e.g. calibration standards) added to prepare the sample
mixture), it is possible to relate the amount of labeled analyte in
the sample mixture back to the amount of that labeled analyte in
the sample from which it originated.
[0168] The labeling reagents can also be isomeric. Although isomers
can sometimes be chromatographically separated, there are
circumstances, that are condition dependent, where the separation
process can be operated to co-elute all of the identical analytes
that are differentially labeled wherein the amount of all of the
labeled analytes exist in the eluent in proportion to their
concentration and/or quantity in the sample mixture.
[0169] As used herein, isobars differ from isomers in that isobars
are structurally and chemically indistinguishable compounds (except
for isotopic content and/or distribution) of the same gross mass
(See for example, FIG. 1) whereas isomers are structurally and/or
chemically distinguishable compounds of the same gross mass.
Workflows:
[0170] In some embodiments, the labeling of the analytes of a
sample can be performed prior to performing sample processing
steps. In some embodiments, the labeling of analytes can be
performed amongst other sample processing steps. In some
embodiments, the labeling of analytes is the last step of sample
processing and/or immediately precedes the preparation of a sample
mixture.
[0171] Using proteomic analysis as a non-limiting example, there
are at least several possible workflows that might be used. To aid
in understanding of the following discussion a distinction is
sometimes made between the precursor protein and the analyte
peptide. However, it should be understood that either, or both, of
the protein and the peptide can be considered analytes as described
herein.
[0172] In one type of workflow, the precursor proteins can be
digested to peptide analytes that can thereafter be labeled with
labeling reagent. In another type of workflow, the precursor
proteins can be labeled with the labeling reagent and then digested
to labeled peptide analytes. In another type of workflow, the
precursor proteins can be captured on a solid support, digested and
then the support bound peptides can be labeled. Optionally the flow
through peptides can also labeled. In another type of workflow, the
precursor proteins can be captured on a solid support, labeled and
then the support bound protein can be digested to produce labeled
peptides. Optionally the flow through peptides can also analyzed.
Regardless of the workflow, additional sample processing (e.g.
separation steps) can be performed on the labeled peptides as
desired before MS analysis.
[0173] In summary, the analyte, can be labeled before or after one
or more separation and/or sample processing steps have been
performed. It is not a limitation of this invention when the
labeling of the analyte takes place so long as the analytes of one
or more samples can be labeled and one or more sample mixtures can
be prepared from differentially labeled samples.
Relative and Absolute Quantitation of Analytes:
[0174] In some embodiments, the relative quantitation of
differentially labeled identical analytes of a sample mixture is
possible. Relative quantitation of differentially labeled identical
analytes is possible by comparison of the relative amounts of
reporter (e.g. intensity, area and/or height of the peak reported)
that are determined in the second mass analysis for a selected,
labeled analyte observed in a first mass analysis. Put differently,
where each reporter can be correlated with information for a
particular sample used to produce a sample mixture, the relative
amount of that reporter, with respect to other reporters observed
in the second mass analysis, is the relative amount of that analyte
in the sample mixture. Where components combined to form the sample
mixture is known, the relative amount of the analyte in each sample
used to prepare the sample mixture can be back calculated based
upon the relative amounts of reporter observed for the ions of the
labeled analyte selected from the first mass analysis. This process
can be repeated for all of the different labeled analytes observed
in the first mass analysis. In this way, the relative amount (often
expressed in terms of concentration and/or quantity) of each
reactive analyte, in each of the different samples used to produce
the sample mixture, can be determined.
[0175] In some embodiments, absolute quantitation of analytes can
be determined. For these embodiments, a known amount of one or more
differentially labeled analytes (the calibration standard or
calibration standards) can be added to the sample mixture. The
calibration standard can be an expected analyte that is labeled
with an isomeric or isobaric label of the set of labels used to
label the analytes of the sample mixture provided that the reporter
for the calibration standard is unique as compared with any of the
samples used to form the sample mixture. Once the relative amount
of reporter for the calibration standard, or standards, is
determined with relation to the relative amounts of the reporter
for the differentially labeled analytes of the sample mixture, it
is possible to calculate the absolute amount (often expressed in
concentration and/or quantity) of all of the differentially labeled
analytes in the sample mixture. In this way, the absolute amount of
each differentially labeled analyte (for which there is a
calibration standard in the sample from which the analyte
originated) can also be determined based upon the knowledge of how
the sample mixture was prepared.
[0176] Notwithstanding the foregoing, corrections to the intensity
(or area or height) of the reporter ions (i.e. signature ions) can
be made, as appropriate, for any naturally occurring, or
artificially created, isotopic abundance within the reporters. A
more sophisticated example of these types of corrections can also
be found in copending and co-owned U.S. Provisional Patent
Application Ser. No. 60/524,844, entitled: "Method and Apparatus
For De-Convoluting A Convoluted Spectrum", filed on Nov. 26, 2003.
The more care taken to accurately quantify the intensity of each
reporter, the more accurate will be the relative and absolute
quantification of the analytes in the original samples.
[0177] In brief, using these methods, the intensity of up mass and
down mass isotope peaks associated with a particular signature ion
can be added to the major intensity peak associated with the
signature ion (i.e. the reporter) so that the contribution of all
intensities can be properly attributed to the correct reporter.
Peak intensities not associated with a particular signature ion can
be deducted as appropriate. By allocating all peak intensities to
the proper signature ions, the relative and absolute quantification
information associated with a signature ion can be quite accurate.
The more accurately intensities are allocated to the correct
reporter, the more accurate the quantitative determinations can
be.
Proteomic Analysis:
[0178] The methods, mixtures, kits and/or compositions of this
invention can be used for complex analysis because samples can be
multiplexed, analyzed and reanalyzed in a rapid and repetitive
manner using mass analysis techniques. For example, sample mixtures
can be analyzed for the amount of individual analytes in one or
more samples. The amount (often expressed in concentration and/or
quantity) of those analytes can be determined for the samples from
which the sample mixture was comprised. Because the sample
processing and mass analyses can be performed rapidly, these
methods can be repeated numerous times so that the amount of many
differentially labeled analytes of the sample mixture can be
determined with regard to their relative and/or absolute amounts in
the sample from which the analyte originated.
[0179] One application where such a rapid multiplex analysis is
useful is in the area of proteomic analysis. Proteomics can be
viewed as an experimental approach to describe the information
encoded in genomic sequences in terms of structure, function and
regulation of biological processes. This may be achieved by
systematic analysis of the total protein component expressed by a
cell or tissue. Mass spectrometry, used in combination with the
method, mixture, kit and/or composition embodiments of this
invention is one possible tool for such global protein
analysis.
[0180] For example, with a set of four isobaric labeling reagents,
it is possible to obtain four time points in an experiment to
determine up or down regulation of protein expression, for example,
based upon response of growing cells to a particular stimulant. It
is also possible to perform fewer time points but to incorporate
one or two controls. In all cases, up or down regulation of the
protein expression, optionally with respect to the controls, can be
determined in a single multiplex experiment. Moreover, because
processing is performed in parallel the results are directly
comparable, since there is no risk that slight variations in
protocol may have affected the results.
4. Description of Various Embodiments of the Invention
[0181] Various embodiments include one or more of kits, arrays,
libraries, mixtures, compounds, labeled analytes, and methods as
described in the following sections.
A. Compounds
[0182] Each of the various embodiments can employ one or more
compounds represented by structural formula I.sup.w: ##STR7## or a
salt form and/or hydrate form thereof. The variable m can be an
integer from one to 3, typically 1, wherein the compound can be
represented by structural formula I.sup.#: ##STR8##
[0183] The variables in the above structural formulas can be
independently selected for each compound as follows: [0184] RG can
be a nucleophilic group or an electrophilic group, or a reaction
product of an analyte with a nucleophilic group or an electrophilic
group; [0185] r and t can be both 0 or one of r and t can be 1 and
the other can be 0; [0186] When one of r and t is 1, S' can be a
linker, e.g., a cleaveable linker coupled to a solid support or an
affinity ligand; [0187] X and Y can be each a bond, wherein X can
couple an atom or an optional substituent of each of RP and LK to
thereby link RP to LK and Y can couple an atom or an optional
substituent of LK to RG; [0188] RP and LK can be each optionally
and independently substituted, wherein [0189] RP and LK can be each
independently a heteroaryl or heterocycloalkyl, or a linear or
branched aliphatic or heteroaliphatic group substituted or
interrupted with a heteroaryl or heterocycloalkyl; or [0190] LK can
be a linking moiety and RP can be a tertiary amine, a 4-9 membered
nitrogenous heteroaryl or heterocycloalkyl bonded at a ring
nitrogen to X, a 5-6 membered arylmethylene, a 5-6 membered
heteroarylmethylene, or a 5-6 membered heterocycloalkyl.
[0191] In some embodiments, the above values can be subject to one
or more provisios selected from: 1) RP--X-LK--Y-- is not a polymer;
2) RP and LK do not both comprise piperazinyl; RP and LK are not
both selected from the group consisting of amino acids (such as
naturally occurring amino acids), nucleotides, oligonucleotides,
peptides, and proteins; and 3) when t is 0, the group RP is not an
optionally substituted 5, 6 or 7 membered heterocycloalkyl
comprising a ring nitrogen atom that is N-alkylated with a
substituted or unsubstituted moiety of the formula
--C(J).sub.2-LK'-- such that LK' is --C(O)--, --C(S)--, --C(NH)--,
or --C(NRz)-, wherein Rz is an alkyl group comprising one to eight
carbon atoms which may optionally contain a heteroatom or
optionally substituted aryl group wherein the carbon atoms of the
alkyl and aryl groups independently comprise linked hydrogen,
deuterium and/or fluorine atoms and each J is the same or different
and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz).sub.2, fluorine,
chlorine, bromine or iodine.
[0192] In various embodiments, RG can be a nucleophilic group or an
electrophilic group represented by RG, and each compound can be a
labeling reagent; and a plurality of the compounds can be a
labeling reagent kit, a library of labeling reagents, or the
like.
[0193] In some embodiments, RG can refer to the reaction product of
an analyte with the nucleophilic groups or electrophilic groups
defined for RG, wherein each compound can be a labeled analyte. A
plurality of such compounds can be a mixture of labeled analytes, a
library of labeled analytes, and the like. For precision of
reference in certain depictions of such embodiments, the reaction
product of an analyte with the nucleophilic groups or electrophilic
groups defined for RG is represented by -Analyte.
[0194] Some embodiments can be a single isotopically enriched
compound represented by Strucutral Formulas I.sup.w or I.sup.#. In
various embodiments, a plurality of compounds can be isotopically
enriched. A compound that is isotopically enriched can be enriched
in one, two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, up to fifteen, up to twenty, up to twenty five, or
more of the same or different heavy atom isotopes.
[0195] Some embodiments can include a plurality of different
compounds, e.g. two or more, wherein the plurality of compounds can
be, for example, a kit, a library, an array, a mixture, and the
like. In such embodiments, RP and LK can each have a unique gross
mass for each different compound that can compensate for the
difference in unique gross mass between the RP for each compound
such that the aggregate gross mass of the RP and LK for each
compound can be the same. In some embodiments, two or more
different compounds can be isobaric isomers, wherein the compounds
have isomeric chemical structures but the same gross mass. In some
embodiments, two or different compounds can be isobaric
isotopologues, wherein the compounds have the same chemical
structure and same gross mass but different isotopic compositions,
e.g., at least one isobaric isotopologues is isotopically
enriched.
[0196] In various embodiments, one of r and t can be 1, and S' can
be a cleavable linker coupled to a solid support or an affinity
ligand. Thus, when S' is a solid support, various embodiments can
include solid supported libraries of labeling reagents, solid
supported libraries of labeled analytes, and the like.
[0197] In some embodiments, for each different compound, the
cleavable linker represented by S' can be coupled to the solid
support at a separate array location on the solid support, the
solid support comprising polystyrene, polyethylene, polypropylene,
polyfluoroethylene, polyethyleneoxy, polyacrylamide, glass, silica,
controlled-pore-glass (CPG), or reverse phase silica, the substrate
in the form of a gel, a membrane or a surface, whereby the kit is
an array library of the different compounds. In some embodiments,
RG can be a nucleophilic group or an electrophilic group, whereby
the kit is an array library of labeling reagents; or RG can be a
reaction product of an analyte with a nucleophilic group or an
electrophilic group; whereby the kit is an array library of labeled
analytes.
[0198] In some embodiments, for each different compound, the
cleavable linker represented by S' can be coupled to the solid
support at a separate solid support bead, sphere, particle, or
granule, the solid support comprising polystyrene, polyethylene,
polypropylene, polyfluoroethylene, polyethyleneoxy, polyacrylamide,
glass, silica, controlled-pore-glass (CPG), or reverse phase
silica, whereby the kit is a solid support library of the different
compounds. In some embodiments, RG can be a nucleophilic group or
an electrophilic group, whereby the kit is a solid support library
of labeling reagents; or RG can be a reaction product of an analyte
with a nucleophilic group or an electrophilic group; whereby the
kit is a solid support library of labeled analytes.
[0199] In some embodiments, for each different compound, the
cleavable linker represented by S' can be coupled to a different
affinity ligand selected from the group consisting of an antigen,
an antibody, an antibody fragment, an avidin, biotin, streptavidin,
a protein A, a lectin, and a carbohydrate, whereby the kit is an
affinity ligand library. In some embodiments, RG can be a
nucleophilic group or an electrophilic group, whereby the kit is an
affinity ligand library of labeling reagents; or RG can be a
reaction product of an analyte with a nucleophilic group or an
electrophilic group; whereby the kit is an affinity ligand library
of labeled analytes.
[0200] In various embodiments, compounds in the kits, arrays,
libraries, labeled analyte mixtures, and methods, and the
isotopically enriched compound can be further represented by one of
Structural Formulas I-S'to VI-S' or I to VI: ##STR9## or
isotopologues thereof. The variables r, s, S', X, and X are as
described above or as further detailed below and can be subject to
the corresponding provisos above. The variables RP.sup.1, RP.sup.2,
RP.sup.3, RP.sup.4, RP.sup.5, RP.sup.6, LK.sup.1, LK.sup.2,
LK.sup.3, LK.sup.4, LK.sup.5, and LK.sup.6 are as described in
greater detail below and can be subject to the corresponding
provisos above for RP--X-LK--Y-- and its variables RP/RP' and
LK/LK'. For example, for compounds that can be represented by
structural formula V or V-S', LK.sup.5 can be a linking moiety
represented by structural formula E (it will be understood that the
points of attachment to the remainder of the labeling reagent are
identified in the structure by the wavy line):
RP.sup.5--X-LK.sup.5--Y--RG V wherein: [0201] RP.sup.5 can be the
reporter group RP (as defined above) and LK.sup.5 can be a linking
moiety represented by structural formula E: ##STR10## [0202]
wherein each n, independently, can be an integer from 1 to 3; and
[0203] each R, independently, can be H, D, an alkyl, a heteroalkyl,
an aryl, a heteroaryl, or a halo group.
[0204] In various embodiments, at least one compound can be
represented by structural formula D: ##STR11##
[0205] In another example, for compounds that can be represented by
structural formula I or I-S', RP.sup.1 can be a reporter group
represented by structural formula A (it will be understood that the
point of attachment to the remainder of the labeling reagent is
identified in the structure by the wavy line): ##STR12## [0206]
Ring A can be aromatic; [0207] each Z can be independently CH,
CR.sup.2, or N, provided that no more than two Z groups are N;
[0208] n can be 1 or 2, typically 2 so that Ring A is a six
membered ring; [0209] each R.sup.2 can be independently selected
from the suitable substituents described in the Definitions, or
more typically, can be selected from hydrogen, deuterium, --OH,
halogen, --CN, --NO.sub.2, alkyl, alkenyl, alkynyl, aryl,
heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl,
heterocycloalkyl, --R.sup.3, or -T-R.sup.3; [0210] each R.sup.3 can
be independently hydrogen, deuterium, alkyl, alkenyl, alkynyl,
aryl, arylalkyl, heteroalkyl, heterocycloalkyl, heteroaryl, or
heteroaralkyl; [0211] T can be --O--, --NR.sup.4--, --S--,
--C(O)--, --S(O)--, --SO.sub.2--, --NR.sup.4C(O)--,
--C(O)NR.sup.4--, --NR.sup.4SO.sub.2--, --SO.sub.2NR.sup.4--,
--C(O)O--, --OC(O)--, --NR.sup.4C(O)O--, or --OC(O)NR.sup.4--;
[0212] each R.sup.4 is independently hydrogen, deuterium, alkyl,
heteroalkyl, aryl, or aralkyl; [0213] LK.sup.1 is a linking moiety;
[0214] X is a bond between an atom of the reporter and LK.sup.1;
and [0215] Y is a bond between an atom of the linker and an atom of
RG.
[0216] In various embodiments, at least one of RP.sup.1 and
LK.sup.1 can be isotopically enriched with one or more heavy atom
isotopes, for example, RP.sup.1. In some embodiments, both RP.sup.1
and LK.sup.1 can each be isotopically enriched with one or more
heavy atom isotopes. In some embodiments, each of RP.sup.1 and
LK.sup.1 comprise at least two heavy atom isotopes. In some
embodiments, each of RP.sup.1 and LK.sup.1 each comprise at least
three heavy atom isotopes.
[0217] In some embodiments, n is 2 whereby Ring A can be a six
membered ring. In some embodiments, either of the Z groups in the
ortho or para positions of Ring A can be C-T-R.sup.3. In various
embodiments, either of the Z groups in the ortho or para positions
of Ring A can be C--NHC(O)--R.sup.3 or C--NHSO.sub.2--R.sup.3 and
each R.sup.3 can be independently an optionally substituted alkyl
group. In some embodiments, n is 2 and each Z is independently CH
or CR.sup.2, and thus RP.sup.1 can be represented by Structural
Formula A-1: ##STR13##
[0218] In some embodiments, at least one atom in formula A is
isotopically enriched with a heavy atom isotope.
[0219] In some embodiments, LK.sup.1 can comprise an amino acid,
peptide, a C.sub.1-12 alkylene chain wherein 1-4 methylene units of
said chain are independently replaced by an amino acid, --O--,
--NR--, --S--, --C(O)--, --S(O)--, --SO.sub.2--, --NRC(O)--,
--C(O)NR--, --NRSO.sub.2--, --SO.sub.2NR--, --C(O)O--, --OC(O)--,
--NRC(O)O--, --OC(O)NR--, or an arylene, arylalkylene,
heteroalkylene, heterocycloalkylene, heteroarylene, or
heteroaralkylene, wherein each R is independently hydrogen,
deuterium, or an optionally substituted C.sub.1-6 alkyl group. The
amino acid moiety can be a glycine, aspartic acid, serine,
cysteine, lysine, proline, or ornithine.
[0220] In some embodiments, LK.sup.1 can be an optionally
substituted C.sub.1-12 alkylene chain wherein 1-4 methylene units
of said chain can be independently replaced by --C(O)O--, --C(O)--,
--O--, --NH--, --C(O)NH--, --S--, --NH--, --S(O)--, --SO.sub.2--,
or an amino acid, wherein the methylene unit .alpha. to group A can
be replaced by --O--, --S--, or --NH--.
[0221] In some embodiments, one of the methylene units of LK.sup.1
can be replaced by an optionally substituted azaalkylene,
azacycloalkylene, or azaarylene.
[0222] In various embodiments, at least one compound can be
represented by structural formula I-1: ##STR14##
[0223] In various embodiments, at least one compound can be
represented by a structural formula selected from: ##STR15##
##STR16## wherein the symbol "*" next to a carbon atom can indicate
that the carbon can be a .sup.13C isotope and the symbol "*" next
to a nitrogen atom can indicates that the nitrogen can be a
.sup.15N isotope.
[0224] In some embodiments, at least one compound can be
represented by structural formula I-1: ##STR17##
[0225] In some embodiments, at least one compound can be
represented by a structural formula selected from: ##STR18##
[0226] In various embodiments, at least one compound can be
represented by a structural formula selected from: ##STR19##
wherein R.sup.8 can be a valence bond, an alkylene, or
--(CH.sub.2).sub.s--(O--CH.sub.2CH.sub.2).sub.p--(CH.sub.2).sub.s--;
p can be 1, 2, 3, or 4; and each s can be independently 0, 1, 2, or
3.
[0227] In some embodiments, the compound can be represented by
structural formula II or II-S', wherein RP.sup.2 can be a reporter
group represented by structural formula B ##STR20## [0228] Ring B
can be non-aromatic; [0229] n can be 1 or 2; [0230] each W can be
independently O, S, or NR.sup.4; in some embodiments, each W in
structural formula II can be O or an isotope thereof; [0231] each
W' can be independently CH.sub.2, CHR.sup.2, C(R.sup.2).sub.2,
C(O), S(O), S(O).sub.2, or C.dbd.N--R.sup.4; [0232] Q can be CH or
CR.sup.2; [0233] each R.sup.2 can be independently selected from
the suitable substituents described in the Definitions, or more
typically, can be selected from hydrogen, deuterium, --OH, halogen,
--CN, --NO.sub.2, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
arylalkyl, heteroarylalkyl, heteroalkyl, heterocycloalkyl,
--R.sup.3, or -T-R.sup.3; [0234] each R.sup.3 can be independently
hydrogen, deuterium, or optionally substituted alkyl, alkenyl,
alkynyl, aryl, arylalkyl, heteroalkyl, heterocycloalkyl,
heteroaryl, or heteroaralkyl; [0235] T can be --O--, --NR.sup.4--,
--S--, --C(O)--, --S(O)--, --SO.sub.2--, --NR.sup.4C(O)--,
--C(O)NR.sup.4--, --NR.sup.4SO.sub.2--, --SO.sub.2NR.sup.4--,
--C(O)O--, --OC(O)--, --NR.sup.4C(O)O--, or --OC(O)NR.sup.4--;
[0236] each R.sup.4 can be independently hydrogen, deuterium, an
alkyl, a heteroalkyl, an aryl, or an aralkyl; [0237] LK.sup.2 can
be a linking moiety; [0238] X can be a bond between an atom of the
reporter and LK.sup.2; and [0239] Y can be a bond between an atom
of the linker and an atom of RG.
[0240] In some embodiments, at least one W moiety is O and at least
one W' moiety is CHR.sup.2.
[0241] In various embodiments, at least one of RP.sup.2 and
LK.sup.2 can be isotopically enriched with one or more heavy atom
isotopes, for example, RP.sup.2. In some embodiments, both RP.sup.2
and LK.sup.2 can each be isotopically enriched with one or more
heavy atom isotopes. In some embodiments, each of RP.sup.2 and
LK.sup.2 comprise at least two heavy atom isotopes. In some
embodiments, each of RP.sup.2 and LK.sup.2 comprise at least three
heavy atom isotopes. In various embodiments, LK.sup.2 can be as
defined for the various embodiments of LK.sup.1.
[0242] In some embodiments, the reporter group is of formula B
wherein n is 2 and each W is O. Thus, a reporter group of formula
B-1 is provided: ##STR21## wherein each R.sup.2 is as defined above
and herein.
[0243] In some embodiments, the reporter group is of formula B
wherein n is 1 and each W is O. Thus, a reporter group of formula
B-2 is provided: ##STR22## wherein each R.sup.2 is as defined above
and herein. In some embodiments, the compound can be represented by
structural formula II-d: ##STR23##
[0244] In some embodiments, the compound can be represented by
structural formula II-e: ##STR24##
[0245] In some embodiments, at least one atom in RP.sup.1 is
isotopically enriched with a heavy atom isotope.
[0246] In various embodiments, the compound can be represented by
structural formula III or III-S', wherein RP.sup.3 can be a
reporter group represented by structural formula C: ##STR25##
[0247] each of R.sup.x and R.sup.y can be independently alkyl,
alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, or
heteroalkyl, wherein suitable optional substituents for R.sup.x and
R.sup.y can be independently selected from the suitable
substituents described in the Definitions, or more typically, can
be selected from hydrogen, deuterium, --OH, halogen, --CN,
--NO.sub.2, alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, heteroalkyl, heterocycloalkyl, --R.sup.3,
-T-R.sup.3, ribose, deoxyribose or phosphate, or R.sup.x and
R.sup.y can be taken together to form Ring C': ##STR26## [0248]
Ring C' can be optionally substituted heteroaryl or
heterocycloalkyl, wherein suitable optional substituents for Ring C
can be independently selected from the suitable substituents
described in the Definitions, or more typically, can be selected
from hydrogen, deuterium, --OH, halogen, --CN, --NO.sub.2, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,
heteroalkyl, heterocycloalkyl, --R.sup.3, -T-R.sup.3, ribose,
deoxyribose or phosphate; [0249] each R.sup.3 can be independently
hydrogen, deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl,
heteroalkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl; [0250]
T can be --O--, --NR.sup.4--, --S--, --C(O)--, --S(O)--,
--SO.sub.2--, --NR.sup.4C(O)--, --C(O)NR.sup.4--,
--NR.sup.4SO.sub.2--, --SO.sub.2NR.sup.4--, --C(O)O--, --OC(O)--,
--NR.sup.4C(O)O--, or --OC(O)NR.sup.4--; [0251] each R.sup.4 can be
independently hydrogen, deuterium, alkyl, heteroalkyl, aryl, or
aralkyl; [0252] LK.sup.3 can be a linking moiety, provided that
when R.sup.x and R.sup.y are taken together to form Ring C', then
the ring nitrogen that links R.sup.x and R.sup.y is linked to a
group other than a substituted or unsubstituted moiety of the
formula --C(J).sub.2-LK'-- such that LK' is --C(O)--, --C(S)--,
--C(NH)--, or --C(NRz)-, wherein Rz is is an alkyl group comprising
one to eight carbon atoms which may optionally contain a heteroatom
or optionally substituted aryl group wherein the carbon atoms of
the alkyl and aryl groups independently comprise linked hydrogen,
deuterium and/or fluorine atoms and J is the same or different and
is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz).sub.2, fluorine,
chlorine, bromine or iodine; [0253] X can be a bond between an atom
of the reporter and LK.sup.3; and [0254] Y can be a bond between an
atom of the linker and an atom of RG.
[0255] In various embodiments, at least one of RP.sup.3 and
LK.sup.3 can be isotopically enriched with one or more heavy atom
isotopes, for example, RP.sup.3. In some embodiments, both RP.sup.3
and LK.sup.3 can each be isotopically enriched with one or more
heavy atom isotopes. In some embodiments, each of RP.sup.3 and
LK.sup.3 comprise at least two heavy atom isotopes. In some
embodiments, each of RP.sup.3 and LK.sup.3 each comprise at least
three heavy atom isotopes.
[0256] In some embodiments, LK.sup.3 is a linking moiety subject to
the proviso that when R.sup.x and R.sup.y are taken together to
form Ring C, then LK can be other than --C(J).sub.2C(O)--,
--C(J).sub.2C(S)--, --C(J).sub.2=NH--, or --C(J).sub.2=NR.sup.4--,
wherein each J can be independently hydrogen, deuterium, R.sup.4,
OR.sup.4, SR.sup.4, NHR.sup.4 or N(R.sup.4).sub.2. In various
embodiments, LK.sup.3 can be as defined for the various embodiments
of LK.sup.1.
[0257] In some embodiments, the reporter group is of formula C
wherein R.sup.x and R.sup.y are taken together to form Ring C'.
Thus, a reporter group of formula C'' is provided: ##STR27##
wherein q is 0-6 and Ring C is as defined as above and herein.
[0258] In some embodiments, the reporter group can be represented
by C wherein Ring C'' is heterocycloalkyl and q is 2, 3 or 4. Thus,
a reporter group of formula C-1 is provided: ##STR28##
[0259] In some embodiments, at least one atom in formula C-1 is
isotopically enriched with a heavy atom isotope. Is some
embodiments, at least one atom in formula C-1 is isotopically
enriched with two heavy atom isotopes.
[0260] In some embodiments, the above-described structures for the
reporter group C require that the linker not be a substituted or
unsubstituted acetic acid moiety that is N-alkylated to the
nitrogen atom through bond X.
[0261] In some embodiments, the compound can be represented by
structural formula III-c: ##STR29## wherein q can be an integer
from 0 to 6 and LK can contain a carbonyl.
[0262] In some embodiments, the compound can be represented by a
structural formula selected from: ##STR30##
[0263] In various embodiments, RP can comprise an optionally
substituted piperazinyl and LK can be an aryl or cycloalkyl, or a
linear or branched aliphatic or heteroaliphatic group substituted
or interrupted with an aryl or cycloalkyl.
[0264] Also, for compounds that can be represented by structural
formula VI or VI-S', at least one of RP.sup.6 or LK.sup.6 can
comprises an optionally substituted nucleobase, or a linear or
branched aliphatic or heteroaliphatic group substituted or
interrupted with an optionally substituted nucleobase; [0265]
optional substituents for RP.sup.6 and LK.sup.6 can be
independently selected from hydrogen, deuterium, --OH, halogen,
--CN, --NO.sub.2, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
arylalkyl, heteroarylalkyl, heteroalkyl, heterocycloalkyl,
--R.sup.3, -T-R.sup.3, ribose, deoxyribose, or phosphate; [0266]
each R.sup.3 can be independently hydrogen, deuterium, alkyl,
alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, heterocycloalkyl,
heteroaryl, or heteroaralkyl; [0267] T can be --O--, --NR.sup.4--,
--S--, --C(O)--, --S(O)--, --SO.sub.2--, --NR.sup.4C(O)--,
--C(O)NR.sup.4--, --NR.sup.4SO.sub.2--, --SO.sub.2NR.sup.4--,
--C(O)O--, --OC(O)--, --NR.sup.4C(O)O--, or --OC(O)NR.sup.4--;
[0268] each R.sup.4 can be independently hydrogen, deuterium,
alkyl, heteroalkyl, aryl, or aralkyl; [0269] X is a bond between an
atom of the reporter and LK.sup.6; and [0270] Y is a bond between
an atom of the linker and an atom of RG, [0271] wherein at least
one of RP.sup.6 and LK.sup.6 can be isotopically enriched with one
or more heavy atom isotopes; [0272] provided that if RP.sup.6 is a
heterocycloalkyl, the heterocycloalkyl is not a 5, 6 or 7 membered
heterocycloalkyl comprising a ring nitrogen atom that is
N-alkylated with a substituted or unsubstituted moiety of the
formula --C(J).sub.2-LK'-- such that LK' is --C(O)--, --C(S)--,
--C(NH)--, or --C(NRz)-, wherein Rz is an alkyl group comprising
one to eight carbon atoms which may optionally contain a heteroatom
or optionally substituted aryl group wherein the carbon atoms of
the alkyl and aryl groups independently comprise linked hydrogen,
deuterium and/or fluorine atoms and J is the same or different and
is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz).sub.2, fluorine,
chlorine, bromine or iodine.
[0273] Also, for compounds that can be represented by structural
formula IV or IV-S', RP.sup.4 and LK.sup.4 can be each
independently a heteroaryl or heterocycloalkyl, or a linear or
branched aliphatic or heteroaliphatic group substituted or
interrupted with a heteroaryl or heterocycloalkyl, wherein [0274]
suitable optional substituents for RP.sup.4 and LK.sup.4 can be
independently selected from the suitable substituents described in
the Definitions, or more typically, can be selected from hydrogen,
deuterium, --OH, halogen, --CN, --NO.sub.2, alkyl, alkenyl,
alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl,
heterocycloalkyl, --R.sup.3, -T-R.sup.3, ribose, deoxyribose or
phosphate; [0275] each R.sup.3 can be independently hydrogen,
deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl,
heterocycloalkyl, heteroaryl, or heteroaralkyl; [0276] T can be
--O--, --NR.sup.4--, --S--, --C(O)--, --S(O)--, --SO.sub.2--,
--NR.sup.4C(O)--, --C(O)NR.sup.4--, --NR.sup.4SO.sub.2--,
--SO.sub.2NR.sup.4--, --C(O)O--, --OC(O)--, --NR.sup.4C(O)O--, or
--OC(O)NR.sup.4--; [0277] each R.sup.4 can be independently
hydrogen, deuterium, alkyl, aryl, or aralkyl; [0278] X can be a
bond between an atom of the reporter and LK.sup.4; and [0279] Y can
be a bond between an atom of the linker and an atom of RG.
[0280] In various embodiments, at least one of RP.sup.4 and
LK.sup.4 can be isotopically enriched with one or more heavy atom
isotopes, for example, RP.sup.4. In some embodiments, both RP.sup.4
and LK.sup.4 can each be isotopically enriched with one or more
heavy atom isotopes. In some embodiments, each of RP.sup.4 and
LK.sup.4 comprise at least two heavy atom isotopes. In some
embodiments, each of RP.sup.4 and LK.sup.4 each comprise at least
three heavy atom isotopes.
[0281] In various embodiments, the heteroaryl or heterocycloalkyl
groups in RP.sup.4 and LK.sup.4 can be each independently selected
from optionally substituted imidazolyl, furyl, pyrrolyl, thienyl,
oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, thiadiazolyl,
oxadiazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl,
quinolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzisooxazolyl,
benzofuryl, benzothiazolyl, indolizinyl, imidazopyridinyl,
pyrazolyl, triazolyl, isothiazolyl, oxazolyl, tetrazolyl,
benzimidazolyl, benzothiazolyl, benzoisothiazolyl,
benzothiadiazolyl, benzoxadiazolyl, indolyl, tetrahydroindolyl,
azaindolyl, imidazopyridyl, quinazolinyl, purinyl,
pyrrolo[2,3]pyrimidyl, pyrazolo[3,4]pyrimidyl, benzo(b)thienyl,
morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, and
thiomorpholinyl.
[0282] In various embodiments, at least one of RP.sup.4 or LK.sup.4
can comprise an optionally substituted piperazinyl, or a linear or
branched aliphatic or heteroaliphatic group substituted or
interrupted with piperazinyl, or in some embodiments, RP.sup.4 can
be an optionally substituted piperazinyl, for example, N-methyl
piperazinyl.
[0283] In various embodiments, at least one of RP.sup.4 or LK.sup.4
can comprise an optionally substituted nucleobase (e.g., optionally
substituted purinyl or pyrimidinyl), or a linear or branched
aliphatic or heteroaliphatic group substituted or interrupted with
an optionally substituted nucleobase. In some embodiments, LK.sup.4
can be an optionally substituted nucleobase, or a linear or
branched aliphatic or heteroaliphatic group substituted or
interrupted with an optionally substituted nucleobase.
[0284] The nucleobases, e,g. the nucleobase in LK.sup.4 can be an
optionally substituted 9H-purin-6-amine,
2-amino-1H-purin-6(9H)-one, 4-aminopyrimidin-2(1H)-one,
5-methylpyrimidine-2,4(1H,3H)-dione, or the like. The nucleobase
can be substituted or unsubstituted.
[0285] In various embodiments, the compound can be represented by a
structural formula selected from: ##STR31## A bond drawn across a
ring, as above, indicates that the bond can be attached to any
substitutable atom in that ring; a bond drawn across two rings can
be attached to any substitutable atom in either of those two
rings.
[0286] The group R.sup.5 can be --C(J).sub.2-C(O)--,
--C(J).sub.2-C(S)--, --C(J).sub.2-C(NH)--, or
--C(J).sub.2-C(NR.sup.z)--, wherein R.sup.z is an alkyl group
comprising one to eight carbon atoms that may optionally contain a
heteroatom or optionally substituted aryl group wherein the carbon
atoms of the alkyl and aryl groups independently comprise linked
hydrogen, deuterium and/or fluorine atoms; and each J is the same
or different and is H, deuterium (D), Rz, ORz, SRz, NHRz,
N(Rz).sub.2, fluorine, chlorine, bromine or iodine.
[0287] R.sup.6 and R7 can each independently be alkyl, alkenyl,
alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl,
heterocycloalkyl, --R.sup.3, -T-R.sup.3, ribose, deoxyribose, or
phosphate, wherein each R.sup.3 is independently hydrogen,
deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl,
heterocycloalkyl, heteroaryl, or heteroaralkyl.
[0288] R.sup.8 and R.sup.9 can each independently be H, deuterium
(D), fluorine, chlorine, bromine, iodine, or a halogenated alkyl
(e.g., a CF.sub.3 group).
[0289] In some embodiments, the compound can be: ##STR32##
##STR33## B. Methods
[0290] According to the methods of this invention, the analyte to
be determined can be labeled by reacting the analyte with a
disclosed compound, e.g., the compounds as represented by one of
Structural Formulas I-S' to IV-S' or I to IV, wherein RG is a
reagtive group that is a nucleophilic group or electrophilic group.
The labeled analyte, the analyte itself, one or more fragments of
the analyte and/or fragments of the label, can be determined by
mass analysis. In some embodiments, methods of this invention can
be used for the analysis of different analytes in the same sample
as well as for the multiplex analysis of the same and/or different
analytes in two or more different samples. The two or more samples
can be mixed to form a sample mixture. In the multiplex analysis,
labeling reagents can be used to determine from which sample of a
sample mixture an analyte originated. The absolute and/or relative
(with respect to the same analyte in different samples) amount
(often expressed in concentration or quantity) of the analyte, in
each of two or more of the samples combined to form the sample
mixture, can be determined. Moreover, the mass analysis of
fragments of the analyte (e.g. daughter fragment ions) can be used
to identify the analyte and/or the precursor to the analyte; such
as where the precursor molecule to the analyte was degraded.
[0291] One distinction of the described approach lies in the fact
that analytes from different samples can be differentially
isotopically labeled (i.e. isotopically coded) with unique labels
that are chemically isomeric or isobaric (have equal mass) and that
identify the sample from which the analyte originated. The
differentially labeled analytes are not distinguished in MS mode of
a mass spectrometer because they all have identical (gross) mass to
charge ratios. However, when subjected to dissociative energy
levels, such as through collision induced dissociation (CID), the
labels can fragment to yield unique reporters that can be resolved
by mass (mass to charge ratio) in a mass spectrometer. The relative
amount of reporter observed in the mass spectrum can correlate with
the relative amount of a labeled analyte in the sample mixture and,
by implication, the amount of that analyte in a sample from which
it originated. Thus, the relative intensities of the reporters
(i.e. signature ions) can be used to measure the relative amount of
an analyte or analytes in two or more different samples that were
combined to form a sample mixture. From the reporter information,
absolute amounts (often expressed as concentration and/or quantity)
of an analyte or analytes in two or more samples can be derived if
calibration standards for the each analyte, for which absolute
quantification is desired, are incorporated into the sample
mixture.
[0292] For example, the analyte might be a peptide that resulted
from the degradation of a protein using an enzymatic digestion
reaction to process the sample. Protein degradation can be
accomplished by treatment of the sample with a proteolytic enzyme
(e.g. trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN,
pronase, chymotrypsin or carboxypeptidase C). By determination of
the identity and amount of a peptide in a sample mixture and
identifying the sample from which it originated, optionally coupled
with the determination of other peptides from that sample sample,
the precursor protein to the degraded peptide can be identified
and/or quantified with respect to the sample from which it
originated. Because this method allows for the multiplex
determination of a protein, or proteins, in more than one sample
(i.e. from a sample mixture), it is a multiplex method.
[0293] In some embodiments, this invention pertains to a method
comprising reacting each of two or more samples, each sample
containing one or more reactive analytes, with a different labeling
reagent of a set of labeling reagents wherein the different
labeling reagents of the set each comprise the formula:
RP--X-LK--Y--RG. Consequently, one or more analytes of each sample
are labeled with the moiety "RP--X-LK--Y--" by reaction of a
nucleophilic group or electrophilic group of the analyte with the
electrophilic or nucleophilic reactive group (RG), respectively, of
the different labeling reagents. The labeling process can produce
two or more differentially labeled samples each comprising one or
more labeled analytes. The labeling reagents of the set can be
isomeric or isobaric. The reporter of each labeling reagent can be
identified with, and therefore used to identify, the sample from
which each labeled analyte originated.
[0294] RG is a reactive group the characteristics of which have
been previously described. RP is a reporter moiety the
characteristics of which have been previously described. The gross
mass of each reporter can be different for each reagent of the set.
LK is a linker moiety the characteristics of which have been
previously described. The gross mass of the linker can compensate
for the difference in gross mass between the reporters for the
different labeling reagents such that the aggregate gross mass of
the reporter-linker combination is the same for each reagent of the
set. X is a bond between an atom of the reporter and an atom of the
linker. Y is a bond between an atom of the linker and an atom of
the reactive group (or after reaction with an analyte, Y is a bond
between the an atom of the linker and an atom of the analyte).
Bonds X and Y fragment in at least a portion of the labeled
analytes when subjected to dissociative energy levels in a mass
spectrometer. The characteristics of bonds X and Y have been
previously described.
[0295] Once the analytes of each sample are labeled with the
labeling reagent that is unique to that sample, the two or more
differentially labeled samples, or a portion thereof, can be mixed
to produce a sample mixture. Where quantitation is desired, the
volume and/or quantity of each sample combined to produce the
sample mixture can be recorded. The volume and/or quantity of each
sample, relative to the total sample volume and/or quantity of the
sample mixture, can be used to determine the ratio necessary for
determining the amount (often expressed in concentration and/or
quantity) of an identified analyte in each sample from the analysis
of the sample mixture. The sample mixture can therefore comprise a
complex mixture wherein relative amounts of the same and/or
different analytes can be identified and/or quantitated, either by
relative quantitation of the amounts of analyte in each of the two
or more samples or absolutely where a calibration standard is also
added to the sample mixture.
[0296] The mixture can then be subjected to spectrometry techniques
wherein a first mass analysis can be performed on the sample
mixture, or fraction thereof, using a first mass analyzer. Ions of
a particular mass to charge ratio from the first mass analysis can
then be selected. The selected ions can then be subjected to
dissociative energy levels (e.g. collision-induced dissociation
(CID)) to thereby induce fragmentation of the selected ions. By
subjecting the selected ions, of a particular mass to charge ratio,
of the labeled analytes to dissociative energy levels, bonds X
and/or Y can be fragmented in at least a portion of the selected
ions. Fragmentation of both bonds X and Y can cause fragmentation
of the reporter-linker moiety as well as cause release the charged
or ionized reporter from the analyte. Ions subjected to
dissociative energy levels can also cause fragmentation of the
analyte to thereby produce daughter fragment ions of the analyte.
The ions (remaining selected ions, daughter fragment ions and
charged or ionized reporters), or a fraction thereof, can then be
directed to a second mass analyzer.
[0297] A second mass analysis can be performed on the selected
ions, and the fragments thereof. The second mass analysis can
determine the gross mass (or m/z) and relative amount of each
unique reporter that is present at the selected mass to charge
ratio as well as the gross mass of the daughter fragment ions of at
least one reactive analyte of the sample mixture. For each analyte
present at the selected mass to charge ratio, the daughter fragment
ions can be used to identify the analyte or analytes present at the
selected mass to charge ratio. For example, this analysis can be
done as previously described in the section entitled: "Analyte
Determination By Computer Assisted Database Analysis".
[0298] In some embodiments, certain steps of the process can be
repeated one or more times. For example, in some embodiments, ions
of a selected mass to charge ratio from the first mass
spectrometric analysis, different from any previously selected mass
to charge ratio, can be treated to dissociative energy levels to
thereby form ionized reporter moieties and ionized daughter
fragment ions of at least some of the selected ions, as previously
described. A second mass analysis of the selected ions, the ionized
reporter moieties and the daughter fragment ions, or a fraction
thereof, can be performed. The gross mass and relative amount of
each reporter moiety in the second mass analysis and the gross mass
of the daughter fragment ions can also be determined. In this way,
the information can be made available for identifying and
quantifying one or more additional analytes from the first mass
analysis.
[0299] In some embodiments, the whole process can be repeated one
or more times. For example, it may be useful to repeat the process
one or more times where the sample mixture has been fractionated
(e.g. separated by chromatography or electrophoresis). By repeating
the process on each sample, it is possible to analyze all the
entire sample mixture. It is contemplated that in some embodiments,
the whole process will be repeated one or more times and within
each of these repeats, certain steps will also be repeated one or
more times such as described above. In this way, the contents of
sample mixture can be interrogated and determined to the fullest
possible extent.
[0300] Those of ordinary skill in the art of mass spectrometry will
appreciate that the first and second mass analysis can be performed
in a tandem mass spectrometer. Instruments suitable for performing
tandem mass analysis have been previously described herein.
Although tandem mass spectrometers are preferred, single-stage mass
spectrometers may be used. For example, analyte fragmentation may
be induced by cone-voltage fragmentation, followed by mass analysis
of the resulting fragments using a single-stage quadrupole or
time-of-flight mass spectrometer. In other examples, analytes may
be subjected to dissociative energy levels using a laser source and
the resulting fragments recorded following post-source decay in
time-of-flight or tandem time-of-flight (TOF-TOF) mass
spectrometers. It is to be understood that in some embodiments, an
instrument with a single analyzer can perform both the first and
the second mass analysis.
[0301] According to the preceding disclosed multiplex methods, in
some embodiments, bond X can be more or less prone to, or
substantially equal to, fragmentation as compared with
fragmentation of bonds of the analyte (e.g. an amide (peptide) bond
in a peptide backbone). In some embodiments, bond Y can be more or
less prone to fragmentation as compared with fragmentation of bonds
of the analyte (e.g. an amide (peptide) bond in a peptide
backbone). In some embodiments, the linker for each reagent of the
set is neutral in charge after the fragmentation of bonds X and Y
(i.e. the linker fragments to produce a neutral loss of mass and is
therefore not observed in the MS/MS spectrum). In some embodiments,
the position of bonds X and Y does not vary within the labeling
reagents of a set, within the labeled analytes of a mixture or
within the labeling reagents of a kit. In some embodiments, the
reporter for each reagent of the set does not substantially
sub-fragment under conditions that are used to fragment the analyte
(e.g. an amide (peptide) bond of a peptide backbone). In some
embodiments, bond X is less prone to fragmentation as compared with
bond Y. In some embodiments, bond Y is less prone to fragmentation
as compared with bond X. In some embodiments, bonds X and Y are of
approximately the same lability or otherwise are selected such that
fragmentation of one of bonds X or Y results in the fragmentation
of the other of bonds X or Y.
[0302] In some embodiments, the method of the invention comprises:
reacting two or more samples, each sample comprising one or more
analytes, with a different labeling reagent to thereby produce two
or more differently labeled samples each comprising one or more
labeled analytes, and mixing two or more of the labeled samples, or
a portion thereof, and optionally one or more calibration standards
to thereby produce the mixture comprising analytes labeled with the
labeling reagents described herein. In some embodiments, each
sample used to produce the mixture was labeled with a labeling
reagent comprising a unique reporter that can be used to identify
the analyte and quantify it relative or absolute amount in the
mixture and/or in the sample from which it originated.
[0303] In various embodiments, the labeling reagents or "isobaric
mass tags" can be represented by any of Structural Formulas
I.sup.w, I.sup.#, I-S' to IV-S' or I to IV, typically one of I to
IV, wherein RG represents a nucleophilic group or an electrophilic
group, and the remaining variables are as described above for the
compounds.
[0304] For example, in some embodiments, the method of the
invention comprises reacting two or more samples, each sample
comprising one or more reactive analytes, with a set of isobaric
mass tags to thereby produce two or more differentially labeled
samples each comprising one or more labeled analytes, and mixing
two or more of the differentially labeled samples, or a portion
thereof, and optionally one or more calibration standards to
thereby produce a sample mixture.
[0305] Once the labeling reagent is reacted with the reactive
analyte, bond Y links the linker to the analyte; at least one of RP
(respectively represented by RP.sup.1, RP.sup.2, RP.sup.3,
RP.sup.4, RP.sup.5, and RP.sup.6 in the various formula) and LK
(respectively represented by LK.sup.1, LK.sup.2, LK.sup.3,
LK.sup.4, LK.sup.5 and LK.sup.6 in the various formula) can be
isotopically enriched with one or more heavy atom isotopes; upon
reaction of the isobaric mass tag with an analyte, each mass tag
can add the same mass to the analyte; and upon fragmentation, RP
(respectively represented by RP.sup.1, RP.sup.2, RP.sup.3,
RP.sup.4, RP.sup.5 and RP.sup.6 in the various formula) of each
isobaric mass tag can yield a signature ion having a different mass
from the signature ions of the other isobaric mass tags in the
set.
[0306] According to some embodiments, the analytes from a sample
can be reacted with the solid support (each sample being reacted
with a different solid support and therefore a different reporter)
and the resin bound components of the sample that do not react with
the reactive group can be optionally washed away. The labeled
analyte or analytes can then be removed from each solid support by
treating the support under conditions that cleave the cleavable
linker S' and thereby release the reporter-linker-analyte complex
from the support. Each support can be similarly treated under
conditions that cleave the cleavable linker to thereby obtain two
or more different samples, each sample comprising one or more
labeled analytes wherein the labeled analytes associated with a
particular sample can be identified and/or quantified by the unique
reporter linked thereto. The collected samples can then be mixed to
form a sample mixture, as previously described.
[0307] For example, each different labeling reagent of the set used
in the previously described method can be attached to a solid
support.
[0308] The support comprising a labeling reagent can be prepared by
any of several methods (see the Example section below). In some
embodiments, the amino, hydroxyl or thiol group of an isobaric mass
tag can be reacted with the cleavable linker of a suitable support.
The cleavable linker can be a "sterically hindered cleavable
linker". Cleavage of the cleavable linker will release the labeled
analyte from the support.
[0309] Non-limiting examples of sterically hindered solid supports
include: Trityl chloride resin (trityl-Cl, Novabiochem, P/N
01-64-0074), 2-Chlorotrityl chloride resin (Novabiochem, P/N
01-64-0021), DHPP (Bachem, P/N Q-1755), MBHA (Applied Biosystems
P/N 400377), 4-methyltrityl chloride resin (Novabiochem, P/N
01-64-0075), 4-methoxytrityl chloride resin (Novabiochem, P/N
01-64-0076), Hydroxy-(2-chorophnyl)methyl-PS (Novabiochem, P/N
01-64-0345), Rink Acid Resin (Novabiochem P/Ns 01-64-0380,
01-64-0202), NovaSyn TGT alcohol resin (Novabiochem, P/N
01-64-0074).
[0310] In some embodiments, methods of the invention can further
comprise digesting each sample with at least one enzyme to
partially, or fully, degrade components of the sample prior to
performing the labeling of the analytes of the sample as more fully
described above in the section entitled: "Sample Processing". For
example, the enzyme can be a protease (to degrade proteins and
peptides) or a nuclease (to degrade oligonucleotides). The enzymes
may also be used together to thereby degrade sample components. The
enzyme can be a proteolytic enzyme such as trypsin, papain, pepsin,
ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin or
carboxypeptidase C.
[0311] In some embodiments, methods can further comprise separating
the sample mixture prior to performing the first mass analysis as
more fully described above in the section entitled: "Separations".
In this manner the first mass analysis can be performed on only a
fraction of the sample mixture. The separation can be performed by
any separations method, including by chromatography or by
electrophoresis. For example, liquid chromatography/mass
spectrometry (LC/MS) can be used to effect such a sample separation
followed by mass analysis. Moreover, any chromatographic separation
process suitable to separate the analytes of interest can be used.
Non-limiting examples of suitable chromatographic and
electrophoretic separations processes have been described
herein.
[0312] In still other embodiments, the methods of the invention can
comprise both an enzyme treatment to degrade sample components and
a separations step.
[0313] As described previously, it is possible to determine the
analyte associated with the selected ions by analysis of the gross
mass of the daughter fragment ions. One such method of
determination is described in the section entitled: "Analyte
Determination By Computer Assisted Database Analysis".
[0314] Once the analyte has been determined, information regarding
the gross mass and relative amount of each reporter moiety in the
second mass analysis and the gross mass of daughter fragment ions
provides the basis to determine other information about the sample
mixture. The amount of reporter can be determined by peak intensity
in the mass spectrum. In some embodiments, the amount of reporter
can be determined by analysis of the peak height or peak width of
the reporter (signature ion) signal obtained using the mass
spectrometer. Because each sample can be labeled with a different
labeling reagent and each labeling reagent can comprise a unique
reporter that can be correlated with a particular sample,
determination of the different reporters in the second mass
analysis identifies the sample from which the ions of the selected
analyte originated. Where multiple reporters are found (e.g.
according to the multiplex methods of the invention), the relative
amount of each reporter can be determined with respect to the other
reporters. Because the relative amount of each reporter determined
correlates with the relative amount of an analyte in the sample
mixture, the relative amount (often expressed as concentration
and/or quantity) of the analyte in each sample combined to form the
sample mixture can be determined. As appropriate, a correction of
peak intensity associated with the reporters can be performed for
naturally occurring, or artificially created, isotopic abundance,
as previously discussed in the section entitled: "Relative and
Absolute Quantitation of Analytes". More specifically, where the
volume and/or quantity of each sample that is combined to the
sample mixture is known, the relative amount (often expressed as
concentration and/or quantity) of the analyte in each sample can be
calculated based upon the relative amount of each reporter
determined.
[0315] This analysis can be repeated one or more times on selected
ions of a different mass to charge ratio to thereby obtain the
relative amount of one or more additional analytes in each sample
combined to form the sample mixture. As appropriate, a correction
of peak intensity associated with the reporters can be performed
for naturally occurring, or artificially created, isotopic
abundance.
[0316] Where a calibration standard comprising a unique reporter
linked to an analyte, having the selected mass to charge ratio, has
been added to the sample mixture in a known amount (often expressed
as a concentration and/or quantity), the amount of the unique
reporter associated with the calibration standard can be used to
determine the absolute amount (often expressed as a concentration
and/or quantity) of the analyte in each of the samples combined to
form the sample mixture. This is possible because the amount of
analyte associated with the reporter for the calibration standard
is known and the relative amounts of all other reporters can be
determined for the labeled analyte associated with the selected
ions. Since the relative amount of reporter, determined for each of
the unique reporters (including the reporter for the calibration
standard), is proportional to the amount of the analyte associated
with each sample combined to form the sample mixture, the absolute
amount (often expressed as a concentration and/or quantity) of the
analyte in each of the samples can be determined based upon a ratio
calculated with respect to the formulation used to produce the
sample mixture. As appropriate, a correction of peak intensity
associated with the reporters can be performed for naturally
occurring, or artificially created, isotopic abundance.
[0317] This analysis can be repeated one or more times on selected
ions of a different mass to charge ratio to thereby obtain the
absolute amount of one or more additional analytes in each sample
combined to form the sample mixture. As appropriate, a correction
of peak intensity associated with the reporters can be performed
for naturally occurring, or artificially created, isotopic
abundance.
[0318] In some embodiments, the methods can be practiced with
digestion and/or separation steps. In some embodiments, the steps
of the methods, with or without the digestion and/or separation
steps, can be repeated one or more times to thereby identify and/or
quantify one or more other analytes in a sample or one or more
analytes in each of the two or more samples (including samples
labeled with support bound labeling reagents). Depending of whether
or not a calibration standard is present in the sample mixture for
a particular analyte, the quantitation can be relative to the other
labeled analytes, or it can be absolute. Such an analysis method
can be particularly useful for proteomic analysis of multiplex
samples of a complex nature, especially where a preliminary
separation of the labeled analytes (e.g. liquid chromatography or
electrophoretic separation) precedes the first mass analysis.
[0319] In some embodiments, the analytes can be peptides in a
sample or sample mixture. Analysis of the peptides in a sample, or
sample mixture, can be used to determine the amount (often
expressed as a concentration and/or quantity) of identifiable
proteins in the sample or sample mixture wherein proteins in one or
more samples can be degraded prior to the first mass analysis.
Moreover, the information from different samples can be compared
for the purpose of making determinations, such as for the
comparison of the effect on the amount of the protein in cells that
are incubated with differing concentrations of a substance that may
affect cell growth. Other, non-limiting examples may include
comparison of the expressed protein components of diseased and
healthy tissue or cell cultures. This may encompass comparison of
expressed protein levels in cells, tissues or biological fluids
following infection with an infective agent such as a bacteria or
virus or other disease states such as cancer. In other examples,
changes in protein concentration over time (time-course) studies
may be undertaken to examine the effect of drug treatment on the
expressed protein component of cells or tissues. In still other
examples, the information from different samples taken over time
may be used to detect and monitor the concentration of specific
proteins in tissues, organs or biological fluids as a result of
disease (e.g. cancer) or infection.
[0320] In some embodiments, the analyte can be a nucleic acid
fragment in a sample or sample mixture. The information on the
nucleic acid fragments can be used to determine the amount (often
expressed as a concentration and/or quantity) of identifiable
nucleic acid molecules in the sample or sample mixture wherein the
sample was degraded prior to the first mass analysis. Moreover, the
information from the different samples can be compared for the
purpose of making determinations as described above.
C. Mixtures
[0321] In some embodiments, this invention pertains to mixtures
(e.g. sample mixtures). The mixtures can comprise at least two
differentially labeled analytes, wherein each of the two-labeled
analytes can originate from a different sample and comprise the
formula: RP--X-LK--Y-Analyte. For each different label, some of the
labeled analytes of the mixture can be the same and some of the
labeled analytes can be different. The atoms, moieties or bonds, X,
Y, RP and LK have been previously described and their
characteristics disclosed. The mixture can be formed by mixing all,
or a part, of the product of two or more labeling reactions wherein
each labeling reaction uses a different labeling reagent of the
general formula: RP--X-LK--Y--RG, wherein atoms, moieties or bonds
X, Y, RP, LK RG have been previously described and their
characteristics disclosed. The labeling reagents can be
isotopically coded isomeric or isobaric labeling reagents. The
unique reporter of each different labeling reagent can indicate
from which labeling reaction each of the two or more labeled
analytes is derived. The labeling reagents can be isomeric or
isobaric. Hence, two or more of the labeled analytes of a mixture
can be isomeric or isobaric. The mixture can be the sample mixture
as disclosed in any of the above-described methods. Characteristics
of the labeling reagents and labeled analytes associated with those
methods have been previously discussed.
[0322] The analytes of the mixture can be peptides. The analytes of
the mixture can be proteins. The analytes of the mixture can be
peptides and proteins. The analytes of the mixture can be nucleic
acid molecules. The analytes of the mixture can be carbohydrates.
The analytes of the mixture can be lipids. The analytes of the
mixture can be steroids. The analytes of the mixture can be small
molecules of less than 1500 daltons. The analytes of the mixture
comprise two or more analyte types. The analyte types can, for
example, be selected from peptides, proteins, oligonucleotides,
carbohydrates, lipids, steroids and/or small molecules of less than
1500 daltons.
[0323] In various embodiments, a mixture of the invention comprises
at least two labeled analytes, wherein at least one of the labeled
analytes originates from a different sample from the other labeled
analytes, combined to form the mixture. For example, the analyte
can be a protein, a peptide, a nucleotide, a carbohydrate, a lipid,
a steroid or a small molecule of less than 1500 daltons.
[0324] In various embodiments, the labeled analytes can be
represented by any of Structural Formulas I.sup.w, I.sup.#, I-S' to
VI-S' or I to VI, typically one of I to VI, wherein RG represents
the reaction product of a nucleophilic group or electrophilic group
and the analyte, e.g., the labeled analytes can be represented by
one of the following formulas: ##STR34## or a salt form and/or
hydrate form thereof, wherein the variables are as defined above.
Typically at least one of RP/LK (or RP.sup.1/LK.sup.1,
RP.sup.2/LK.sup.2, RP.sup.3/LK.sup.3, RP.sup.4/LK.sup.4,
RP.sup.5/LK.sup.5 or RP.sup.6/LK.sup.6) can be isotopically
enriched with one or more heavy atom isotopes; and the group
RP--X-LK-- (or RP.sup.1--X-LK.sup.1--, RP.sup.2--X-LK.sup.2--,
RP.sup.3--X-LK.sup.3--, RP.sup.4--X-LK.sup.4--,
RP.sup.5--X-LK.sup.5--, or RP.sup.6--X-LK.sup.6--) of each labeled
analyte can have the same mass.
[0325] Upon fragmentation of the moiety added to the analyte by
reaction of the labeling reagent with the analyte, RP of each
labeled analyte can then yield a signature ion that identifies the
sample from which the analyte originated. Accordingly, the
intensity of the signature ion relates to the amount of the analyte
in the mixture as well as the amount of analyte in the original
sample added to form the sample mixture. In some embodiments, each
of RP and LK comprise at least two heavy atom isotopes. In some
embodiments, each of RP and LK comprise at least three heavy atom
isotopes.
[0326] For example, in some embodiments, the method of the
invention comprises reacting two or more samples, each sample
comprising one or more reactive analytes, with a set of labeling
reagents or "isobaric mass tags" to thereby produce two or more
differentially labeled samples each comprising one or more labeled
analytes, and mixing two or more of the differentially labeled
samples, or a portion thereof, and optionally one or more
calibration standards to thereby produce a sample mixture.
[0327] Once the labeling reagent is reacted with the reactive
analyte, bond Y can link the linker to the analyte; at least one of
RP (respectively represented by RP.sup.1, RP.sup.2, RP.sup.3,
RP.sup.4, RP.sup.5, and RP.sup.6 in the various formula) and LK
(respectively represented by LK.sup.1, LK.sup.2, LK.sup.3,
LK.sup.4, LK.sup.5 and LK.sup.6 in the various formula), e.g. RP
can be isotopically enriched with one or more heavy atom isotopes;
upon reaction of the isobaric mass tag with an analyte, each mass
tag can add the same mass to the analyte; and upon fragmentation,
RP (respectively represented by RP.sup.1, RP.sup.2, RP.sup.3,
RP.sup.4, RP.sup.5 and RP.sup.6 in the various formula) of each
isobaric mass tag can yield a signature ion having a different mass
from the signature ions of the other isobaric mass tags in the
set.
[0328] Exemplary compounds (e.g. mass tags/labeling reagents) that
can be used to label analytes according to the method describe
above have been previously discussed under the heading:
"Compounds".
D. Kits
[0329] In various embodiments, a kit of the invention can comprise
one or more labeling reagents or "isobaric mass tags", at least one
of which can be represented by any of Structural Formulas I.sup.w,
I.sup.#, I-S' to VI-S' or I to VI, typically one of I to VI, or a
salt form and/or hydrate form thereof, wherein RG represents a
nucleophilic group or electrophilic group and wherein the remaining
variables are as defined above.
[0330] Compounds selected for use in the kits typically will be
"isotopically encoded". By "isotopically encoded" we mean that the
distribution of isotopes in each of the compounds of the kit is
selected to produce, for each different compound (i.e. labeling
reagent) a reporter that comprises a unique mass.
[0331] Typically at least one of the reporter group and the linker
group (e.g., RP/LK, RP.sup.1/LK.sup.1, RP.sup.2/LK.sup.2,
RP.sup.3/LK.sup.3, RP.sup.4/LK.sup.4, RP.sup.5/LK.sup.5 or
RP.sup.6/LK.sup.6 in the various formulas) can be isotopically
enriched with one or more heavy atom isotopes; and the group
RP--X-LK-- (or RP.sup.1--X-LK'--, RP.sup.2--X-LK.sup.2--,
RP.sup.3--X-LK.sup.3--, RP.sup.4--X-LK.sup.4--,
RP.sup.5--X-LK.sup.5--, or RP.sup.6--X-LK.sup.6--) of each labeled
analyte has the same mass. Typically, upon fragmentation, RP of
each labeled analyte can then yield a signature ion having a
different mass from the signature ions of the other isobaric mass
tags in the kit.
[0332] Other properties of the labeling reagents have likewise been
disclosed. For example, the labeling reagents can be useful for the
multiplex analysis of one or more analytes in the same sample, or
in two or more different samples.
[0333] Each isobaric labeling reagent (i.e. mass tag) of the kit is
isotopically enriched (coded) with at least one heavy atom isotope.
The labeling reagents can be isotopically enriched to comprise two
or more heavy atom isotopes. The labeling reagents can be
isotopically enriched to comprise three or more heavy atom
isotopes. The labeling reagents can be isotopically enriched to
comprise four or more heavy atom isotopes. In some embodiments, at
least one heavy atom isotope can be incorporated into a carbonyl or
thiocarbonyl group of the labeling reagent and at least one other
heavy atom isotope cam be incorporated into the reporter group of
the labeling reagent.
[0334] The labeling reagents comprise a reporter group that
contains a fixed charge or that is ionizable. The reporter group
therefore can include basic or acidic moieties that are easily
ionized. In some embodiments, the reporter can be a carboxylic
acid, sulfonic acid or phosphoric acid group containing compound.
Accordingly, is some embodiments, the labeling reagents can be
isolated in their salt form.
[0335] In some embodiments, the labeling reagents can comprise a
carbonyl or thiocarbonyl linker. Labeling reagents comprising a
carbonyl or thiocarbonyl linker can be used in active ester form
for the labeling of analytes. In an active ester, an alcohol group
forms a leaving group (LG), e.g., in some embodiments, the leaving
groups depicted in FIG. 9. In some embodiments, the active ester
can be an N-hydroxysuccinimidyl ester.
EXAMPLES
[0336] Aspects of the present teachings may be further understood
in light of the following examples, which should not be construed
as limiting the scope of the present invention in any way.
[0337] Definitions for some of the abbreviations that are used in
the examples are as follows: HMI stands for Hexamethyleneimine, Pbf
stands for 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl, Fmoc
stands for 9-Fluorenylmethoxycarbonyl, Trt stands for Trityl, Mpe
stands for 3-Methyl-pent-3-yl, HATU stands for
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, NMP stands for 1-Methyl-2-pyrrolidinone,
FmocOSu stands for (9-Fluorenylmethoxycarbonyloxy)succinimide, DMAP
stands for 4-Dimethylaminopyridine, THF stands for Tetrahydrofuran,
HBTU stands for 0-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, and HOBT stands for 1-Hydroxybenzotriazole
hydrate.
[0338] General Protocols of Amine Acylations to Generate a Reactive
Group on a Mass Tag:
[0339] FIG. 10 illustrates Protocol I and Protocol II for amine
acylation to generate a reactive group on a mass tag suitable for
reacting with the thiol group of cystine aminoe acids.
[0340] Protocol I: A respective amine (1-400 .mu.mol) was dissolved
in aqueous sodium bicarbonate (0.2 M) and acetonitrile (v/v 2:1 or
1:1). Typically, the concentration of the amine was in the range of
between about 0.01 to about 0.1 M. N-Hydroxysuccinimidyl
iodoacetate in acetonitrile (about 0.4 M, around 10 fold excess
relative to the free amine) was added while vortexing the reaction
mixture. The mixture was shaken at room temperature for about 10
min. to about 30 min. The product was purified with HPLC, and
confirmed with mass spectrometry (MS).
[0341] Protocol II: Iodoacetic anhydride (0.74 g, 2.1 mmol) in
CH.sub.2Cl.sub.2 (3 mL) was added to a stirred solution of a
respective amine (1.9 mmol) with N, N-diisopropylethylamine (DIEA,
1.9 mmol) at room temperature. The reaction solution was further
stirred at room temperature for 1.5-3 hour, then partitioned
between methylene chloride and water. The organic layer was dried
with anhydrous Na.sub.2SO.sub.4, concentrated in vacuo, and
purified with silica gel flash chromatography. The product was
characterized with NMR and/or MS.
Syntheses of Mass Tags (Labeling Reagents)
[0342] I. Synthesis of Mass Tag (1) ##STR35##
[0343] Commercially available 3,4-dimethoxybenzyl amine (Aldrich)
was acylated according to Protocol II to form Mass Tag (1). .sup.1H
NMR (CDCl.sub.3): .delta.3.72 (s, 2H), 3.90 (s, 6H), 4.60 (d, 2H),
6.61 (d, 2H), 7.28 (t, 1H). [M+H].sup.+ in MS: 336.0, calculated;
336.0, found.
II. Synthesis of Mass Tag (2)
[0344] FIG. 11 illustrates the synthesis of Mass Tag (2).
4-Amino-benzylamine (Aldrich, 1 mmol), N-succinimidyl iodoacetate
(Pierce, 1 mmol), and N,N-diisopropyl ethylamine (DIEA, 100 .mu.L)
were mixed in dichloromethane (10 mL) and stirred at room
temperature for 1 hr. Solvent was removed under reduced pressure.
The product was purified by silica gel column chromatography,
eluting with hexane, ethyl acetate (20%-60%), to give
4-amino-N-iodoacetylbenzylamine (62.2% yield). .sup.1H NMR (MeOD):
7.1(d, 2H), 6.75(d, 2H), 4.21(s, 2H), 3.65(s,2H). [M+H].sup.+ in
MS: 291.0, calculated; 291.0, found.
[0345] 4-amino-N-iodoacetylbenzylamine (0.172 mmol), acetic
anhydride (0.2 ml), and DIEA (0.2 ml) were stirred in acetonitrile
(3 ml) for 1.5 hours. The solvent was evaporated under reduced
pressure. The residual was partitioned between dichloromethane and
water. The organic layer was dried with anhydrous sodium sulfate,
filtered and concentrated in vacuo. The brownish residual was
purified with silica gel column chromatography, eluting with
dichloromethane and methanol to give the product, Mass Tag (2) (35
mg, 61% yield). [M+H].sup.+ in MS: 333.0 calculated; 333.0,
found.
III. Synthesis of Mass Tag (3)
[0346] FIG. 12 illustrates the synthesis of Mass Tag (3). To a
mixture of N-Boc-.beta.-tert-butyl-.alpha.-succinimido-aspartic
acid (Boc-Asp(But)-OSu) (Bacchem, 0.1 mmol) in DMF (1 ml) was added
hexamethyleneimine (Aldrich, 0.4 mmol). More DMF (2 ml) was added.
The mixture was shaken at room temperature for 2 hours to form
Boc-Asp(But)-HMI. Boc-Asp(But)-HMI was purified with preparative
HPLC, and characterized with MS ([M+H].sup.+: 371.3, calculated;
371.1, found).
[0347] The Boc-protected amine group of Boc-Asp(But)-HMI was
deprotected by exposure to a solution of methylene chloride (0.1
ml) and trifluoroacetic acid (TFA, 0.1 ml) at room temperature for
20 minutes. The solvents were evaporated in vacuo at 40.degree. C.
to dryness. The free amine was then acylated with Protocol I to
furnish Mass Tag (3) ([M+H].sup.+: 383.0, calculated; 383.0,
found).
IV. Syntheses of Mass Tag (4) and Mass Tag (5)
[0348] FIG. 13 illustrates Mass Tags (4) and (5).
[0349] Mass Tag (4) was prepared by acylating the amine group of
commercially available O-benzyl serine using Protocol I.
([M+H].sup.+ in MS: 364.0, calculated; 364.0, found).
[0350] Mass Tag (5) was prepared by acylating the amine group of
commercially available S-(p-methyl benzyl)cysteine using Protocol
I. ([M+H].sup.+ in MS: 394.0, calculated; 394.0, found).
V. Syntheses of Mass Tags (6), (7) and (8).
[0351] FIG. 14 illustrates the syntheses of Mass Tags (6), (7) and
(8).
[0352] A. Synthesis of Mass Tag (6)
[0353] A solution of a BocNH--OH (1-2 mmol) in DMF (2-4 ml) was
cooled with an ice-water bath. Sodium hydride (1.5-2 equivalent to
the BocNH--OH) was added. After evolution of hydrogen gas ceased,
benzyl bromide (Aldrich, 1 equivalent to the BocNH--R--OH) was
added while vortexing the mixture. The mixture was shaken at room
temperature for 5 hours. After centrifugation, the product,
BocNH--O(Bzl), was purified with preparative HPLC, and
characterized with MS.
[0354] The Boc-protected amine group of BocNH--O(Bzl) was
deprotected by exposure to 4-8 ml of 25% TFA in methylene chloride
at room temperature for 30 minutes. The deprotected compound,
NH.sub.2--O(Bzl), was extracted with water twice, and then either
purified with preparative HPLC or used directly in the acylation
reaction after evaporation of solvents.
[0355] NH.sub.2--O(Bzl) was acylated with Protocol I to furnish
Mass Tag (6) ([M+H].sup.+: 292.0, calculated; 292.0, found).
[0356] B. Synthesis of Mass Tag (7)
[0357] A solution of a BocNH--CH.sub.2CH.sub.2--OH (1-2 mmol) in
DMF (2-4 ml) was cooled with an ice-water bath. Sodium hydride
(1.5-2 equivalent to the BocNH--CH.sub.2CH.sub.2--OH) was added.
After evolution of hydrogen gas ceased, benzyl bromide (Aldrich, 1
equivalent to the BocNH--CH.sub.2CH.sub.2--OH) was added while
vortexing the mixture. The mixture was shaken at room temperature
for 5 hours. After centrifugation, the product,
BocNH--CH.sub.2CH.sub.2--O(Bzl), was purified with preparative
HPLC, and characterized with MS.
[0358] The Boc-protected amine group of
BocNH--CH.sub.2CH.sub.2--O(Bzl) was deprotected by exposure to 4-8
ml of 25% TFA in methylene chloride at room temperature for 30
minutes. The deprotected compound,
NH.sub.2--CH.sub.2CH.sub.2--O(Bzl), was extracted with water twice,
and then either purified with preparative HPLC or used directly in
the acylation reaction after evaporation of solvents.
[0359] NH.sub.2--CH.sub.2CH.sub.2--O(Bzl) was acylated with
Protocol I to furnish Mass Tag (7) ([M+H].sup.+: 320.0, calculated;
320.0, found).
[0360] C. Synthesis of Mass Tag (8)
[0361] A solution of a BocNH--(CH.sub.2).sub.5--OH (1-2 mmol) in
DMF (2-4 ml) was cooled with an ice-water bath. Sodium hydride
(1.5-2 equivalent to the BocNH--(CH.sub.2).sub.5--OH) was added.
After evolution of hydrogen gas ceased, benzyl bromide (Aldrich, 1
equivalent to the BocNH--(CH.sub.2).sub.5--OH) was added while
vortexing the mixture. The mixture was shaken at room temperature
for 5 hours. After centrifugation, the product,
BocNH--(CH.sub.2).sub.5--O(Bzl), was purified with preparative
HPLC, and characterized with MS.
[0362] The Boc-protected amine group of
BocNH--(CH.sub.2).sub.5--O(Bzl) was deprotected by exposure to 4-8
ml of 25% TFA in methylene chloride at room temperature for 30
minutes. The deprotected compound,
NH.sub.2--(CH.sub.2).sub.5--O(Bzl), was extracted with water twice,
and then either purified with preparative HPLC or used directly in
the acylation reaction after evaporation of solvents.
[0363] NH.sub.2--(CH.sub.2).sub.5--O(Bzl) was acylated with
Protocol I to furnish Mass Tag (8) ([M+H].sup.+: 362.1, calculated;
362.2, found).
VI. Syntheses of Mass Tags (9), (10) and (11)
[0364] FIG. 15 illustrates the syntheses of Mass Tags (9), (10) and
(11).
[0365] A. Synthesis of Mass Tag (9)
[0366] FmocGly (Applied Biosystems, 1 mmol),
N,N,N',N'-tetramethyl(succinimido)-uranium tetrafluoroborate (TSTU,
Advanced ChemTech, 1 mmol) and N,N-diisopropylethylamine (DIEA,
Aldrich, 2 mmol) were dissolved in N,N-dimethylformamide (DMF,
Burdick & Jackson, 6 ml). The mixture was shaken at room
temperature for half an hour. The solvent was evaporated to form
FmocGly-OSu, which was used directly in the following steps.
[0367] To L-Serine(Bzl) (NovaBiochem, 0.4 mmol) in DMF (0.8 ml) and
0.2 M aqueous sodium bicarbonate (2.8 ml) was added FmocGly-OSu
(0.4 mmol) in DMF (2.4 ml) while vortexing. The mixture was shaken
at room temperature for 20 minutes. The compound, FmocGly-Ser(Bzl),
was purified with preparative HPLC, and characterized with MS
([M+H].sup.+: 475.2, calculated; 475.2, found).
[0368] FmocGly-Ser(Bzl) (0.17 mmol) was exposed to 4 ml of 20%
piperidine in DMF at room temperature for 15 minutes to remove the
Fmoc-protecting group. The solvents were evaporated in vacuo at
40.degree. C., and the residual was purified with preparative HPLC.
The compound, Gly-Ser(Bzl), was characterized with MS ([M+H].sup.+:
253.1, calculated; 253.2, found).
[0369] FmocGly-OSu (0.08 mmol) in DMF (0.48 ml) was added to
Gly-Ser(Bzl) in DMF (2.6 ml) and 0.2 M aqueous sodium bicarbonate
(0.26 ml) while vortexing. The mixture was shaken at room
temperature for 20 minutes. The compound formed,
FmocGly-Gly-Ser(Bzl), was purified with preparative HPLC, and
characterized with MS ([M+H].sup.+: 532.2, calculated; 532.2,
found).
[0370] FmocGly-Gly-Ser(Bzl) (0.5 mg) was exposed to 0.2 ml of 20%
piperidine in DMF at room temperature for 10 minutes to remove the
Fmoc-protecting group. The solvent was evaporated in vacuo at
40.degree. C. to dryness. The deprotected amine was acylated using
Protocol I to furnish Mass Tag (9) ([M+H].sup.+ in MS: 478.0,
calculated; 478.0, found).
[0371] B. Synthesis of Mass Tag (10)
[0372] Gly-Ser(Bzl) was prepared as in Section A and was acylated
using Protocol I to furnish Mass Tag (10) ([M+H].sup.+: 421.0,
calculated; 421.0, found).
[0373] C. Synthesis of Mass Tag (11)
[0374] FmocGly-Ser(Bzl) (0.01 mmol), TSTU (0.02 mmol), and DIEA
(0.02 mmol) were dissolved in DMF (0.1 ml). The mixture was shaken
at room temperature for 40 minutes, and then transferred to glycine
(0.1 mmol) and sodium bicarbonate (0.2 mmol) in water (0.05 ml)
while vortexing. The mixture was shaken at room temperature for 30
minutes. The product, FmocGly-Ser(Bzl)-Gly, was purified with
semi-preparative HPLC, and characterized with MS ([M+H].sup.+:
532.2, calculated; 532.2, found).
[0375] FmocGly-Ser(Bzl)-Gly (1 mg) was exposed to a solution of 0.2
ml of 20% piperidine in DMF for 10 minutes to remove the
Fmoc-protecting group. After evaporation of solvents in vacuo at
40.degree. C., the deprotected amine was acylated using Protocol I
to furnish Mass Tag (11) ([M+H].sup.+: 478.0, calculated; 478.0,
found).
VII. Synthesis of Mass Tag (12)
[0376] FIG. 16 illustrates the synthesis of Mass Tag (12).
[0377] FmocGly (1 mmol), TSTU (1 mmol), and DIEA (1.5 mmol) were
dissolved in DMF (5 ml). The mixture was shaken at room temperature
for 40 minutes, and then transferred to a solution of glycine (4
mmol) in 5 ml of 0.2 M aqueous sodium bicarbonate while vortexing.
The mixture was shaken at room temperature for 20 minutes. The
product, FmocGly-Gly, was purified with preparative HPLC, and
characterized with MS ([M+H].sup.+: 355.2, calculated; 355.2,
found).
[0378] BocNH--O(Bzl) (see Section V.A. and FIG. 14 for preparation)
(0.2 mmol) was exposed to a solution of 5 ml of 25% TFA in
methylene chloride for 30 minutes to remove the Boc-protecting
group to form NH.sub.2--O(Bzl). NH.sub.2--O(Bzl) was extracted with
water, purified with preparative HPLC, and characterized with MS
([M+H].sup.+: 124.1, calculated; 124.2, found).
[0379] FmocGly-Gly (0.02 mmol), TSTU (0.02 mmol), and DIEA (0.03
mmol) were dissolved in DMF (0.2 ml). The mixture was shaken at
room temperature for 40 minutes, and then transferred to a solution
of NH.sub.2--O(Bzl) (2 mg) in DMF (0.1 ml) and 0.2 M aqueous sodium
bicarbonate (0.1 ml) while vortexing. The mixture was shaken at
room temperature for 20 minutes. The product,
FmocGly-Gly-NH--O(Bzl), was purified with HPLC, and characterized
with MS ([M+H].sup.+: 406.0, calculated; 405.8, found).
[0380] FmocGly-Gly-NH--O(Bzl) was exposed to a solution of 0.2 ml
of 20% piperidine in DMF at room temperature for 10 minutes to
remove the Fmoc-protecting group. After evaporation of all the
solvents, the deprotected amine was acylated using Protocol I to
furnish Mass Tag (12) ([M+H].sup.+: 406.0, calculated; 405.8,
found). VIII. Synthesis of Mass Tag (13) ##STR36##
[0381] O-Benzyl tyrosine was acylated using Protocol I to form Mass
Tag (13). ([M+H].sup.+ in MS: 440.0, calculated; 440.2, found).
IX. Syntheses of Mass Tags (14) and (15)
[0382] FIG. 17 illustrates Mass Tags (14) and (15).
[0383] The .alpha.-amine group of
.epsilon.-N-(benzyloxycarbonyl)-lysine was acylated using Protocol
I to form Mass Tag (14). ([M+H].sup.+ in MS: 435.0, calculated;
435.0, found).
[0384] The .epsilon.-amine group of
.alpha.-N-(benzyloxycarbonyl)-lysine was acylated using Protocol I
to form Mass Tag (15). ([M+H].sup.+ in MS: 463.1, calculated;
463.0, found).
X. General Protocol for Syntheses of Mass Tags (16), (17), (18),
(19) and (20)
[0385] FIG. 18 illustrates a general protocol for syntheses of Mass
Tags (16), (17), (18), (19) and (20).
[0386] To a diamine (NH.sub.2--R'--NH.sub.2) (0.4-4 mmol) in 0.2 M
aqueous sodium bicarbonate (1-4 ml) was added benzyl chloroformate
(Alfa Aesar, 0.1-2 mmol) in DMF (1-4 ml) while vortexing. R' is
defined in FIG. 18. The molar ratio for benzyl chloroformate versus
diamine was 1: 2-6. The mixture was shaken at room temperature for
5-20 minutes. The product, NH.sub.2--R'--NH(Z), was purified with
preparative HPLC, and characterized with MS. The monoamine was then
acylated using Protocol I to furnish an appropriate mass tag.
[0387] Mass Tag (16) ([M+H].sup.+ in MS: 335.0, calculated; 335.0,
found). Mass Tag (17) ([M+H].sup.+ in MS: 377.0, calculated; 377.0,
found). Mass Tag (18) ([M+H].sup.+ in MS: 407.1, calculated; 407.2,
found). Mass Tag (19) ([M+H].sup.+ in MS: 451.1, calculated; 451.0,
found). Mass Tag (20) ([M+H].sup.+ in MS: 479.1, calculated; 479.2,
found).
XI. Syntheses of Mass Tags (21), (22), (23), and (24)
[0388] FIG. 19 illustrates the syntheses of Mass Tags (21), (22),
(23) and (24).
[0389] A. Synthesis of Mass Tag (21)
[0390] .alpha.-N-Fmoc-.gamma.-N-(benzyloxycarbonyl)ornithine
(FmocOrn(Z)) (Advanced ChemTech, 0.25 mmol), TSTU (0.25 mmol), and
DIEA (0.375 mmol) were dissolved in DMF (3 mL). The mixture was
shaken at room temperature for 1 hour, and then transferred to a
solution of 1 mmol of glycine with sodium bicarbonate in water (3
mL). The mixture was shaken at room temperature for 30-60 minutes.
The product, FmocOrn(Z)-Gly, was purified with preparative HPLC,
and characterized with MS (FmocOrn(Z)-Gly: [M+H].sup.+: 546.2,
calculated; 546.4, found).
[0391] FmocOrn(Z)-Gly (2 mg) was exposed to a solution of 0.1 mL of
20% piperidine in DMF for 10 minutes to remove the Fmoc-protecting
group. After evaporation of all the solvents, the deprotected amine
was acylated using Protocol I to furnish Mass Tag (21) [M+H].sup.+:
492.1, calculated; 492.0, found).
[0392] B. Synthesis of Mass Tag (22)
[0393] .alpha.-N-Fmoc-.gamma.-N-(benzyloxycarbonyl)ornithine
(FmocOrn(Z)) (Advanced ChemTech, 0.25 mmol), TSTU (0.25 mmol), and
DIEA (0.375 mmol) were dissolved in DMF (3 mL). The mixture was
shaken at room temperature for 1 hour, and then transferred to a
solution of 1 mmol of L-alanine with sodium bicarbonate in water (3
mL). The mixture was shaken at room temperature for 30-60 minutes.
The product, FmocOrn(Z)-Ala, was purified with preparative HPLC,
and characterized with MS (FmocOrn(Z)-Ala: [M+H].sup.+: 560.2,
calculated; 560.2, found).
[0394] FmocOrn(Z)-Ala (2 mg) was exposed to a solution of 0.1 mL of
20% piperidine in DMF for 10 minutes to remove the Fmoc-protecting
group. After evaporation of all the solvents, the deprotected amine
was acylated using Protocol I to furnish Mass Tag (22)
([M+H].sup.+: 506.1, calculated; 505.8, found).
[0395] C. Synthesis of Mass Tag (23)
[0396] FmocOrn(Z)-Gly (0.1 mmol), TSTU (0.2 mmol), and DIEA (0.3
mmol) were dissolved in DMF (1 ml). The mixture was shaken at room
temperature for 1 hour, and then transferred to a solution of
sodium bicarbonate (1.5 mmol) and L-alanine (1 mmol) in water (1
ml). The mixture was shaken at room temperature for 30 minutes. The
product, FmocOrn(Z)-Gly-Ala, was purified with preparative HPLC,
and characterized with MS ([M+H].sup.+: 617.2, calculated; 617.2,
found).
[0397] FmocOrn(Z)-Gly-Ala (2 mg) was exposed to a solution of 0.1
ml of 20% piperidine in DMF for 10 minutes to remove the
Fmoc-protecting group. After evaporation of all the solvents, the
deprotected amine was acylated using Protocol I to furnish Mass Tag
(23) ([M+H563.1, calculated; 563.2, found).
[0398] D. Synthesis of Mass Tag (24)
[0399] FmocOrn(Z)-Ala (0.1 mmol), TSTU (0.2 mmol), and DIEA (0.3
mmol) were dissolved in DMF (1 ml). The mixture was shaken at room
temperature for 1 hour, and then transferred to a solution of
sodium bicarbonate (1.5 mmol) and glycine (1 mmol) in water (1 ml).
The mixture was shaken at room temperature for 30 minutes. The
product, FmocOrn(Z)-Ala-Gly, was purified with preparative HPLC,
and characterized with MS ([M+H].sup.+: 617.2, calculated; 617.2,
found).
[0400] FmocOrn(Z)-Ala-Gly (2 mg) was exposed to a solution of 0.1
ml of 20% piperidine in DMF for 10 minutes. After evaporation of
all the solvents, the deprotected amine was acylated using Protocol
I to furnish Mass Tag (24) ([M+H].sup.+: 563.1, calculated; 563.2,
found).
XII. Mass Tag Labeled Glu-Fib Peptide
[0401] [Glu.sup.1]-Fibrinopeptide B human [Glu-Fib, SEQ ID No.: 25
GVNDNEEGFFSAR), CAS#: 103213-49-6]: The peptide was assembled on
trityl chloride resin (P/N: Novabiochem, 01-64-0074) using standard
Fmoc-peptide synthesis protocol (Novabiochem caltalog, 2004-2005).
The following amino acid derivatives were used: Fmoc-Arg(Pbf)-OH
(P/N: Novabiochem, 04-12-1145), Fmoc-Glu(O.sup.tBu)--OH (P/N:
Novabiochem, 04-12-1020), Fmoc-Gly-OH (P/N: Novabiochem,
04-12-1001), Fmoc-Val-OH (P/N: Novabiochem, 04-12-1039),
Fmoc-Asn(Trt)-OH (P/N: Novabiochem, 04-12-1089), Fmoc-Asp(Mpe)-OH
(P/N: Bachem, B-3560), Fmoc-Phe-OH (P/N: Novabiochem, 04-12-1030),
Fmoc-Ser(.sup.tBu)-OH (P/N: Novabiochem, 04-12-1033), Fmoc-Ala-OH
(P/N: Novabiochem, 04-12-1006).
[0402] A. Synthesis of Mass Tag Labeled Glu-Fib Peptide (38) (see
FIG. 30)
[0403] Approximately 10 mg of Fmoc-Glu-Fib-Trityl-chloride resin
was treated with 20% (v/v) piperidine in DMF (2 mL.times.1 min,
filtered, then 2 mL.times.5 min), filtered and washed (NMP).
4-(Fmoc-amino)benzoic acid (P/N: Bachem, B-3260; 10 eqv to Glu-Fib
amount on the resin) was activated with HATU (P/N: Applied
Biosystems 4317033, 9.5 eqv) and N,N-Diisopropylethylamine (30 eqv)
in NMP (.about.1 mL), added to the resin and mixed for 30 min.
Resin was then filtered, washed with NMP, and Fmoc group was
cleaved. Piperazine acetic acid-TFA salt (10 eqv) was then
activated using HATU (9.5 eqv) and N,N-Diisopropylethylamine (60
eqv) in NMP (.about.1.5 mL) and added to the resin. After 30 min
resin was washed with NMP followed by CH.sub.3CN. Conjugated
peptide was cleaved (and deprotected) from resin using 95:5
TFA-water (200 .mu.L, 2 h) and precipitated using Et.sub.2O.
Analysis of compound (38) (see FIG. 30) was performed using
MALDI-TOF (Sinapinic acid matrix, Calculated [M+H].sup.+=1843.8,
Observed [M+H].sup.+=1844.9).
[0404] Further Mass Spectral Analysis: MS/MS analyses of Mass Tag
labeled Glu-Fib peptide (38) were performed on MALDI and
electrospray platforms. Data indicate that the Mass Tag is a good
candidate for MALDI platform but the intensity of the signature ion
peak was very weak in electrospray platforms (data not shown).
Differentially .sup.13C, .sup.15N and/or .sup.2H labeled
aminobenzoic acids are not commercially available and would need to
be synthesized.
[0405] B. Synthesis of Mass Tag Labeled Glu-Fib Peptide (39) (see
FIG. 30)
[0406] Approximately 10 mg of Fmoc-Glu-Fib-Trityl-chloride resin
was treated with 20% (v/v) piperidine in DMF (2 mL.times.1 min,
filtered, then 2 mL.times.5 min), filtered and washed (NMP).
4-(Fmoc-aminomethyl)benzoic acid (P/N: Fluka, 04062; 10 eqv to
Glu-Fib amount on the resin) was individually activated with HATU
(P/N: Applied Biosystems 4317033, 9.5 eqv) and
N,N-Diisopropylethylamine (30 eqv) in NMP (.about.1 mL), added to
the resin and mixed for 30 min. Resin was then filtered, washed
with NMP, and the Fmoc group was cleaved. Piperazine acetic
acid-TFA salt (10 eqv) was then activated using HATU (9.5 eqv) and
N,N-Diisopropylethylamine (60 eqv) in NMP (.about.1.5 mL) and added
to the resin. After 30 min resin was washed with NMP followed by
CH.sub.3CN. Conjugated peptide was cleaved (and deprotected) from
resin using 95:5 TFA-water (200 .mu.L, 2 h) and precipitated using
Et.sub.2O. Analysis of compound (39) (see FIG. 30) was performed
using ES-MS (direct infusion in water, Calculated
[M+H].sup.+=1829.8, Observed [M+H].sup.+=1829.9)
[0407] Further Mass Spectral Analysis: MS/MS analyses of Mass Tag
labeled Glu-Fib peptide (39) were performed on MALDI and
electrospray platforms. Data indicate that the Mass Tag is a good
candidate for MALDI platform but the intensity of the signature ion
peak was very weak in electrospray platforms (data not shown).
Differentially .sup.13C, .sup.15N and/or .sup.2H labeled
aminobenzoic acids are not commercially available and would need to
be synthesized.
[0408] C. Synthesis of Mass Tag Labeled Glu-Fib Peptide (40) (see
FIG. 31)
[0409] Approximately 10 mg of Fmoc-Glu-Fib-Trityl-chloride resin
was treated with 20% (v/v) piperidine in DMF (2 mL.times.1 min,
filtered, then 2 mL.times.5 min), filtered and washed (NMP).
Fmoc-4-carboxymethyl-piperazine (P/N: Chem-Impex, 04960); 10 eqv to
Glu-Fib amount on the resin) was activated with HATU (P/N: Applied
Biosystems 4317033, 9.5 eqv) and N,N-Diisopropylethylamine (30 eqv)
in NMP (.about.1 mL), added to the resin and mixed for 30 min.
Resin was then filtered, washed with NMP, and the Fmoc group was
cleaved. Piperazine acetic acid-TFA salt (10 eqv) then activated
using HATU (9.5 eqv) and N,N-Diisopropylethylamine (60 eqv) in NMP
(.about.1.5 mL) and added to the resin. After 30 min the resin was
washed with NMP followed by CH.sub.3CN. Conjugated peptide was
cleaved (and deprotected) from resin using 95:5 TFA-water (200
.mu.L, 2 h) and precipitated using Et.sub.2O. Analysis of compound
(40) (see FIG. 31) was performed using ES-MS (direct infusion in
water, Calculated [M+H].sup.+=1836.9, Observed
[M+H].sup.+=1837.0)
[0410] Further Mass Spectral Analysis: MS/MS analyses of Mass Tag
labeled Glu-Fib peptide (40) were performed on MALDI and
electrospray platforms. Data indicate that the Mass Tag is a good
candidate for MALDI and electrospray platforms. Signature ion
intensity and fragmentation pattern of the peptide were similar to
current iTRAQ.TM. reagent N-Methyl piperazine acetic acid (data not
shown).
[0411] D. Synthesis of Mass Tag Labeled Glu-Fib Peptide (41) (see
FIG. 32)
[0412] Compound (41b): To a solution of thymine acetic acid propyl
ester (41a) (can be synthesized according to: Alahiane, A.;
Taourirte, M.; Rochdi, A.; Redwane, N.; Sebti, S.; Engels, J. W.;
Lazrek, H. B., "Building blocks for polyamide nucleic acids: Facile
synthesis using potassium fluoride doped natural phosphate as basic
catalyst", Nucleosides, Nucleotides & Nucleic Acids 2003, 22,
109-114; 1.33 g, 5.88 mmol) and 2-Boc-(amino)-ethyl bromide (P/N:
Fluka, 17354, 2.978 g, 13.29 mmol) in DMF (30 mL), K.sub.2CO.sub.3
(3.4 g, 24.60 mmol) was added as solid and stirred for 20 h at
ambient temperature. TLC analysis at this point showed the
formation of a single product (41b) (R.sub.f=0.33, silica plate,
1:1 EtOAc-hexanes; UV 254 nm, TLC was developed by heating with 3%
(w/v) solution of ninhydrin in EtOH). After DMF removal under
reduced pressure, the resulting oil was partitioned between EtOAc
(350 mL) and dilute HCl (150 mL, 0.5 M). EtOAc layer was then
washed with brine (100 mL.times.2), dried over Na.sub.2SO.sub.4 and
concentrated to give a colorless oil. The oil was purified by
flash-chromatography (CombiFlash purification system, 120 g column,
85 mL/min, 270 nm, 0-5 min 20% EtOAc in hexanes, then 80% EtOAc in
hexanes, 18 mL fraction collected, fractions 24-30 had pure
product) to give 2.02 g (93% yield) of product (41b). ES-MS (Direct
infusion in MeOH, Calculated
[M+Na].sup.+=[C.sub.17H.sub.27N.sub.3O.sub.6+Na].sup.+=392.18,
observed [M+Na].sup.+=392.15.)
[0413] Compound (41c): To a solution of compound (41b) (1.10 g,
2.98 mmol) in THF (30 mL) NaOH solution (3.6 mL, 1 M) was added and
stirred for 30 min. Solvent removed under reduced pressure and the
residue was treated with 95% TFA in water (20 mL) for 1 h at
ambient temperature. TFA-water was removed under reduced pressure
and the oil so obtained was dissolved in saturated NaHCO.sub.3 (15
mL, pH=8-9) solution and FmocOSu (1.20 g, 3.58 mmol) was added as a
solution in acetone (80 mL). The reaction mixture stirred for 19 h,
when TLC analysis showed formation of single product (41c)
(R.sub.f=0.22; silica plate, 9:1:0.01 CH.sub.2Cl.sub.2-MeOH--AcOH,
UV 254 nm, TLC was developed by heating with 3% (w/v) solution of
ninhydrin in EtOH). The reaction mixture was then concentrated to
remove acetone and the residue so obtained was diluted with water
(100 mL). Non-polar impurities were removed by extraction with
Et.sub.2O (100 mL.times.3). The aqueous layer was acidified (pH
.about.1, HCl, 1 M) and extracted with CH.sub.2Cl.sub.2 (150
mL.times.3). The CH.sub.2Cl.sub.2 layer was dried over
Na.sub.2SO.sub.4 and concentrated to give 1.41 g of product (41c)
(98% yield) as white solid. ES-MS (MeOH-direct infusion) Calculated
[M+Na].sup.+=[C.sub.24H.sub.23N.sub.3O.sub.6+Na].sup.+=472.15,
Observed [M+Na].sup.+=472.09.
[0414] Approximately 10 mg of Fmoc-Glu-Fib-Trityl-chloride resin
was treated with 20% (v/v) piperidine in DMF (2 mL.times.1 min,
filtered, then 2 mL.times.5 min), filtered and washed (NMP).
Compound (41c) (10 eqv to Glu-Fib amount on the resin) was
activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and
N,N-Diisopropylethylamine (30 eqv) in NMP (.about.1 mL), added to
the resin and mixed for 30 min. Resin was then filtered, washed
with NMP, and Fmoc group was cleaved. Piperazine acetic acid-TFA
salt (10 eqv) then activated using HATU (9.5 eqv) and N,
N-Diisopropylethylamine (60 eqv) in NMP (.about.1.5 mL) and added
to the resin. After 30 min resin was washed with NMP followed by
CH.sub.3CN. Conjugated peptide was cleaved (and deprotected) from
resin using 95:5 TFA-water (200 .mu.L, 2 h) and precipitated using
Et.sub.2O. Analysis of compound (41) was performed using ES-MS
(direct infusion in water, Calculated [M+H].sup.+=1919.9, Observed
[M+H].sup.+=1920.3)
[0415] Further Mass Spectral Analysis: MS/MS analyses of the Mass
Tag labeled Glu-Fib peptide (41) were performed on MALDI and
electrospray platforms. Data indicates that the Mass Tag is good
candidate for MALDI and electrospray platform. Signature ion
intensity was strong and desired peptide fragmentation pattern was
observed (data not shown).
[0416] E. Synthesis of Mass Tag Labeled Glu-Fib Peptide (42) (see
FIG. 33)
[0417] Compound (42b): To a solution of 5-Fluorouracil (42a) (P/N:
Oakwood, 003241, 0.5 g, 3.84 mmol) and DMAP (46 mg, 0.384 mmol) in
acetonitrile (25 mL), Di-tert-butyl dicarbonate (P/N: Chem-Impex,
00128, 0.835 g, 3.84 mmol) was added and stirred for 17 h at RT (RT
stands for room temperature). TLC analysis showed formation of
single product N-1-Boc-5-Fluorouracil (42b) (R.sub.f=0.86, silica
plate, 7:3 EtOAc-hexanes, UV 254 nm, Reference: Jaime-Figueroa, S.;
Zamilpa, A.; Guzman, A.; Morgans, D. J., "N-3-Alkylation of Uracil
and Derivatives via N-1-Boc Protection", Synthetic Communications,
2001, 31, 3739-3746). The white solid obtained after removal of
solvent was used in the next reaction without further
purification.
[0418] Compound (42c): Compound (42b) (3.84 mmol) was dissolved in
DMF (20 mL) and cooled to 0.degree. C. To this solution NaH (184
mg, 4.60 mmol, 60% dispersion in oil) was added as solid and
stirred for 30 min at RT. At this point BrCH.sub.2COOMe (P/N:
Acros, 16955, 0.437 mL, 4.60 mmol) was added and the reaction
mixture stirred for 2 h at RT. TLC analysis showed formation of a
new product (R.sub.f=0.64, silica plate, 1:1 EtOAc-hexanes, UV 254
nm). Volatiles were removed using a rotary evaporator and the
resulting oil was partitioned between EtOAc (250 mL) and HCl (0.5
M, 100 mL). EtOAc layer was then washed with brine (100
mL.times.2), dried over Na.sub.2SO.sub.4 and concentrated to a
colorless oil. The oil was purified by flash-chromatography
(CombiFlash purification system, 40 g column, 40 mL/min, 270 nm,
gradient: 10-65% increment of EtOAc in hexanes over 25 min, 18 mL
fraction collected, fractions 15-25 had pure product) to give 0.830
g (71% yield) of product (42c). ES-MS (Direct infusion in MeOH,
Calculated
[M+Na].sup.+=[C.sub.12H.sub.15FN.sub.2O.sub.6+Na].sup.+=325.08,
observed [M+Na].sup.+=325.12.
[0419] Compound (42d): Compound (42c) (0.403 mg, 1.33 mmol) was
treated with TFA-CH.sub.2Cl.sub.2 (1:1, 10 mL) solution for 15 min
and the volatiles were removed to give compound (42d) (0.250 g, 93%
yield, R.sub.f=0.40, silica plate, 1:1 EtOAc-hexanes, UV 254 nm) as
a white solid. ES-MS (Direct infusion in MeOH, Calculated
[M+H].sup.+=[C.sub.7H.sub.7FN.sub.2O.sub.4+H].sup.+=203.05,
observed [M+H].sup.+=203.09).
[0420] Compound (42e): To a solution of (42d) (0.250 g, 1.23 mmol)
and BrCH.sub.2CH.sub.2NHBoc (P/N: Fluka, 17354, 0.482 g, 2.15 mmol)
in DMF (15 mL), K.sub.2CO.sub.3 (0.509 g, 3.69 mmol) was added as
solid and the suspension stirred for 23 h at RT. TLC analysis
showed the formation of a single product (42e) (R.sub.f=0.28,
silica plate, 1:1 EtOAc-hexanes, UV 254 nm, TLC was developed by
heating with 3% (w/v) ninhydrin solution in EtOH). Solvents and
volatiles were removed using a rotary evaporator and the resulting
oil was partitioned between EtOAc (200 mL) and dilute HCl (100 mL,
pH=3-4). EtOAc layer was then washed with brine (50 mL.times.2),
dried over Na.sub.2SO.sub.4 and concentrated to a colorless oil.
The oil was purified by flash-chromatography (CombiFlash
purification system, 40 g column, 40 mL/min, 270 nm, gradient:
40-60% increment of EtOAc in hexanes over 25 min, 18 mL fraction
collected, fractions 12-20 had pure product) to give 0.382 g (90%
yield) of product (42e). ES-MS (Direct infusion in MeOH, Calculated
[M+Na].sup.+=[C.sub.14H.sub.20FN.sub.3O.sub.6+Na.sup.+]=368.12,
observed [M+Na].sup.+=368.23.
[0421] Compound (42f): LiOH.H.sub.2O (55 mg, 1.32 mmol dissolved in
2 mL water) was added to a solution of (42e) (0.226 g, 0.66 mmol)
in DMF (10 mL) and stirred for 20 min, when TLC analysis showed
complete hydrolysis of methyl ester (R.sub.f=0, silica plate, 1:1
EtOAc-hexanes, UV 254 nm, TLC was developed by heating with 3%
(w/v) ninhydrin solution in EtOH). Volatiles were removed under
reduced pressure and the residue was treated with TFA-water (9:1,
10 mL) for 1 h at RT. After removal of TFA-water, the residue was
dissolved in saturated NaHCO.sub.3 (30 mL, pH=8-9). A solution of
Fmoc-OSu (P/N: Advance ChemTech RC8015, 0.268 g, 0.79 mmol in
acetone (30 mL)) was then added to the aqueous solution and stirred
for 1 hour at ambient temperature. TLC analysis showed formation of
a product (42f) (R.sub.f=0.20, silica plate, 9:1:0.01
CH.sub.2Cl.sub.2-MeOH--AcOH, UV 254 nm, TLC was developed by
heating with 3% (w/v) solution of ninhydrin in EtOH). The reaction
mixture was then concentrated to remove acetone and the residue so
obtained was diluted with water (150 mL). Non-polar impurities were
removed by extraction with Et.sub.2O (100 mL.times.2). The aqueous
layer was acidified (pH .about.1, HCl, 1 M) and extracted with
EtOAc (250 mL). EtOAc layer was washed with brine (50 mL.times.2),
dried over Na.sub.2SO.sub.4 and concentrated to give 0.107 g (35%
yield over three steps) of product (42f) as colorless viscous oil.
ES-MS (MeOH-direct infusion) Calculated
[M+Na].sup.+=[C.sub.23H.sub.20FN.sub.3O.sub.6+Na].sup.+=476.12,
Observed [M+Na].sup.+ 476.20.
[0422] Approximately 10 mg of Fmoc-Glu-Fib-Trityl-chloride resin
was treated with 20% (v/v) piperidine in DMF (2 mL.times.1 min,
filtered, then 2 mL.times.5 min), filtered and washed (NMP).
Compound (42f) (10 eqv to Glu-Fib amount on the resin) was
activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and
N,N-Diisopropylethylamine (30 eqv) in NMP (.about.1 mL), added to
the resin and mixed for 30 min. Resin was then filtered, washed
with NMP, and Fmoc group was cleaved. Piperazine acetic acid-TFA
salt (10 eqv) was then activated using HATU (9.5 eqv) and
N,N-Diisopropylethylamine (60 eqv) in NMP (.about.1.5 mL) and added
to the resin. After 30 min the resin was washed with NMP followed
by CH.sub.3CN. Conjugated peptide was cleaved (and deprotected)
from the resin using 95:5 TFA-water (200 .mu.L, 2 h) and
precipitated using Et.sub.2O. Analysis of compound (42) was
performed using ES-MS (direct infusion in water, Calculated
[M+H].sup.+=1923.8, Observed [M+H].sup.+=1924.0)
[0423] Further Mass Spectral Analysis: MS/MS analyses of the Mass
Tag labeled Glu-Fib peptide (42) were performed on MALDI and
electrospray platforms. Data indicates that the Mass Tag is good
candidate for MALDI and electrospray platform. Signature ion
intensity was strong and desired peptide fragmentation pattern was
observed (data not shown).
[0424] F. Synthesis of Mass Tag Labeled Glu-Fib Peptide (43) (see
FIG. 34)
[0425] Compound (43b): To a solution of (43a) (P/N: Oakwood,
003333, 1.0 g, 5.55 mmol) and DMAP (67 mg. 0.55 mmol) in
acetonitrile (40 mL), Di-tert-butyl dicarbonate (P/N: Chem-Impex,
00128, 1.21 g, 5.55 mmol) was added and stirred for 2 h at RT. TLC
analysis showed formation of single product (43b) (R.sub.f=0.81,
silica plate, 1:1 EtOAc-hexanes, UV 254 nm, Reference:
Jaime-Figueroa, S.; Zamilpa, A.; Guzman, A.; Morgans, D. J.,
"N-3-Alkylation of Uracil and Derivatives via N-1-Boc Protection",
Synthetic Communications, 2001, 31, 3739-3746). White solid
obtained after removal of solvent was used in next reaction without
further purification.
[0426] Compound (43c): Compound (43b) (5.55 mmol) was dissolved in
DMF (35 mL) and cooled to 0.degree. C. To this solution NaH (267
mg, 6.66 mmol, 60% dispersion in oil) was added as solid and
stirred for 30 min at RT. At this point BrCH.sub.2COO.sup.tBu (P/N:
Aldrich, 124230, 0.984 mL, 6.66 mmol) was added and the reaction
mixture stirred for 1 h at RT. TLC analysis showed formation of one
major product (43c) (R.sub.f=0.60, silica plate, 1:4 EtOAc-hexanes,
UV 254 nm). Volatiles were removed using a rotary evaporator and
the resulting oil was partitioned between EtOAc (250 mL) and HCl
(0.5 M, 100 mL). EtOAc was layer then washed with brine (50
mL.times.2), dried over Na.sub.2SO.sub.4 and concentrated to give
colorless oil. The oil was purified by flash-chromatography
(CombiFlash purification system, 120 g column, 85 mL/min, 270 nm,
25% EtOAc in hexanes, 18 mL fraction collected) to give 0.528 g
(20% yield) of product (43c). ES-MS (Direct infusion in MeOH,
Calculated
[M.sub.2+Na].sup.+=[C.sub.32H.sub.42F.sub.6N.sub.4O.sub.12+Na].sup.+=811.-
26, observed [M.sub.2+Na].sup.+=811.42
[0427] Compound (43d): Compound (43c) (0.528 g, 1.34 mmol) was
treated with TFA-CH.sub.2Cl.sub.2 (1:9, 15 mL) solution for 5 min
and the volatiles were removed to give compound (43d) (0.390 g, 99%
yield, R.sub.f=0.22, silica plate, 1:4 EtOAc-hexanes, UV 254 nm) as
a white solid. ES-MS (Direct infusion in MeOH, Calculated
[M+Na].sup.+=[C.sub.11H.sub.13F.sub.3N.sub.2O.sub.4+Na].sup.+=317.07,
observed [M+Na].sup.+=317.15.
[0428] Compound (43e): To a solution of (43d) (0.390 g, 1.32 mmol)
and BrCH.sub.2CH.sub.2NHBoc (P/N: Fluka, 17354, 1.112 g, 4.62 mmol)
in DMF (15 mL) K.sub.2CO.sub.3 (1.094 g, 7.92 mmol) was added as
solid and the suspension was stirred for 23 h at RT. TLC analysis
showed the formation of a single product (R.sub.f=0.70, silica
plate, 1:1 EtOAc-hexanes, UV 254 nm, TLC was developed by heating
with 3% (w/v) ninhydrin solution in EtOH). Volatiles were removed
using a rotary evaporator and the resulting oil was partitioned
between EtOAc (200 mL) and dilute-HCl (100 mL, pH=3-4). EtOAc layer
was then washed with brine (50 mL.times.2), dried over
Na.sub.2SO.sub.4 and concentrated to give a colorless oil. The oil
was purified by flash-chromatography (CombiFlash purification
system, 40 g column, 40 mL/min, 270 nm, 35% EtOAc in hexanes, 18 mL
fraction collected, fractions 10-15 had pure product) to give 0.256
g (45% yield) of product (43e). ES-MS (Direct infusion in MeOH,
Calculated
[M+Na].sup.+=[C.sub.18H.sub.26F.sub.3N.sub.3O.sub.6+Na].sup.+=460.17,
observed [M+Na].sup.+=460.30.
[0429] Compound (43f): Compound (43e) (0.256 g, 0.59 mmol) was
treated with TFA-water (95:5, 10 mL) and stirred for 1 h. Volatiles
were removed under reduced pressure and the residue was washed with
Et.sub.2O. The white precipitate so obtained was dissolved in
saturated NaHCO.sub.3 (30 mL, pH=8-9). A solution of Fmoc-OSu (P/N:
Advance ChemTech RC8015, 0.239 g, 0.71 mmol in acetone (2 mL)) was
then added to the aqueous solution and stirred for 1 hour at
ambient temperature. TLC analysis showed formation of a product
(43f) (R.sub.f=0.24; silica plete, 9:1:0.01
CH.sub.2Cl.sub.2-MeOH--AcOH, UV 254 nm, TLC was developed by
heating with 3% (w/v) solution of ninhydrin in EtOH). The reaction
mixture was then concentrated to remove acetone and the residue so
obtained was diluted with water (150 mL). Non-polar impurities were
removed by extraction with Et.sub.2O (100 mL.times.2). The aqueous
layer was acidified (pH .about.1, HCl, 1 M) and extracted with
EtOAc (250 mL.times.2). EtOAc layer was dried over Na.sub.2SO.sub.4
and concentrated to give 0.266 g of (43f) as white solid. ES-MS
(MeOH-direct infusion) Calculated
[M+Na].sup.+=[C.sub.24H.sub.20F.sub.3N.sub.3O.sub.6+Na].sup.+=526.12,
Observed [M+Na].sup.+ 526.20.
[0430] Approximately 10 mg of Fmoc-Glu-Fib-Trityl-chloride resin
was treated with 20% (v/v) piperidine in DMF (2 mL.times.1 min,
filtered, then 2 mL.times.5 min), filtered and washed (NMP).
Compound (43f) (10 eqv to Glu-Fib amount on the resin) was
activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and
N,N-Diisopropylethylamine (30 eqv) in NMP (.about.1 mL), added to
the resin and mixed for 30 min. Resin was then filtered, washed
with NMP, and the Fmoc group was cleaved. Piperazine acetic
acid-TFA salt (10 eqv) was then activated using HATU (9.5 eqv) and
N,N-Diisopropylethylamine (60 eqv) in NMP (.about.1.5 mL) and added
to the resin. After 30 min resin was washed with NMP followed by
CH.sub.3CN. Conjugated peptide was cleaved (and deprotected) from
resin using 95:5 TFA-water (200 .mu.L, 2 h) and precipitated using
Et.sub.2O. Analysis of compound (43) was performed using ES-MS
(direct infusion in water, Calculated [M+H].sup.+=1973.8, Observed
[M+H].sup.+=1974.0)
[0431] Further Mass Spectral Analysis: MS/MS analyses of the Mass
Tag labeled Glu-Fib peptide (43) were performed on MALDI and
electrospray platforms. Data indicates that the Mass Tag is good
candidate for MALDI and electrospray platform. Signature ion
intensity was strong and desired peptide fragmentation pattern was
observed (data not shown).
[0432] G. Synthesis of Mass Tag Labeled Glu-Fib Peptide (46) (see
FIG. 36)
[0433] Compound (46b): To a solution of (46a) (P/N: Chem-Impex
01343, 2.73 g, 5.88 mmol) in THF (50 mL) at 0.degree. C.,
BH.sub.3.THF (14.7 mL, 1 M) solution was added and allowed to react
for 18 h at RT. TLC analysis of a small aliquot (quenched with
MeOH) showed formation of a new product and some Boc-deprotected
product (R.sub.f(46b)=0.50, silica plate, 1:1 EtOAc-hexanes, UV 254
nm, TLC was developed by heating with 3% (w/v) ninhydrin solution
in EtOH, Boc deprotected product was identified from the base-line
spot on TLC which was intensely ninhydrin positive (unlike compound
(46a)). Reaction was quenched with MeOH, Di-tert-butyl dicarbonate
(P/N: Chem-Impex, 00128, 1.28 g, 5.88 mmol) was added and stirred
for 1 h at RT. TLC analysis at this stage showed the presence of
compound (46b) only. Reaction mixture was concentrated and the
resulting oil was purified by flash chromatography (CombiFlash
purification system, 120 g column, 85 mL/min, 270 nm, 0-15 min 10%
EtOAc in hexanes, then 60% EtOAc in hexanes, 18 mL fraction
collected) to give 2.12 g (80% yield) of product (46b). ES-MS
(MeOH-direct infusion) Calculated
[M+Na].sup.+=[C.sub.27H.sub.31NO.sub.3S+Na].sup.+=472.19, Observed
[M+Na].sup.+=472.17.
[0434] Compound (46c): To a solution of (46b) (1.07 g, 2.38 mmol)
and CBr.sub.4 (P/N: Aldrich C11081, 1.18 g, 3.57 mmol) in
CH.sub.2Cl.sub.2 (10 mL) PPh.sub.3 solution (P/N: Aldrich T84409,
0.685 g, 2.39 mmol in 3 mL CH.sub.2Cl.sub.2) was added over 4 h at
RT (using syringe pump). After completion of PPh.sub.3 addition,
the reaction was stirred for another 1 h. TLC showed formation of a
major product (46c) (R.sub.f=0.50, silica plate, 1:4 EtOAc-hexanes,
UV 254 nm, TLC was developed by heating with 3% (w/v) ninhydrin
solution in EtOH). Solvent was removed under reduced pressure and
the oil was purified by flash chromatography (CombiFlash
purification system, 120 g column, 85 mL/min, 270 mm, 0-5 min 5%
EtOAc in hexanes, then 20% EtOAc in hexanes, 18 mL fraction
collected) to give 0.690 g (56% yield) of product (46c). ES-MS
(MeOH-direct infusion) Calculated
[M+Na].sup.+=[C.sub.27H.sub.30BrNO.sub.2S+Na].sup.+=534.11,
Observed [M+Na].sup.+=534.06.
[0435] Compound (46e): To a solution of (46c) (0.461 g, 0.90 mmol)
and (46d) (0.244 g, 1.08 mmol) in DMF (25 mL) solid K.sub.2CO.sub.3
(0.372 g, 2.70 mmol) was added and stirred for 68 h at RT. TLC
showed presence of a product (46e) (R.sub.f=0.50, silica plate, 1:1
EtOAc-hexanes, UV 254 nm, TLC was developed by heating with 3%
(w/v) ninhydrin solution in EtOH), unreacted (46c) and (46d). After
DMF removal under reduced pressure, the resulting oil was
partitioned between EtOAc (200 mL) and dilute HCl (150 mL, 0.5 M).
EtOAc layer was then washed with brine (50 mL.times.2), dried over
Na.sub.2SO.sub.4 and concentrated to give colorless oil. The oil
was purified by flash-chromatography (CombiFlash purification
system, 40 g column, 40 mL/min, 265 nm, 0-1 min 20% EtOAc in
hexanes, 1-10 min then 35% EtOAc in hexanes then 50% EtOAc in
hexanes, 18 mL fraction collected, fractions 19-27 had pure
product) to give 0.200 g (34% yield) of product (46e). ES-MS
(Direct infusion in MeOH, Calculated
[M+Na].sup.+=[C.sub.37H.sub.43N.sub.3O.sub.6S+Na].sup.+=680.28,
observed [M+Na].sup.+=680.23.
[0436] Compound (46f): Compound (46e) (0.200 g, 0.304 mmol) was
treated with TFA-CH.sub.2Cl.sub.2 (9:1, 10 mL) for 30 min at RT and
then TFA-CH.sub.2Cl.sub.2 removed under reduced pressure. The
yellow oil so obtained was co-evaporated with THF until the oil was
colorless (re-tritylation of thiol group). The oil was then
dissolved in DMF (5 mL) and basified with N,N-Diisopropylethylamine
(P/N: Applied Biosystems 400136, pH 9-10, moist pH paper).
N,N-Diisopropylethylamine (0.206 mL, 1.19 mmol) was added to a
solution of N-Me-piperazine acetic acid.2TFA (0.152 g, 0.395 mmol)
and HATU (Applied Biosystems, 4317033, 0.138 g, 0.364 mmol) in DMF
(2 mL); mixed for 1 min and added to the above solution of Boc
deprotected (46c). After 1 h the reaction mixture was acidified
with HCl (1 M), diluted with brine (150 mL) and extracted with
CH.sub.2Cl.sub.2 (150 mL). Dichloromethane layer was dried over
Na.sub.2SO.sub.4 and concentrated to give 0.207 g of product (46f)
as white foam (98%). ES-MS (Direct infusion in MeOH, Calculated
[M+H].sup.+ [C.sub.39H.sub.47N.sub.5O.sub.5S+H].sup.+=698.33,
observed [M+H].sup.+=698.27.
[0437] Compound (46 g): To a solution of (46f) (72 mg, 0.1 mmol) in
THF-water (2:1, 3 mL) NaOH solution (0.20 mL, 1 M) was added and
mixed for 30 min. Reaction was neutralized to pH=3 by TFA solution
(1 M) and dried under vacuum to give product (46 g).
[0438] Approximately 10 mg of Fmoc-Glu-Fib-Trityl-chloride resin
was treated with 20% (v/v) piperidine in DMF (2 mL.times.1 min,
filtered, then 2 mL.times.5 min), filtered and washed (NMP).
Compound (46 g) (10 eqv to Glu-Fib amount on the resin) was
activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and
N,N-Diisopropylethylamine (60 eqv) in NMP (.about.1 mL), added to
the resin and mixed for 30 min. Resin was then filtered, and washed
with NMP followed by CH.sub.3CN. Conjugated peptide was cleaved
(and deprotected) from resin using 95:5 TFA-water (200 mL, 2 h) and
precipitated using Et.sub.2O. To an ice cold solution of product
peptide (1:5 CH.sub.3CN-Water, 0.60 ml) per-formic acid solution
(0.6 mL) was added and mixed at 0.degree. C. for 5 min (Per-formic
(HCOOOH) acid solution preparation: 4 mL HCOOH (99%)+0.45 mL
H.sub.2O.sub.2 (30%)+0.25 mL water were mixed and allowed to stand
for 1 h at RT). ES-MS analysis showed the presence of desired
oxidized product (46). Calculated [M+H].sup.+=2013.8, observed
[M+H].sup.+=2013.7.
[0439] Further Mass Spectral Analysis: MS/MS analyses of the Mass
Tag labeled Glu-Fib peptide (46) were performed on a MALDI
platform. Data indicate that the Mass Tag is a good candidates for
MALDI platform (data not shown). In MS analysis (electrospray) the
amount of +3 charged species was much lower than in the case of
only thymine nucleobase containing tags. This compound gave only
`y` ion series upon peptide fragmentation when analyzed on MALDI
platform (data not shown)--thus substantially reducing the
complexity of the MS/MS spectra for analysis.
[0440] G. Synthesis of Mass Tag Labeled Glu-Fib Peptide (47) (see
FIG. 37)
[0441] To a solution of cysteic acid (P/N: TCI America C0514, 1 g,
5.90 mmol) in aq NaHCO.sub.3 (pH=8-9, .about.50 mL) Fmoc-OSu
solution (2.4 g, 7.08 mmol in 150 mL acetone) was added and stirred
for 18-19 h at RT. Acetone was removed under reduced pressure and
the suspension was diluted with 150 mL of water. Non-polar
impurities were removed by extraction with Et.sub.2O (100
mL.times.3). Aqueous layer was acidified with conc. HCl to pH 1 and
then Amberlite IR-120-H resin (P/N: Aldrich 216534, 12 g, 1.9
mmol/g --SO.sub.3H group) was added, mixed and filtered. Filtrate
was lyophilized to give 2.52 g of Fmoc-cysteic-acid as white
hygroscopic solid. ES-MS (Direct infusion in water, negative mode,
Calculated [M-H].sup.-=[C.sub.18H.sub.17NO.sub.7S--H].sup.-=390.06,
observed [M-H].sup.-=390.03.
[0442] Approximately 10 mg of Fmoc-Glu-Fib-Trityl-chloride resin
was treated with 20% (v/v) piperidine in DMF (2 mL.times.1 min,
filtered, then 2 mL.times.5 min), filtered and washed (NMP).
Fmoc-cysteic-acid (10 eqv to Glu-Fib amount on the resin) was
individually activated with HATU (P/N: Applied Biosystems 4317033,
9.5 eqv) and N, N-Diisopropylethylamine (30 eqv) in NMP (.about.1
mL), added to the resin and mixed for 30 min. Resin was then
filtered, washed with NMP, and Fmoc group was cleaved. Piperazine
acetic acid-TFA salt (10 eqv) was then activated using HATU (9.5
eqv) and N,N-Diisopropylethylamine (60 eqv) in NMP (.about.1.5 mL),
and added to the resin. After 30 min the resin was washed with NMP
followed by CH.sub.3CN. Conjugated peptide was cleaved (and
deprotected) from resin using 95:5 TFA-water (200 .mu.L, 2 h) and
precipitated using Et.sub.2O. Analysis of compound (47) was
performed using ES-MS (direct infusion in water, Calculated
[M+H].sup.+=2070.9, Observed [M+H].sup.+=2071.8)
[0443] Further Mass Spectral Analysis: MS/MS analyses of the Mass
Tag labeled Glu-Fib peptide (47) were performed on a MALDI
platform. Data indicate that the Mass Tag is a good candidate for
MALDI platform (data not shown). In MS analysis (electrospray) the
amount of +3 charged species was much lower than in the case of
only thymine nucleobase containing tags. This compound gave only
`y` ion series upon peptide fragmentation when analyzed on MALDI
platform (data not shown)--thus substantially reducing the
complexity of the MS/MS spectra for analysis.
[0444] H. Synthesis of Mass Tag Labeled Glu-Fib Peptide (49) (see
FIG. 38)
[0445] To a solution of Br(CH.sub.2).sub.3CO.sub.2Et (P/N: Aldrich
167118, 0.645 mL, 4.5 mmol) in EtOAc (20 mL) 1-Methyl piperazine
(P/N: Aldrich 130001, 1 mL, 9.0 mmol) was added and stirred
overnight. After filtration of the precipitate, EtOAc layer was
concentrated and the oil so formed was purified by flash
chromatography (CombiFlash purification system, 12 g silica column,
30 mL/min, 20% methanol in CH.sub.2Cl.sub.2, 18 mL fraction
collected) to give product (49a). Product (49a) was dissolved in
water (4 mL) and heated at 95.degree. C. for 4 h. After removal of
water the solid (49b) was washed with THF and dried under vacuum.
ES-MS (Direct infusion in water, Calculated
[M+H].sup.+=[C.sub.9H.sub.18N.sub.2O.sub.2+H].sup.+=187.14,
observed [M+H].sup.+=187.18.
[0446] Approximately 10 mg of Fmoc-Glu-Fib-Trityl-chloride resin
was treated with 20% (v/v) piperidine in DMF (2 mL.times.1 min,
filtered, then 2 mL.times.5 min), filtered and washed (NMP).
Compound (49b) (10 eqv to Glu-Fib amount on the resin) was
activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and
N,N-Diisopropylethylamine (30 eqv, 60 eqv for compound 21) in NMP
(.about.1 mL), added to the resin and mixed for 30 min. Resin was
then filtered, washed with NMP followed by CH.sub.3CN. Conjugated
peptide was cleaved (and deprotected) from resin using 95:5
TFA-water (200 .mu.L, 2 h) and precipitated using Et.sub.2O.
Analysis of compound (50) was performed using ES-MS (direct
infusion in water, Calculated [M+H].sup.+=1738.8, Observed
[M+H].sup.+=1739.2)
[0447] Further Mass Spectral Analysis: MS/MS analyses of the Mass
Tag labeled Glu-Fib peptide (49) was performed on MALDI and
electrospray platforms. Data indicate that propionic acid
derivative (49) could be a suitable candidate (data not shown).
However a close analysis of the MS/MS pattern revels presence of
some peptide fragments in which part of the tag was still
attached.
[0448] The foregoing synthetic methods could be applied to the
preparation of isotopically coded labeling reagents by the
incorporation of starting materials comprising heavy atom isotopes.
The following examples demonstrate various methods for generating
isotopically encoded isobaric labeling reagents.
Syntheses of Isobaric Mass Tags (Labeling Reagents)
I. Isobaric Mass Tags Isotopically Coded with Deuterium
Isotopes.
A. Synthesis of Mass Tag (25)
[0449] FIG. 20 illustrates the synthesis of Mass Tag (25).
[0450] FmocSer(Bzl) (1 mmol), TSTU (1 mmol), and DIEA (2 mmol) were
dissolved in DMF (6 mL). The mixture was shaken at room temperature
for half an hour. The solvent was evaporated to form
FmocSer(Bzl)-OSu, which was used directly in the following
steps.
[0451] To a solution of glycine (0.4 mmol) in DMF (0.8 ml) and 0.2
M aqueous sodium bicarbonate (2.8 ml) was added Fmoc-Ser(Bzl)-OSu
(0.4 mmol) in DMF (2.4 ml) while vortexing. The mixture was shaken
at room temperature for 20 minutes. The compound, FmocSer(Bzl)-Gly,
was purified with preparative HPLC.
[0452] FmocSer(Bzl)-Gly (50 mg) was exposed to 20% piperidine in
DMF (5 ml) for 10 minutes to remove the Fmoc-protecting group.
After evaporation of solvents, the product, Ser(Bzl)-Gly, was
purified with preparative HPLC, and characterized with MS
([M+H].sup.+: 253.1, calculated; 253.0, found).
[0453] FmocGlycine-2,2-d.sub.2 (ISOTEC, 0.14 mmol), TSTU (0.21
mmol), and DIEA (0.28 mmol) were dissolved in DMF (1 ml). The
mixture was shaken at room temperature for 45 minutes, and then
transferred to a solution of Ser(Bzl)-Gly (0.14 mmol) in 0.2 M
aqueous sodium bicarbonate (2 ml). More DMF (1 ml) was added. The
mixture was shaken at room temperature for 20 minutes. The
compound, FmocGly(d.sub.2)-Ser(Bzl)-Gly, was purified with
preparative HPLC, and characterized with MS ([M+H].sup.+: 534.2,
calculated; 534.2, found).
[0454] FmocGly(d.sub.2)-Ser(Bzl)-Gly was exposed to 20% piperidine
in DMF (5 ml) for 15 minutes to remove the Fmoc-protecting group.
After evaporation of solvents, the compound,
Gly(d.sub.2)-Ser(Bzl)-Gly, was purified with preparative HPLC, and
characterized with MS ([M+H].sup.+: 312.1, calculated; 312.0,
found).
[0455] Gly(d.sub.2)-Ser(Bzl)-Gly was acylated using Protocol I to
furnish Mass Tag (25) ([M+H].sup.+: 480.1, calculated; 480.0,
found).
[0456] B. Synthesis of Mass Tag (26)
[0457] FIG. 21 illustrates the synthesis of Mass Tag (26).
[0458] A solution of Boc-L-Ser (NovaBichem, 2 mmol) in DMF (4 ml)
was cooled with an ice-water bath. Sodium hydride (Aldrich, 6 mmol)
was added. After the evolution of hydrogen gas ceased,
benzyl-.alpha.,.alpha.-d.sub.2 bromide (ISOTEC, 2 mmol) was added
while vortexing. The mixture was shaken at room temperature for 5
hours. The product, BocSer(Bzl-d.sub.2), was purified with
preparative HPLC, and characterized with MS ([M+H].sup.+: 298.2,
calculated; 298.2, found).
[0459] BocSer(Bzl-d.sub.2) (0.4 mmol), TSTU (0.6 mmol), and DIEA
(0.8 mmol) were dissolved in DMF (2 ml). The mixture was shaken at
room temperature for 1 hour, and then transferred dropwise to a
solution of glycine (2 mmol) in 3 mL of 1M aqueous sodium
bicarbonate. The mixture was shaken at room temperature for 30
minutes. The product, BocSer(Bzl-d.sub.2)-Gly, was purified with
preparative HPLC, and characterized with MS ([M+H].sup.+: 355.3,
calculated; 355.2, found).
[0460] BocSer(Bzl-d.sub.2)-Gly (64 mg) was exposed to a solution of
trifluoroacetic acid (TFA, Applied Biosystems, 1 ml) and methylene
chloride (2 ml) at room temperature for 30 minutes to remove the
Boc-protecting group. The mixture was extracted with water twice
(1.5 ml each). The extracts were combined, and purified with
preparative HPLC. The product, Ser(Bzl-d.sub.2)-Gly, was
characterized with MS ([M+H].sup.+: 255.1, calculated; 255.2,
found).
[0461] FmocGly (0.3 mmol), TSTU (0.3 mmol), and DIEA (0.45 mmol)
were dissolved in DMF (2 ml). The mixture was shaken at room
temperature for 1 hour, and then transferred dropwise to a solution
of Ser(Bzl-d.sub.2)-Gly in 0.2 M in aqueous sodium bicarbonate (2
ml). More DMF (1 ml) was added. The mixture was shaken at room
temperature for 20 minutes. The product,
FmocGly-Ser(Bzl-d.sub.2)-Gly, was purified with preparative HPLC,
and characterized with MS ([M+H].sup.+: 534.3, calculated; 534.4,
found).
[0462] FmocGly-Ser(Bzl-d.sub.2)-Gly was exposed to a solution of 5
ml of 20% piperidine in DMF at room temperature for 10 minutes to
remove the Fmoc-protecting group. After evaporation of solvents,
the product, Gly-Ser(Bzl-d.sub.2)-Gly, was purified with
preparative HPLC, and characterized with MS ([M+H].sup.+: 312.2,
calculated; 312.4, found).
[0463] Gly-Ser(Bzl-d.sub.2)-Gly was acylated using Protocol I to
furnish Mass Tag (26) ([M+H].sup.+: 480.1, calculated; 480.2,
found).
II. Isobaric Mass Tags Isobarically Coded with .sup.12C/.sup.13C
and .sup.14N/.sup.15N
[0464] A. Synthesis of Mass Tag (27)
[0465] FIG. 22 illustrates the synthesis of Mass Tag (27).
[0466] FmocGly(.sup.13C.sub.2, .sup.15N) (ISOTEC, 0.33 mmol), TSTU
(0.66 mmol) and DIEA (0.66 mmol) were dissolved in DMF (2 ml). The
mixture was shaken at room temperature for 40 minutes, and then
transferred dropwise to a solution of L-Serine(Bzl) (NovaBiochem, 2
mmol) in DIEA (4 mmol), DMSO (8 ml) and water (2 ml) while
vortexing. The mixture was shaken at room temperature for 20
minutes. After filtration, the filtrate, which contained the
product, was purified with preparative HPLC. The product,
FmocGly(.sup.13C.sub.2, .sup.15N)-Ser(Bzl), was characterized with
MS ([M+H].sup.+: 478.2, calculated; 478.2, found).
[0467] FmocGly(.sup.13C.sub.2, .sup.15N)-Ser(Bzl), TSTU (0.6 mmol)
and DIEA (0.6 mmol) were dissolved in DMF (2 ml). The mixture was
shaken at room temperature for 1 hour, and transferred dropwise to
a solution of Gly(.sup.13C.sub.2, .sup.15N) (ISOTEC, 1 mmol) in
water (2 ml) with sodium bicarbonate (2 mmol) while vortexing. More
DMF (4 ml) was added. The mixture was shaken at room temperature
for 30 minutes. After centrifugation, the supernatant, which
contained the product, was purified with preparative HPLC. The
product, FmocGly(.sup.13C.sub.2,
.sup.15N)-Ser(Bzl)-Gly(.sup.13C.sub.2, .sup.15N), was characterized
with MS ([M+H].sup.+: 538.2, calculated; 538.2, found).
[0468] FmocGly(.sup.13C.sub.2,
.sup.15N)-Ser(Bzl)-Gly(.sup.13C.sub.2, .sup.15N) (4 mg) was exposed
to 0.2 ml of 20% piperidine in DMF at room temperature for 10
minutes to remove the Fmoc-protecting group. After removal of all
the solvents, the deprotected amine was acylated using Protocol I
to furnish Mass Tag (27) ([M+H].sup.+: 484.1, calculated; 484.0,
found).
[0469] B. Synthesis of Mass Tag (28)
[0470] FIG. 23 illustrates the synthesis of Mass Tag (28).
[0471] Boc-L-Ser (NovaBiochem, 5.82 mmol) was dissolved in DMF (6
ml), and cooled with an ice-water bath. Sodium hydride (17.46 mmol)
was added while vortexing. The mixture was shaken at room
temperature for 15 minutes. After no more gas was released, benzyl
(.alpha.-.sup.13C) bromide (ISOTEC, 2.91 mmol) was added while
vortexing. The mixture was shaken at room temperature for 4 hours,
and then purified with preparative HPLC. The product,
BocSer(Bzl-.alpha.-.sup.13C), was characterized with MS
([M+H].sup.+: 297.1, calculated; 297.2, found).
[0472] BocSer(Bzl-.alpha.-.sup.13C) (300 mg) was deprotected with
10 ml of 30% TFA in methylene chloride for 30 minutes, and then
extracted with water twice (3 mL each). The aqueous layers were
combined, and purified with preparative HPLC. The product,
Ser(Bzl-.alpha.-.sup.13C), was characterized with MS ([M+H].sup.+:
197.1, calculated; 197.0, found).
[0473] FmocGly(2-.sup.13C, .sup.15N) (ISOTEC, 1 mmol), TSTU (2
mmol) and DIEA (2 mmol) were dissolved in DMF (3 ml). The mixture
was shaken at room temperature for 1 hour, and then transferred to
Ser(Bzl-.alpha.-.sup.13C) in 3 ml of 0.2 M aqueous sodium
bicarbonate solution while vortexing. The mixture was shaken at
room temperature for 20 minutes, and purified with preparative
HPLC. The product, FmocGly(2-.sup.13C,
.sup.15N)-Ser(Bzl-.alpha.-.sup.13C), was characterized with MS
([M+H].sup.+: 478.2, calculated; 478.2, found).
[0474] FmocGly(2-.sup.13C, .sup.15N)-Ser(Bzl-.alpha.-.sup.13C)
(0.021 mmol), TSTU (0.042 mmol) and DIEA (0.042 mmol) were
dissolved in DMF (0.5 ml). The mixture was shaken at room
temperature for 1 hour, and transferred to Glycine(.sup.13C.sub.2,
.sup.15N) (ISOTEC, 0.1 mmol) in 0.5 ml of 0.2 M aqueous sodium
bicarbonate solution. The mixture was shaken at room temperature
for 20 minutes, and purified with preparative HPLC. The product,
FmocGly(2-.sup.13C,
.sup.15N)-Ser(Bzl-.alpha.-.sup.13C)-Gly(.sup.13C.sub.2, .sup.15N),
was characterized with MS ([M+H].sup.+: 538.2, calculated; 538.0,
found).
[0475] FmocGly(2-.sup.13C,
.sup.15N)-Ser(Bzl-.alpha.-.sup.13C)-Gly(.sup.13C.sub.2, .sup.15N)
(12 mg) was deprotected with 0.8 ml of 20% piperidine in DMF at
room temperature for 10 minutes. After evaporation of all the
solvents, the free amine was acylated using Protocol I to furnish
Mass Tag (28) ([M+H].sup.+: 484.1, calculated; 484.0, found).
Solid Supports with Isobaric Mass Tags
I. Synthesis of FmocGly-Ser(Bzl-.sup.13C.sub.6)
[0476] FIG. 24 illustrates the synthesis of
FmocGly-Ser(Bzl-.sup.13C.sub.6) (29)
[0477] The compound was prepared with the same procedures as those
for preparing FmocGly(2-.sup.13C,
.sup.15N)-Ser(Bzl-.alpha.-.sup.13C) ([M+H].sup.+ in MS: 481.2,
calculated; 481.2, found) (see: Syntheses of Isobaric Mass Tags
Isotopically Coded with heavy atom isotopes, .sctn. IIB).
II. Syntheses of Resin Bound Mass Tags
[0478] FIG. 25 illustrates the syntheses of resin bound isobaric
isotopically coded Mass Tags (30), (31) and (32).
[0479] A. Synthesis of Resin Bound Isobaric Isotopically Coded Mass
Tag (30)
[0480] 1 g of wet amino PEGA resin (NovaBiochem, 0.05 mmol
substitution) was washed with water, DMF, DCM, methanol, DCM and
DMF. The resin was typically washed twice with each solvent
(approximately 5 ml of each). FmocPAL linker (Applied Biosystems,
0.15 mmol), TSTU (0.15 mmol) and DIEA (0.225) were dissolved in DMF
(1 ml). The mixture was shaken at room temperature for 20 minutes,
and then transferred to the resin suspended in around 1 ml of DMF.
The mixture was shaken at room temperature for 1 hour. After
filtration, the resin was washed twice with DMF, DCM, methanol, DCM
and DMF.
[0481] The resin was washed with 5 ml of 20% piperidine in DMF
once, and then fully deprotected with 5 ml of 20% piperidine at
room temperature for 10 minutes. After filtration, the resin was
washed twice with DMF, DCM, methanol, DCM and DMF.
[0482] FmocGly(.sup.13C.sub.2, .sup.15N) (ISOTEC, 0.1 mmol), TSTU
(0.1 mmol) and DIEA (0.15 mmol) were dissolved in DMF (1 ml). The
mixture was shaken at room temperature for 20 minutes, and then
transferred to the resin suspended in approximately 1 ml of DMF.
The mixture was shaken at room temperature for 2 hours. After
filtration, the resin was washed twice with DMF, DCM, methanol, DCM
and DMF.
[0483] The resin was washed with 5 ml of 20% piperidine in DMF
once, and then fully deprotected with 5 ml of 20% piperidine at
room temperature for 10 minutes. After filtration, the resin was
washed twice with DMF, DCM, methanol, DCM and DMF.
[0484] FmocGly(.sup.13C.sub.2, .sup.15N)-Ser(Bzl) (0.1 mmol),
HBTU/HOBT (Applied Biosystems, 0.1 mmol) and DIEA (0.15 mmol) were
dissolved in DMF (1 ml). The mixture was shaken at room temperature
for 2 hours. After filtration, the resin was washed twice with DMF,
DCM, methanol, DCM and DMF.
[0485] The resin was washed with 5 ml of 20% piperidine in DMF
once, and then fully deprotected with 5 ml of 20% piperidine at
room temperature for 10 minutes. After filtration, the resin was
washed twice with DMF, DCM, methanol, DCM and DMF.
[0486] Iodoacetic acid (0.15 mmol) and N-hydroxysuccinimide (0.15
mmol) were dissolved in DMF (0.5 ml). DCC (Aldrich, 0.15 mmol) in
DMF (0.5 ml) was added while vortexing. The mixture was shaken at
room temperature for 1 hour. After filtration, the solution was
added to the resin suspended in 1 ml DMF with sodium bicarbonate
(0.15 mmol). The mixture was shaken at room temperature for 1 hour.
After filtration, resin bound Mass Tag (30) was washed twice with
water, DMF, DCM, methanol, DCM, DMF and DCM. The resin bound mass
tag was split into equal portions within cartridges (Millipore
UFC3OLG25), dried with a SpeedVac, and stored in a freezer
(-30.degree. C.) for future uses. Each cartridge had around 4 mg of
dry resin bound Mass Tag (30).
[0487] B. Synthesis of Resin Bound Isobaric Isotopically Coded Mass
Tag (31)
[0488] The procedure for synthesizing resin bound Mass Tag (31) was
same as that for synthesizing resin bound Mass Tag (30), except
that FmocGly(.sup.13C.sub.2, .sup.15N)-Ser(Bzl) was replaced with
FmocGly(2-.sup.13C, .sup.15N)-Ser(Bzl-.alpha.-.sup.13C).
[0489] C. Synthesis of Resin Bound Isobaric Isotopically Coded Mass
Tag (32)
[0490] The procedure for synthesizing resin bound Mass Tag (31) was
same as that for synthesizing resin bound Mass Tag (30), except
that FmocGly(.sup.13C.sub.2, .sup.15N) and FmocGly(.sup.13C.sub.2,
.sup.15N)-Ser(Bzl) were replaced with FmocGlycine and
FmocGly-Ser(Bzl-.sup.13C.sub.6), respectively.
[0491] Synthesis of Nucleobase Comprising Labeling Reagents: FIG.
26 illustrates the incorporation of the nucleobase (thymine) into
Mass Tag (36a) starting from compound (33) and proceeding through
intermediate compounds (34) and (35). A procedure for such
conversion was performed and is described as follows:
[0492] Synthesis of Compound (34):
[0493] To a solution of thymine acetic acid ethyl ester (33) (500
mg, 2.35 mmol) and 2-Boc-(amino)-ethyl bromide (634 mg, 2.82 mmol)
in DMF (50 mL), was added K.sub.2CO.sub.3 (974 mg, 7.05 mmol). The
reaction was stirred for 18 h at ambient temperature. Thin layer
chromatography (TLC) analysis indicated the formation of a single
product (Silica plate, EtOAc solvent; R.sub.f=0.7; UV, ninhydrin).
After the DMF was removed under reduced pressure, the product was
purified by flash chromatography (ISCO Companion purification
system; 40 g SiO.sub.2 column, detection at 260 nm, Flow=40 mL/min;
0-7 min 50% EtOAc in hexanes to remove unreacted
2-Boc-(amino)-ethyl bromide, then 100% EtOAc to elute the product).
ES-MS (Direct infusion in methanol) [M+H].sup.+ 356.18 calculated,
356.18 found).
[0494] Note: Compound (33) can be prepared according to: "Building
blocks for polyamide nucleic acids: Facile synthesis using
potassium fluoride doped natural phosphate as basic catalyst.
Alahiane, A.; Taourirte, M.; Rochdi, A.; Redwane, N.; Sebti, S.;
Engels, J. W.; Lazrek, H. B. Nucleosides, Nucleotides & Nucleic
Acids (2003), 22(2), 109-114", the entire teachings of which are
incorporated herein by reference for all purposes.
[0495] Synthesis of Compound 35:
[0496] Compound (34) (465 mg, 1.3 mmol) was treated with 90% TFA in
dichoromethane (DCM) for 30 min at ambient temperature, when TLC
analysis showed complete Boc deprotection. The TFA-DCM solution was
removed under reduced pressure and the foam so obtained was
dissolve in DMF (25 mL). The solution was then neutralized by
addition of di-isopropylethylamine (checked with moist pH paper).
To this neutral solution was added a mixture of piperazine acetic
acid (206 mg, 1.3 mmol), HATU (494 mg, 1.3 mmol) and
di-isopropylethylamine (0.679 mL, 3.9 mmol) in DMF (25 mL). After
30 minutes, TLC analysis indicated the formation of product (Silica
plate, EtOAc-MeOH (1:1) solvent; R.sub.f=0.2; UV, ninhydrin). After
DMF removal under reduced pressure, the product was purified by
flash chromatography (ISCO Companion purification system; 40 g
SiO.sub.2 column, detection at 260 nm, Flow=40 mL/min; 0-1 min 95%
EtOAc in MeOH, 1-10 min 50% EtOAc in MeOH, 10-30 min 10% EtOAc in
MeOH). ES-MS (Direct infusion in water) [M+H].sup.+ 396.22
calculated, 396.28 found).
[0497] Synthesis of Compound (36a):
[0498] To a solution of compound (35) (300 mg, 0.76 mmol) in water
(20 mL) was added NaOH solution (1.14 mL, 1N). The solution was
stirred for 3 h at ambient temperature. TLC analysis indicated
completion of ethyl ester hydrolysis. The reaction mixture was then
acidified with TFA and then concentrated under reduced pressure.
The oil so obtained was used directly without any further
purification. ES-MS (Direct infusion in water) [M+Na].sup.+ 390.18
calculated, 390.40 found).
[0499] Synthesis of Compound (37a):
[0500] Compound (37a), which comprises a reactive group RG, can be
prepared by well known methods discussed in the section entitled
"The Reactive Group."
[0501] Preparation of other labeling reagents comprising
nucleobases & methods for isotopically coding said labeling
reagents: FIG. 27A illustrates a known synthetic procedure for the
synthesis of 6-methyl uracil in greater than 90% yield. The general
procedure outlined in FIG. 26 can be used to convert the 6-methyl
uracil to the isomer (36b) analogous to Compound (36a), and
similarly to compounds (37a) and (37b) containing reactive groups
RG. Compounds (36a) and (37b) are embodiments of compounds of the
general formula RP--X-LK--Y--RG wherein the nucleobase is a
component of the linker (LK) and the N-methyl piperazine is a
component of the reporter (RP).
[0502] FIGS. 27B and 27C identify commercially available
isotopically substituted starting materials (Cambridge Isotope
Labs, Andover Mass.) that can be used to produce isotopically
enriched versions of 6-methyl uracil as illustrated in FIG. 27A. As
illustrated, the symbol "*" next to a carbon atom indicates that
the carbon is a .sup.13C isotope and the symbol "*" next to a
nitrogen atom indicates that the nitrogen is a .sup.15N isotope.
Thus, by employing known synthetic procedures and isotopically
substituted starting materials, a variety of isotopically
substituted labeling reagents, and precursors thereto, can be
created.
[0503] FIGS. 28A-28B illustrate numerous isotopically enriched
versions of 6-methyl uracil that can be prepared using these
commercially available isotopically substituted starting materials
and the procedure illustrated in FIG. 27A. In FIGS. 28A and 28B,
the designations +1, +2, +3, +4, +5, +6 and +7, are used to denote
versions of 6-methyl uracil comprising 1, 2, 3, 4, 5, 6 and 7 heavy
atom isotopes, respectively. Because versions of 6-methyl uracil
can be prepared with any where from no heavy atom isotopes to those
with up to 7 heavy atom isotopes, it is possible to prepare at
least 8 different isobaric labeling reagents of the general formula
(37b). Some exemplary isotopically coded labeling reagents are
illustrated in FIG. 28C.
[0504] Note: 6-methyl uracil can be prepared according to: 1.
Donleavy, J. J.; Kise, M. A. 6-Methyl Uracil, Organic Syntheses,
Coll. Vol. 2, p. 422; Vol. 17, p. 63; 2. Jiang, Z.; Wang, Z.; Ma,
D.; Zhou, Y. Improved synthesis of 6-methyluracil. Tongji Daxue
Xuebao, Ziran Kexueban, 2003, 31(2), 250-252: 3. 6-Methyluracil.
SAIJIYOU SHIGEYA; NISHINAKA TOSHIYOSHI (Yodogawa Pharmaceutical
Co., Ltd., Japan). Jpn. Kokai Tokkyo Koho (1981), 2 pp. JP
56139467; Patent written in Japanese. Abstract: Refluxing
MeCOCH.sub.2CO.sub.2Me with urea and p-MeC.sub.6H.sub.4SO.sub.3H in
hexane 6 h with azeotropic removal of H.sub.2O gave
Me.sub.3-ureidocrotonate, which was heated with 10% NaOH 0.5 h at
95.degree. to give 92.6% 6-methyluracil.
III. Protocol for One Step Solid-Phase iTRAQ
[0505] A. Protein Digestion [0506] a. A protein sample (50-100
.mu.g) was dissolved in 50 .mu.l of Denaturing Buffer (0.2 M
aqueous NH.sub.4HCO.sub.3, containing 8 M urea and 20 mM
CaCl.sub.2). [0507] b. 2 .mu.l of tris[2-carboxyethyl]phosphine
(TCEP, Sigma, 50 mM) was added to the sample solution and incubated
for 1 hour at 37.degree. C. [0508] c. 1 .mu.l of the methyl
methanethiosulfonate (MMTS) reagent (Aldrich, 200 mM) was added and
the sample solution was vortexed for 10 minutes. [0509] d. The
sample solution was diluted with 0.1 M N.sub.4HCO.sub.3 (1:1, 50
.mu.l). [0510] e. 2 .mu.l of LysC (Wako, 1 .mu.g/.mu.l) was added
to the sample solution and the sample solution was incubated at
37.degree. C. for 1 hour to digest the protein. [0511] f. The
digest solution was diluted with water (1:1, 100 .mu.l). [0512] g.
10 .mu.l (.about.5 .mu.g) of the Trypsin (Promega V5113, 0.5
.mu.g/.mu.l) was added to the digest solution and the digest
solution was incubated at 37.degree. C. for 4-6 hours. [0513] h. 4
.mu.l of the TCEP was added to the digest solution and the digest
solution was incubated at 37.degree. C. for 1 hour.
[0514] B. Capturing and Tagging Peptides having Cysteine Amino
Acids [0515] a. A resin bound isobaric isotopically coded mass tag
in the Millipore Cartridge (UFC3OLG 25, as prepared above) was
washed with 50 mM Tris buffer (pH 8) (3.times.300 .mu.l). [0516] b.
A protein digestion solution (.about.200 .mu.l) was transferred
into the pre-conditioned cartridge of step a. [0517] c. The
cartridge was vortexed at low speed for 30-60 minutes. [0518] d.
The cartridge was spun to remove the unbound peptides. The filtrate
was analyzed by HPLC (to determine the capturing completion).
[0519] e. The resin in the cartridge was washed with 0.1% aqueous
TFA solution (3.times.300 .mu.l). [0520] f. The resin was further
dried in a SpeedVac.
[0521] C. Release of the Tagged Peptides from the Resin [0522] a.
200 .mu.l of a cleavage cocktail of TFA (95%) and TIPS (Aldrich,
5%) was added to the cartridge. [0523] b. The cartridge was allowed
to stand at room temperature for 90 minutes. [0524] c. The
cartridge was spun down at low speed (6.times.1000 g) and the
filtrate was retained. [0525] d. An additional 100 .mu.l of 0.1%
TFA was added to the cartridge and the cartridge was spun down the
tube again and the filtrate was retained. [0526] e. The filtrates
were pooled and then dry down in the SpeedVac yielding a residue
containing the mass tagged peptides.
Analyses of Peptides Labeled with Isobaric Mass Tags Using MS and
LC/MS/MS
[0527] Mass tags (38) and (39) are a pair of mass tags that were
tested extensively. Mass tags (38) and (39) have the following
structural formulae: ##STR37## Mass tags (38) and (39) can be
synthesized by employing appropriate isotopically substituted
starting materials with any known amino acid syntheses, for
example, appropriate isotopically substituted starting materials
can be employed with the methods shown in FIG. 25 in combination
with a cleavage step to release the mass tags from the solid resin
support.
[0528] Both mass tags (38) and (39) have a mass of 479.05 Da and
are expected to lose a benzyl group when subjected to dissociative
energy levels. However, because of the placement of the deuterium
substituents on each mass tag, mass tag (38) will have a signature
ion having a mass of 91.05 Da and mass tag (39) will have a
signature ion of 93.07 Da.
[0529] A. QTRAP.TM. 2000 Analysis of Peptides Alkylated with Mass
Tag (38)
[0530] The cysteine amino acid residues of synthetic peptides SEQ
ID No.: 1 and SEQ ID No.: 2 having the following formulae:
TABLE-US-00002 IAVAAQNCYK SEQ ID No.:1 IIYGGSVTGATCK SEQ ID
No.:2
were alkylated with a mass tag (38) and were purified by RP-HPLC.
The purified tagged-peptides were reconstituted in 0.1% TFA with a
concentration at 1 .mu.M. A mass spectra was generated by infusion
experiment on the QTRAP.TM. 2000 System using TurboIonSpray
operation. Total 0.5 min (40 scans) were collected. As shown in
FIGS. 2A and 2B, there were small percentages of fragmentations
(losing 91 Da) of the molecular ions for tagged SEQ ID Nos.: 1 and
2 (approximately 3% and 10%, respectively). In the MS/MS mode in
QTRAP.TM. 2000, tagged SEQ ID Nos.: 1 and 2 generated signature
ions of 91 Da (see FIGS. 3A and 3B, respectively). Their
intensities were peptide dependent and were typically at least
about as intense as those of immonium ions. The sequence ions of
the tagged peptides were comparable with those of corresponding
peptides alkylated with iodoacetic acid in both presence and
intensities.
[0531] B. Analysis of Peptides Alkylated with Mass Tag (38) or (39)
Using a 4700 Proteomic Analyzer.
[0532] SEQ ID No.: 1 and SEQ ID No.: 3 (DCGATWVVLGHSER) were
alkylated with mass tag (38), purified by RP-HPLC, and diluted to
100 .mu.L with 0.1% aqueous TFA. Each sample (1 .mu.L) was mixed
with the matrix (1 .mu.L saturated solution), and each mixture (1
.mu.L) was then loaded on a MALDI plate for analysis. The parent
ions for tagged SEQ ID No.: 1 and SEQ ID No.: 3 were m/z 1431.7 and
1880.8, respectively. Both peptides were stable, and loss of the
signature ion by the parent ion in the MS stage was not observed
(see FIGS. 4A and 4B).
[0533] The MS/MS spectra for tagged SEQ ID No.: 1 at M/z 1431.7 and
tagged SEQ ID No.: 3 at m/z 1880.8 were generated using CID gas
pressure set at 1.times.10.sup.-5 Torr and a total of 2,000 shots
were collected (see FIGS. 5A and 5B). The signature ions and the
sequence ions are indicated on the figure. The intensities of the
signature ions in the MS/MS stage were peptide dependent and were
typically less than the intensities obtained using QTRAP.TM. 2000.
However, the intensities could be enhanced significantly when CID
was increased (data not shown). The sequence coverage was
consistent with that obtained for corresponding peptides alkylated
with iodoacetic acid.
[0534] C. Quantitation of Peptides Using Mass Tags with QTRAP.TM.
2000
[0535] To evaluate the relative quantifications of protein
expressions in a sample, two samples having five peptides were
prepared. The HPLC chromatogram for the five peptides is as listed
in FIGS. 6A-C. These peptides are named as SEQ ID Nos.: 1-5, based
on their retention time from the earliest to the longest
TABLE-US-00003 IAVAAQNCYK SEQ ID No.:1 IIYGGSVTGATCK SEQ ID No.:2
DCGATWVVLGHSER SEQ ID No.:3 VPADTEVVCAPPTAYIDFAR SEQ ID No.:4
VAHALSEGLGVIACIGEK SEQ ID No.:5
[0536] The first sample was reduced, alkylated with mass tag (38),
and digested with trysin. The second sample contained the same five
peptides as the first sample, but was alkylated with mass tag (39)
instead of mass tag (38). The first sample was aliquoted (5 pmoles
each), and each aliquote was combined with varied amounts of the
second sample from 250 fmoles to 50 pmoles. Each sample mixture was
analyzed by LC-MS/MS experiment on QTRAP.TM. 2000 using the MRM
scan mode. Peptides tagged with mass tag (38) generated a signature
ion at 91 Da while peptides tagged with mass tag (39) generated a
signature ion at 93 Da at MS/MS. In MRM experiments, the specific
molecular ion-to-fragment ion transition was measured. As each
sample mixture contained 5 cys-peptides which were alkylated with 2
different tags, a total of 10 MRM transition (or pairs) were
monitored: 716.5/91 and 716.5/93 for SEQ ID No.: 1; 811.5/91 and
811.5/93 for SEQ ID No.: 2; 628.5/91 and 628.5/93 for SEQ ID No.:
3; 829.9/91 and 829.9/93 for SEQ ID No.: 4 and 707.5/91 and
707.5/93 for SEQ ID No.: 5. The spectra seen in FIG. 6 represented
the abundance of the specific fragment ions (i.e., ions of 91 Da
and 93 Da) from the corresponding molecular ions (i.e., intact
peptide ions) as a function of RPLC-retention time.
[0537] As can be seen from Table 1, the expected ratios were
consistent with the ratios obtained experimentally. The dynamic
range was from 1/0.05 to 1/10, spanning more than 2 orders of
magnitude. Since the 91 Da ion from mass tag (38) for the ratio
1/0.1 and the 93 Da ion from mass tag (39) for the ratio 1/10 were
still above the background noise, the dynamic range may be to 3
orders of magnitude or more.
[0538] D. Quantitation of Peptides Using Mass Tags with 4700
Proteomic Analyzer
[0539] A mixture of samples 1 and 2 from section C above was
prepared. In the resulting mixture, the concentrations of peptides
from sample 1 was fixed at 0.2 .mu.M. Each (1 .mu.L) was then mixed
with the matrix (1 .mu.L). Each mixture (1 .mu.L) was then loaded
on a MALDI plate, and analyzed on the 4700 Proteomic Analyzer. The
CID was set at 9.times.10.sup.-6 and total 3,000 shots were taken
per each MS/MS experiment. The peak area intensities of the 91-ion
and the 93-ion were used to calculate the experimental ratios (see
Table 1).
[0540] The dynamic range was from 1/0.05 to 1/10, spanning more
than 2 orders of magnitude. Since the 91 Da ion from mass tag (38)
for the ratio 1/0.1 and the 93 Da ion from mass tag (39) for the
ratio 1/10 were still above the background noise, the dynamic range
may be to 3 orders of magnitude or more.
[0541] For relative quantifications, probes with dynamic ranges of
1-order of magnitude may be sufficient since typical relative
protein expressions are less than 10-fold. However, for absolute
quantifications, probes with a large dynamic range are desirable.
Since the mass tags of the invention have a dynamic range of
greater than 2-orders of magnitude, they can be used for absolute
quantification, as well as relative quantification of proteins.
TABLE-US-00004 TABLE 1 Relative quantification of mass tagged
proteins in a sample using QTRAP .TM. or 4700 Protein Analyzer.
Expected Ratio of Ratio of Signature Ions Signature Ions Ratio of
Signature Ions (91/93) from 4700 (91/93) (91/93) from QTRAP .TM.
Protein Analyzer 1/0.1 1/0.094 1/0.14 1/0.2 1/0.195 1/0.27 1/0.5
1/0.473 1/0.60 1/1 1/1.11 1/1.16 1/2 1/2.06 1/2.3 1/5 1/5.06 1/4.85
1/10 1/9.9 1/10.1
[0542] E. Analysis of a Complexed Protein Mixture
[0543] Two identical protein mixtures, each containing 19 peptides
were alkylated with mass tag (38) or mass tag (39). Thus, the
peptides in the mixture alkylated with mass tag (38) will yield a
signature ion of 91 Da, while the peptides in the mixture alkylated
with mass tag (39) will yield a signature ion of 93 Da. The two
protein mixtures were mixed in either a 1:1 ratio or a 1:2 ratio
and the mixtures were analyzed by QTRAP.TM. to determine the ratio
of each peptide in each sample based on the ratio of the signature
ions (91/93) in the MS/MS stage for each molecular ion. As can be
seen from the data in Table 2, the experimental results for the 1:1
mixture and the 1:2 mixture corresponded closely with the expected
ratio for each of the nineteen peptides. TABLE-US-00005 (91/93)
(91/93) Ratio Ratio 1:1 1:2 Peptide Peptide Sequence Mixture
Mixture BSA CASIQK 1:1.07 1:2.04 (aa 223-228) (SEQ ID No.:6) BSA
QNCDQFEK 1:0.89 1:1.8 (aa 413-420) (SEQ ID No.:7) BSA CCTKPESER
1:0.86 1:2.21 (aa 460-468) (SEQ ID No.:8) BSA YICDNQDTISSK 1:0.73
1:1.96 (aa 286-297) (SEQ ID No.:9) BSA GACLLPK 1:1.06 1:2.21 (aa
198-204) (SEQ ID No.:10) BSA DDPHACYSTVFDK 1:1.07 1:1.44 (aa
387-399) (SEQ ID No.:11) BSA LKPDPNLCDEFK 1:1.16 1:2.24 (aa
139-151) (SEQ ID No.:12) BSA RPCFSALTPDETYVPK 1:0.89 1:1.97 (aa
508-523) (SEQ ID No.:13) Transferrine WCAVSEHEATK 1:1.0 1:1.5 (aa
27-37) (SEQ ID No.:14) Transferrine EGTCPEAPTDECKPVK 1:1.28 1:2.44
(aa 347-362) (SEQ ID No.:15) Transferrine DDTVCLAK 1:1.05 1:1.7 (aa
652-659) (SEQ ID No.:16) .alpha.-Lacta ALCSEK 1:0.75 1:1.66 (aa
128-133) (SEQ ID No.:17) .alpha.-Lacta CEVFR 1:0.69 1:1.88 (aa
25-29) (SEQ ID No.:18) .alpha.-Lacta LDQWLCEK 1:0.94 1:1.93 (aa
134-141) (SEQ ID No.:19) .alpha.-Lacta DDQNPHSSNICNISCDK 1:0.58
1:1.16 (aa 82-98) (SEQ ID No.:20) Lyso CELAAAMK 1:1.62 1:3.24 (aa
6-13) (SEQ ID No.:21) .beta.-Lactoglobulin WENGECAQK 1:1.3 1:1.61
(aa 77-85) (SEQ ID No.:22) AVE 1:1.0226 1:1.8 (SEQ ID No.:23) STDV
0.293 0.367 (SEQ ID No.:24)
[0544] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
25 1 10 PRT Artificial Sequence Synthetic Probe Sequence 1 Ile Ala
Val Ala Ala Gln Asn Cys Tyr Lys 1 5 10 2 13 PRT Artificial Sequence
Synthetic Probe Sequence 2 Ile Ile Tyr Gly Gly Ser Val Thr Gly Ala
Thr Cys Lys 1 5 10 3 14 PRT Artificial Sequence Synthetic Probe
Sequence 3 Asp Cys Gly Ala Thr Trp Val Val Leu Gly His Ser Glu Arg
1 5 10 4 20 PRT Artificial Sequence Synthetic Probe Sequence 4 Val
Pro Ala Asp Thr Glu Val Val Cys Ala Pro Pro Thr Ala Tyr Ile 1 5 10
15 Asp Phe Ala Arg 20 5 18 PRT Artificial Sequence Synthetic Probe
Sequence 5 Val Ala His Ala Leu Ser Glu Gly Leu Gly Val Ile Ala Cys
Ile Gly 1 5 10 15 Glu Lys 6 6 PRT Artificial Sequence BSA (amino
acids 223-228) 6 Cys Ala Ser Ile Gln Lys 1 5 7 8 PRT Artificial
Sequence BSA (amino acids 413-420) 7 Gln Asn Cys Asp Gln Phe Glu
Lys 1 5 8 9 PRT Artificial Sequence BSA (amino acids 460-468) 8 Cys
Cys Thr Lys Pro Glu Ser Glu Arg 1 5 9 12 PRT Artificial Sequence
BSA (amino acids 286-297) 9 Tyr Ile Cys Asp Asn Gln Asp Thr Ile Ser
Ser Lys 1 5 10 10 7 PRT Artificial Sequence BSA (amino acids
198-204) 10 Gly Ala Cys Leu Leu Pro Lys 1 5 11 13 PRT Artificial
Sequence BSA (amino acids 387-399) 11 Asp Asp Pro His Ala Cys Tyr
Ser Thr Val Phe Asp Lys 1 5 10 12 12 PRT Artificial Sequence BSA
(amino acids 139-151) 12 Leu Lys Pro Asp Pro Asn Leu Cys Asp Glu
Phe Lys 1 5 10 13 16 PRT Artificial Sequence BSA (amino acids
508-523) 13 Arg Pro Cys Phe Ser Ala Leu Thr Pro Asp Glu Thr Tyr Val
Pro Lys 1 5 10 15 14 11 PRT Artificial Sequence Transferrine (amino
acids 27-37) 14 Trp Cys Ala Val Ser Glu His Glu Ala Thr Lys 1 5 10
15 16 PRT Artificial Sequence Transferrine (amino acids 347-362) 15
Glu Gly Thr Cys Pro Glu Ala Pro Thr Asp Glu Cys Lys Pro Val Lys 1 5
10 15 16 8 PRT Artificial Sequence Transferrine (amino acids
652-659) 16 Asp Asp Thr Val Cys Leu Ala Lys 1 5 17 6 PRT Artificial
Sequence alpha-Lacta (amino acids 128-133) 17 Ala Leu Cys Ser Glu
Lys 1 5 18 5 PRT Artificial Sequence alpha-Lacta (amino acids
25-29) 18 Cys Glu Val Phe Arg 1 5 19 8 PRT Artificial Sequence
alpha-Lacta (amino acids 134-141) 19 Leu Asp Gln Trp Leu Cys Glu
Lys 1 5 20 17 PRT Artificial Sequence alpha-Lacta (amino acids
82-98) 20 Asp Asp Gln Asn Pro His Ser Ser Asn Ile Cys Asn Ile Ser
Cys Asp 1 5 10 15 Lys 21 8 PRT Artificial Sequence Lyso (amino
acids 6-13) 21 Cys Glu Leu Ala Ala Ala Met Lys 1 5 22 9 PRT
Artificial Sequence Beta-Lactoglobulin (amino acids 77-85) 22 Trp
Glu Asn Gly Glu Cys Ala Gln Lys 1 5 23 3 PRT Artificial Sequence
Synthetic probe sequence 23 Ala Val Glu 1 24 4 PRT Artificial
Sequence Synthetic probe sequence 24 Ser Thr Asp Val 1 25 13 PRT
Artificial Sequence [Glu1]-Fibrinopeptide B human 25 Gly Val Asn
Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg 1 5 10
* * * * *
References