U.S. patent application number 11/319685 was filed with the patent office on 2006-05-18 for methods, compositions and kits pertaining to analyte determination.
This patent application is currently assigned to Applera Corporation. Invention is credited to Michael Bartlet-Jones, Darryl J.C. Pappin.
Application Number | 20060105416 11/319685 |
Document ID | / |
Family ID | 32850790 |
Filed Date | 2006-05-18 |
United States Patent
Application |
20060105416 |
Kind Code |
A1 |
Pappin; Darryl J.C. ; et
al. |
May 18, 2006 |
Methods, compositions and kits pertaining to analyte
determination
Abstract
This invention pertains to methods, 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: |
Pappin; Darryl J.C.;
(Boxborough, MA) ; Bartlet-Jones; Michael;
(Surrey, GB) |
Correspondence
Address: |
APPLIED BIOSYSTEMS
500 OLD CONNECTICUT PATH
FRAMINGHAM
MA
01701
US
|
Assignee: |
Applera Corporation
Framingham
MA
|
Family ID: |
32850790 |
Appl. No.: |
11/319685 |
Filed: |
December 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10765267 |
Jan 27, 2004 |
|
|
|
11319685 |
Dec 28, 2005 |
|
|
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60443612 |
Jan 30, 2003 |
|
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Current U.S.
Class: |
435/23 ;
546/208 |
Current CPC
Class: |
C07D 207/46 20130101;
Y10T 436/24 20150115; G01N 33/6842 20130101; G01N 33/6848 20130101;
Y10T 436/147777 20150115; A61K 51/04 20130101; C07D 401/12
20130101; G01N 2458/15 20130101; Y10T 436/142222 20150115; G01N
33/532 20130101; Y10T 436/145555 20150115 |
Class at
Publication: |
435/023 ;
546/208 |
International
Class: |
C12Q 1/37 20060101
C12Q001/37; C07D 403/02 20060101 C07D403/02 |
Claims
1. An active ester compound that is a 5, 6 or 7 membered
heterocyclic ring comprising a ring nitrogen atom that is
N-alkylated with a substituted or unsubstituted acetic acid moiety
to which the alcohol moiety of the active ester is linked through
the carbonyl carbon of the N-alkyl acetic acid moiety, wherein the
compound is isotopically enriched with one or more heavy atom
isotopes.
2. The compound of claim 1, wherein the compound is isotopically
enriched with three or more heavy atom isotopes.
3. The compound of claim 1, wherein the heterocyclic ring is
substituted with one or more substituents.
4. The compound of claim 3, wherein the one or more substituents
are alkyl, alkoxy or aryl groups.
5. The compound of claim 4, wherein the one or more substituents
are protected or unprotected amine groups, hydroxyl groups or thiol
groups.
6. The compound of claim 1, wherein the heterocyclic ring is
aliphatic.
7. The compound of claim 1, wherein the heterocyclic ring is
aromatic.
8. The compound of claim 1, wherein the heterocyclic ring comprises
one or more additional nitrogen, oxygen or sulfur atoms.
9. The compound of claim 1, wherein active ester is an
N-hydroxysuccinimide ester.
10. The compound of claim 1, wherein the compound is a salt.
11. The compound of claim 1, wherein the compound is a mono-TFA
salt, a mono-HCl salt, a bis-TFA salt or a bis-HCl salt.
12. The compound of claim 1, wherein each incorporated heavy atom
isotope is present in at least 80 percent isotopic purity.
13. The compound of claim 1, wherein each incorporated heavy atom
isotope is present in at least 93 percent isotopic purity.
14. The compound of claim 1, wherein each incorporated heavy atom
isotope is present in at least 96 percent isotopic purity.
15. A kit comprising a compound as claimed in claim 1, further
comprising one or more reagents, containers, enzymes, buffers or
instructions.
16. The kit of claim 15, wherein the kit comprises a proteolytic
enzyme.
17. The kit of claim 15, wherein the kit comprises two or more
compounds as claimed in claim 1, wherein said two or more compounds
are related as a set of isomers and/or isobaric labeling
reagents.
18. The kit of claim 17, wherein each labeling reagent is
independently linked to a solid support through a cleavable
linker.
19. The kit of claim 15, wherein the compound has the formula:
##STR32## wherein; a) RG is a reactive group that is an
electrophile; b) Z is O, S, NH or NR.sup.1; c) each J is the same
or different and is selected from the group consisting of: H,
deuterium (D), R.sup.1, OR.sup.1, SR.sup.1, NHR.sup.1,
N(R.sup.1).sub.2, fluorine, chlorine, bromine and iodine; d) W is
an atom or group that is located ortho, meta or para to the ring
nitrogen and is selected from the group consisting of: NH,
N--R.sup.2, P--R.sup.2, O or S; and e) each carbon of the
heterocyclic ring has the formula CJ.sub.2; f) 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; and g) R.sup.2 is an amino alkyl,
hydroxy alkyl, thio alkyl group or a cleavable linker that
cleavably links the reagent to a solid support wherein the amino
alkyl, hydroxy alkyl or thio alkyl group comprises one to eight
carbon atoms, which may optionally contain a heteroatom or a
substituted or unsubstituted aryl group, and wherein the carbon
atoms of the alkyl and aryl groups independently comprise linked
hydrogen, deuterium and/or fluorine atoms.
20. A method comprising: a) reacting an analyte with the compound
as claimed in claim 1 to thereby produce a labeled analyte; and b)
mixing the labeled analyte with one or more differentially labeled
analytes.
21. The method of claim 20, further comprising analyzing the
mixture in a mass spectrometer.
22. The method of claim 20, wherein the analysis in a mass
spectrometer involves MS/MS analysis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/765,267 filed on Jan. 27, 2004,
incorporated herein by reference, which application claims the
benefit of U.S. Provisional Patent Application Ser. No. 60/443,612,
filed on Jan. 30, 2003, incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention pertains to the field of analyte
determination by mass analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] FIG. 1A illustrates the reaction of an analyte with two
different isobaric labeling reagents (e.g. compounds I and II).
[0004] FIG. 1B illustrates the fragmentation of the labeled analyte
illustrated in FIG. 1A to thereby produce reporter moieties (e.g.
compounds VII and VIII as signature ions) of different masses from
the isobarically labeled analytes.
[0005] FIG. 2 is an expansion plot of a mass spectrum of a labeled
analyte.
[0006] FIG. 3 is the complete mass spectrum obtained from a second
mass analysis of the selected labeled analyte identified in the
expansion plot of FIG. 2.
[0007] FIG. 4 is an expansion plot of a mass spectrum of the
predominate y-ion daughter fragment ion of the analyte as
determined in the second mass analysis.
[0008] FIG. 5 is an expansion plot of a mass spectrum of the
predominate b-ion daughter fragment ion of the analyte as
determined in the second mass analysis.
[0009] FIG. 6 is an expansion plot of a mass spectrum of two
reporters (i.e. signature ions) as determined in the second mass
analysis.
[0010] FIG. 7 is a plot of observed vs. predicted ratios of
reporters determined by a second mass analysis for various mixtures
of a labeled peptide, each peptide of the mixture comprising one of
two different reporters.
[0011] FIG. 8 is an illustration of two sets of isobaric labeling
reagents wherein the same isotopes (compounds X-XM) and different
isotopes (compounds XV-XVIII) are used within the set to thereby
achieve reporter/linker moieties of the same gross mass but each
with a reporter moiety of a different gross mass within the
set.
[0012] FIGS. 9A and 9B are an illustration of synthetic routes to
isotopically labeled piperazine labeling reagents from basic
starting materials. The route can also be used to prepare
non-isotopically labeled piperazine reagents wherein
non-isotopically labeled starting materials are used.
[0013] FIG. 10 is an illustration of a synthetic route to
isotopically labeled and non-isotopically labeled N-alkyl
piperazine labeling reagents from basic starting materials.
[0014] FIG. 11 is an illustration of a synthetic route to
isotopically labeled and non-isotopically labeled N-alkyl
piperazine labeling reagents from basic starting materials.
[0015] FIG. 12 is an illustration of a solid phase based synthetic
route to isotopically labeled and non-isotopically labeled
piperazine labeling reagents from basic starting materials.
1. INTRODUCTION
[0016] 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, nucleic acids, carbohydrates, lipids, steroids
and small molecules of less than 1500 daltons.
[0017] Analytes can be labeled by reaction of the analyte with a
labeling reagent of the formula: RP-X-LK-Y-RG, or a salt thereof,
wherein RG is a reactive group that reacts with the analyte and RP,
X, LK and Y are described in more detail below. A labeled analyte
therefore can have the general formula: RP-X-LK-Y-Analyte. 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 products of 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 proteomic
analysis.
2. Definitions:
[0018] 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:
[0019] a. As used herein, "analyte" refers to a molecule of
interest that may be determined. Non-limiting examples of analytes
can include, but are not limited to, proteins, peptides, nucleic
acids (both DNA or RNA), carbohydrates, lipids, steroids 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.
b. As used herein, "fragmentation" refers to the breaking of a
covalent bond.
c. As used herein, "fragment" refers to a product of fragmentation
(noun) or the operation of causing fragmentation (verb).
[0020] d. 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.
[0021] 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.
[0022] This can be illustrated with reference to FIG. 8. In FIG. 8,
the reporter/linker combination of compound XVII (FIG. 8; chemical
formula: C.sub.5.sup.13CH.sub.10.sup.15N.sub.2O) has two .sup.15N
atoms and one .sup.13C atom and a total theoretical mass of
129.138. By comparison, isobar XV (FIG. 8; chemical formula
C.sub.5.sup.13CH.sub.10N.sub.2.sup.18O) has one .sup.18O atom and
one .sup.13C atom and a total theoretical mass of 129.151.
Compounds XVII and XV are isobars that are structurally and
chemically indistinguishable, except for heavy atom isotope
content, although there is a slight absolute mass difference (mass
129.138 vs. mass 129.151 respectively). However, the gross mass of
compounds XVII and XV is 129.1 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 isobars XVII and XV.
[0023] From FIG. 8, 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.
[0024] e. 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.
[0025] f. 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.
[0026] g. As used herein, "support", "solid support" or "solid
carrier" means any solid phase material upon which a labeling
reagent 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.
[0027] h. 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 will typically contain about 0.6% .sup.13C.
3. General:
The Reactive Group:
[0028] The reactive group "RG" of the labeling reagent or reagents
used in the method, mixture, kit and/or composition embodiments can
be either an electrophile or a nucleophile that is capable of
reacting with one or more reactive analytes of a sample. The
reactive group can be preexisting or it can be prepared in-situ.
In-situ preparation of the reactive group can proceed in the
absence of the reactive analyte or 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 nucleophile 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 (nucleophile)
containing analyte. In some embodiments, the amine (nucleophile)
containing analyte can also be added after the initial reaction
with EDC is performed. In other 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
nucleophiles and/or electrophiles are contemplated by the method,
mixture, kit and/or composition embodiments of this invention.
[0029] Where the reactive group of the labeling reagent is an
electrophile, it can react with a suitable nucleophilic group of
the analyte or analytes. Where the reactive group of the labeling
reagent is a nucleophile, 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, nucleic acids, 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.
[0030] 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. For
example, the alcohol or thiol group of an active ester can have the
formula: ##STR1## wherein X is O or S, but preferably O. All of the
foregoing being 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 N-substituted piperazine acetic
acids compounds that are representative examples of labelling
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, incorporated herein by reference.
[0031] In another embodiment, 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.
[0032] 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.
By halide or halo we mean atoms of fluorine, chlorine, bromine or
iodine.
[0033] 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.
[0034] The reactive group of the labeling reagent can be a
nucleophile such as an amine group, a hydroxyl group or a thiol
group.
The Reporter Moiety:
[0035] 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.15N), 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. These
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.
[0036] A unique reporter can be associated with a sample of
interest thereby labeling one or multiple analytes of that sample
with 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 a calibration standard
(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.
[0037] 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 an 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).
Non-limiting examples of reporters comprising a basic nitrogen
include, substituted or unsubstituted, morpholines, piperidines or
piperazines.
[0038] The reporter can be a 5, 6 or 7 membered heterocyclic ring
comprising a ring nitrogen atom that is N-alkylated with a
substituted or unsubstituted acetic acid moiety to which the
analyte is linked through the carbonyl carbon of the N-alkyl acetic
acid moiety, wherein each different label comprises one or more
heavy atom isotopes. The heterocyclic ring can be substituted or
unsubstituted. The heterocyclic ring can be aliphatic or aromatic.
Possible substituents of the heterocylic moiety include alkyl,
alkoxy and aryl groups. The substituents can comprise protected or
unprotected groups, such as amine, hydroxyl or thiol groups,
suitable for linking the analyte to a support. The heterocyclic
ring can comprise additional heteroatoms such as one or more
nitrogen, oxygen or sulfur atoms.
[0039] 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.
[0040] The reporter can be a small molecule that is non-polymeric.
The reporter does not have to be a biopolymer (e.g. a peptide, a
protein or a nucleic acid) or a component of a biopolymer (e.g. an
amino acid, a nucleoside or a nucleotide). 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 (See for
example: FIG. 6). These factors may prove to be useful advancements
to the state of the art.
The Linker Moiety:
[0041] The linker moiety of the labeling reagent or reagents 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: ##STR2## wherein 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. The
linker can be a polymer or a biopolymer. The linker can be designed
to sub-fragment when subjected to dissociative energy levels;
including sub-fragmentation to thereby produce only neutral
fragments of the linker.
[0042] 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.
[0043] For example, the labeled analytes, or the reagents 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.
[0044] 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.
[0045] 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:
[0046] 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/s ratio) and its intensity can be
observed directly in MS/MS analysis.
[0047] 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 WO00/11208). 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):
[0048] 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.
[0049] 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 ffight)-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
a discussion of TOF-TOF mass analysis techniques.
Fragmentation by Dissociative Energy Levels:
[0050] 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.
[0051] The process of fragmenting bonds by collision-induced
dissociation involves increasing the kinetic energy state of
selected ions, through collision with an inert gas, 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.
[0052] 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.
[0053] 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
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:
[0054] 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.
[0055] 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)).
[0056] 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).
[0057] 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 nucleic acids.
Bonds X and Y:
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
Sample Processing:
[0062] In certain embodiments of this invention, a sample can be
processed prior to, as well as after, labeling of the analytes. The
processing can facilitate the labeling of the analytes. The
processing can facilitate the analysis of the sample components.
The processing can simplify the handling of the samples. The
processing can facilitate two or more of the foregoing.
[0063] 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 nucleic acids) 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.
[0064] 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 trpsin 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.
[0065] 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).
[0066] 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.
Separation of the Sample Mixture:
[0067] In some embodiments the processing of a sample or sample
mixture of labeled analytes can involve separation. 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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 nominal gross
mass (See for example, FIG. 1) whereas isomers are structurally
and/or chemically distinguishable compounds of the same nominal
gross mass.
Relative and Absolute Quantitation of Analytes:
[0074] 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. area 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 are 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.
[0075] In other 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.
[0076] Notwithstanding the foregoing, corrections to the intensity
of the reporters (signature ions) can be made, as appropriate, for
any naturally occurring, or artificially created, isotopic
abundance within the reporters. An example of such a correction can
be found in Example 3. 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.
Proteomic Analysis:
[0077] 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.
[0078] 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.
[0079] 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:
[0080] A. Methods
[0081] According to the methods of this invention, the analyte to
be determined is labeled. 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.
[0082] 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.
[0083] 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, 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.
[0084] 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 nucleophile
or electrophile 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.
[0085] 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.
[0086] 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.
[0087] 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, both bonds X
and 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.
[0088] In the second mass analyzer, 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".
[0089] 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.
[0090] 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 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.
[0091] 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.
[0092] 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 still some other
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 yet some other
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 yet some other embodiments, bond X is less prone to
fragmentation as compared with bond Y. In still some other
embodiments, bond Y is less prone to fragmentation as compared with
bond X. In still some other 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. Other characteristics of the groups
that for the RP-X-LK-Y-moiety of labeled analytes have previously
been described.
[0093] In some embodiments, the label of each isobarically labeled
analyte can be a 5, 6 or 7 membered heterocyclic ring comprising a
ring nitrogen atom that is N-alkylated with a substituted or
unsubstituted acetic acid moiety to which the analyte is linked
through the carbonyl carbon of the N-alkyl acetic acid moiety,
wherein each different label can comprise one or more heavy atom
isotopes. The heterocyclic ring can be substituted or
unsubstituted. The heterocyclic ring can be aliphatic or aromatic.
Possible substituents of the heterocylic moiety include alkyl,
alkoxy and aryl groups. The substituents can comprise protected or
unprotected groups, such as amine, hydroxyl or thiol groups,
suitable for linking the analyte to a support. The heterocyclic
ring can comprise additional heteroatoms such as one or more
nitrogen, oxygen or sulfur atoms.
[0094] In some embodiments, labeled analytes in the sample mixture
can be isobars and each comprise the general formula: ##STR3##
wherein: Z is O, S, NH or NR.sup.1; each J is the same or different
and is H, deuterium (D), R.sup.1, OR.sup.1, SR.sup.1, NHR.sup.1,
N(R.sup.1).sub.2, fluorine, chlorine, bromine or iodine; W is an
atom or group that is located ortho, meta or para to the ring
nitrogen and is NH, N--R.sup.1, N--R.sup.2, P--R.sup.1, P--R.sup.2,
O or S; each carbon of the heterocyclic ring has the formula
CJ.sub.2; 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; and
R.sup.2 is an amino alkyl, hydroxy alkyl, thio alkyl group or a
cleavable linker that cleavably links the reagent to a solid
support wherein the amino alkyl, hydroxy alkyl or thio alkyl group
comprises one to eight carbon atoms, which may optionally contain a
heteroatom or a substituted or unsubstituted aryl group, and
wherein the carbon atoms of the alkyl and aryl groups independently
comprise linked hydrogen, deuterium and/or fluorine atoms.
[0095] For example, the sample mixture can comprise one or more
isobarically labeled analytes of the general formula: ##STR4##
wherein isotopes of carbon 13 and oxygen 18 are used to balance the
gross mass between the morpholine reporter and the carbonyl linker
of the different labeling reagents.
[0096] Morpholine labeling reagents suitable to produce labeled
analytes of this general structure can be prepared by numerous
synthetic routes. For example, isotopically labeled or
non-isotopically morpholine compounds can be reacted with
isotopically labeled or non-isotopically labeled bromoacetic acid
compounds as described in Example 1. It should likewise be apparent
that a ring-substituted morpholine and/or substituted bromoacetic
acid starting materials can also be selected and used by one of
skill in the art without the exercise of undue experimentation
(with little or no change to the above described procedure or other
procedures well-known in the art) to thereby produce various
different morpholine based labeling reagents, of differing heavy
atom isotope content (i.e. isotopically coded), that can be used in
the sets or kits of this invention.
[0097] Instead of morpholine, it is possible to choose a
substituted or unsubstituted piperidine of desired isotopic
distribution. When piperidine is chosen, the isotopes D (deuterium)
.sup.13C or .sup.15N can be substituted for H, .sup.12C and
.sup.14N, respectively, and used to alter the gross mass of the
reagents of a set of labeling reagents in a manner similar to that
illustrated for morpholine except that in the case of piperidine,
.sup.18O is not used in the ring atoms. An exemplary synthesis of a
piperidine, optionally using isotopically enriched starting
materials, is described in Example 6.
[0098] The sample mixture can comprise one or more isobarically
labeled analytes of the formula: ##STR5## wherein isotopes of
carbon 13, oxygen 18 and nitrogen 15 are used to balance the gross
mass between the reporter and the carbonyl linker of the different
labeling reagents. Piperazine labeling reagents suitable to produce
labeled analytes of this general structure can be prepared by
numerous synthetic routes. For example, heavy or light piperazine
compounds can be reacted with heavy or light labeled bromoacetic
acid compounds as described in Example 7. With reference to FIGS.
9A and 9B, a general schematic is shown for two different synthetic
routes to isotopically enriched piperazines using readily available
heavy or light starting materials.
[0099] Specifically with reference to FIG. 9A, two equivalents of
.sup.15N-labeled glycine 1 can be condensed to form the
bis-isotopically labeled di-ketopiperazine 2 (the isotopic label is
represented by the * in the Figure). The di-ketopiperazine can then
be reduced to an isotopically labeled piperazine. The isotopically
labeled piperazine can then be reacted with bromoacetic acid and
converted to an active ester 3 as described in Example 7.
[0100] Specifically with reference to FIG. 9B, bis-.sup.15N-labeled
ethylenediamine 4 can be condensed with oxalic acid 5 to for the
bis-isotopically labeled di-ketopiperazine 6 (the isotopic label is
represented by the * in the Figure). The di-ketopiperazine can then
be reduced to an isotopically labeled piperazine. The isotopically
labeled piperazine can then be reacted with bromoacetic acid and
converted to an active ester 3 as described in Example 7.
[0101] It should likewise be apparent that a ring-substituted
piperazine can be made using the above-described methods by merely
choosing appropriately substituted starting materials. Where
appropriate, a substituted bromoacetic acid (either heavy or light)
can likewise be used. By heavy we mean that the compound is
isotopically enriched with one or more heave atom isotopes). By
light we mean that it is not isotopically enriched. Accordingly,
appropriately substituted starting materials can be selected to
thereby produce various different piperazine based labeling
reagents that can be used in the sets of this invention.
[0102] For example, the sample mixture can comprise one or more
isobarically labeled analytes of the formula: ##STR6## wherein:
isotopes of carbon 13, oxygen 18 and nitrogen 15 are used to
balance the gross mass between the reporter and the carbonyl linker
of the different labeling reagents and wherein; 1) 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; and 2) each K is independently
selected as hydrogen or an amino acid side chain. Substituted
piperazine labeling reagents suitable to produce labeled analytes
of this general structure can be prepared by numerous synthetic
routes.
[0103] For example, with reference to FIG. 10, N-alkyl substituted
piperazine reagents can be prepared in accordance with the
illustrated procedure. The tert-butyloxycarbonyl (t-boc) protected
glycine 10 can be condensed with the ester (e.g. ethyl ester) of
N-methyl-glycine 11 to thereby form the ester of the t-boc
protected glycine-N-methyl-glycine dimer 12. The gly-gly dimer 12
can then be cyclized by removal of the t-boc protecting group
followed by condensation to thereby form the acid salt of the
N-methyl-di-ketopiperazine 13. The acid salt of 13 can be
neutralized and reduced to form the N-methyl-piperazine 14. The
N-methyl-piperazine 14 can then be reacted with bromoacetic acid 15
(or substituted versions thereof) and converted to an active ester
16 as described in Example 7.
[0104] It should be apparent that a ring-substituted piperazine can
be made using the above-described method by merely choosing an
amino acid or N-methyl amino acid (or ester thereof) other than
glycine (e.g. alanine, phenylalanine, leucine, isoleucine, valine,
asparagine, apartic acid, etc). It should likewise be apparent that
the amino acids can be isotopically labeled in a manner suitable
for preparing ring substituted piperazines having the desired
distribution of isotopes necessary to prepare sets of isobaric
labeling reagents.
[0105] N-alkyl substituted piperazine reagents can be prepared in
accordance by still another illustrated procedure. With reference
to FIG. 11, glycine methyl ester 21 can be reacted with the ethyl
ester of bromoacetic acid 22 to form the diethyl iminodiacetate 23.
The diester of the diethyl iminodiacetate 23 can be converted to a
di-acid chloride 24 by treatment an appropriate reagent (e.g.
thionyl chloride). The di-acid chloride 24 can then be reacted
with, for example, an alkyl amine (e.g. methyl amine) to form an
N-alkyl-di-ketopiperazine 25. The N-alkyl-di-ketopiperazine 25 can
then be reduced to form the N-alkyl-piperazine 26. The
N-alkyl-piperazine can then be reacted with bromoacetic acid and
converted to an active ester 27 as described in Example 7.
[0106] It should be apparent that a ring-substituted piperazine can
be made using the above-described method by merely choosing an
ester of an amino acid other than glycine (e.g. alanine,
phenylalanine, leucine, isoleucine, valine, asparagine, apartic
acid, etc) or a substituted version of bromoacetic acid. It should
likewise be apparent that the amino acids and bromoacetic acid (and
its substituted derivatives) can be isotopically labeled in a
manner suitable for preparing ring substituted piperazines having
the desired distribution of isotopes necessary to prepare sets of
isobaric labeling reagents. It should be further apparent that
choosing an alkyl diamine, hydroxyalkyl amine or thioalkylamine, or
isotopically labeled version thereof, instead of an alkyl amine can
be used to produce the support bound labeling reagents as described
in more detail below.
[0107] In yet some other embodiments of the method, labeled
analytes in the sample mixture are isobars and each comprise the
formula: ##STR7## wherein: Z is O, S, NH or NR.sup.1; each J is the
same or different and is selected from the group consisting of: H,
deuterium (D), R.sup.1, OR.sup.1, SR.sup.1, NHR.sup.1,
N(R.sup.1).sub.2, fluorine, chlorine, bromine and iodine; 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.
[0108] For example, the sample mixture can comprise two or more
isobarically labeled analytes of the formula: ##STR8## wherein
isotopes of carbon 13 and oxygen 18 are used to balance the gross
mass between the reporter and the carbonyl linker of the different
labeling reagents. Substituted labeling reagents suitable to
produce labeled analytes of this general structure can be prepared
by the general process described in Example 8.
[0109] In still some other embodiments of this invention, each
different labeling reagent of a set or kit of labeling reagents can
be linked to a support through a cleavable linker such that each
different sample can be reacted with a support carrying a different
labeling reagent. In some embodiments, the supports can themselves
be used for the labeling of reactive analytes. In some embodiments,
the labeling reagents can be removed from the supports and then
used, in some cases after subsequent processing (e.g. protection of
reactive groups), for the labeling of reactive analytes.
[0110] 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 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.
[0111] For example, each different labeling reagent of the set used
in the previously described method can be a solid support of the
formula: E-F-RP-X-LK-Y-RG, wherein; RG, X, Y, RP and LK have been
described previously. E is a solid support and F is a cleavable
linker linked to the solid support and cleavably linked to the
reporter. Supports of this general formula can be prepared as
described in Example 9.
[0112] In some embodiments, a set of support bound labeling
reagents can be based on labeled N-(aminoalkyl), N-(thioalkyl) or
N-(hydroxyalkyl)-piperazine derivatives. Both heavy and light
piperazine derivatives can be prepared. The labeled N-(aminoalkyl),
N-(thioalkyl) or N-(hydroxyalkyl)-piperazine derivatives can be
formed, for example, by using the procedure illustrated in FIG. 11
starting with an alkyl diamine, thioalkyl amine or hydroxyalkyl
amine as the N-alkyl amine (see the discussion of FIG. 11, above).
The alkyl diamine, thioalkyl amine or hydroxyalkyl amine can be
heavy or light where appropriate for synthesis of a desired
N-(aminoalkyl), N-(thioalkyl) or N-(hydroxyalkyl)-piperazine
derivative. The amino, hydroxyl or thiol group of the
N-(aminoalkyl), N-(thioalkyl) or N-(hydroxyalkyl)-piperazine
derivatives can be protected as appropriate. When an alkyl diamine,
thioalkylamine or hydroxyalkyl amine is used, the piperazine can
comprise an N-aminoalkyl, N-thioalkyl or N-hydroxyalkyl moiety
wherein the amino, hydroxyl or thiol group of the moiety can be
reacted with the cleavable linker on a support to thereby cleavably
link the piperazine, prepared from the N-(aminoalkyl),
N-(thioalkyl) or N-(hydroxyalkyl)-piperazine derivative, to the
support.
[0113] The support comprising a labeling reagent can be prepared by
any of several methods. In some embodiments, the amino, hydroxyl or
thiol group of the N-(aminoalkyl), N-(thioalkyl) or
N-(hydroxyalkyl)-piperazine can be reacted with the cleavable
linker of a suitable support. The cleavable linker can be a
"sterically hindered cleavable linker" (See: Example 9). The
piperazine can be reacted with isotopically labeled or
non-isotopically labeled haloacetic acid (substituted or
unsubstituted) depending on the nature of the labeling reagent
desired for the set of labeling reagents. Thereafter the carboxylic
acid can be converted to an active ester. The active ester can be
reacted with analytes of a sample to thereby label the analytes
with the labeling reagent of the support. Cleavage of the cleavable
linker will release the labeled analyte from the support. This
process can be repeated with an unique piperazine based labeling
reagent for the preparation of the different supports of a set of
labeling supports.
[0114] In some embodiments, the N-(aminoalkyl), N-(thioalkyl) or
N-(hydroxyalkyl)-piperazine can be first reacted with isotopically
labeled or non-isotopically labeled haloacetic acid (substituted or
unsubstituted), or an ester thereof. Preferably, the amino,
hydroxyl or thiol group of the N-(aminoalkyl), N-(thioalkyl) or
N-(hydroxyalkyl)-piperazine can be protected with a suitable
protecting reagent (For a list of suitable protecting groups See:
Green et al., Protecting Groups In Organic Synthesis, Third
Edition, John Wiley & Sons, Inc. New York, 1999). The
unprotected amino, thiol or hydroxyl group of the resulting
bis-alkylated piperazine can then be reacted with the cleavable
linker of a suitable support. Thereafter the carboxylic acid can be
converted to an active ester. If the haloacetic acid compound was
an ester, the ester can be saponified prior to conversion to an
active ester. The active ester can be reacted with analytes of a
sample to thereby label the analytes with the labeling reagent of
the support. Cleavage of the cleavable linker will release the
labeled analyte from the support. This process can be repeated with
a unique piperazine based labeling reagent for the preparation of
the different supports of a set of labeling supports.
[0115] Therefore, in some embodiments, the set of labeling reagents
can comprise one or more of the following support bound labeling
reagents: ##STR9## wherein RG, E and F have been previously
described. According to the method, G can be an amino alkyl,
hydroxy alkyl or thio alkyl group, cleavably linked to the
cleavable linker wherein the amino alkyl, hydroxy alkyl or thio
alkyl group comprises one to eight carbon atoms, which may
optionally contain a heteroatom or a substituted or unsubstituted
aryl group, and wherein the carbon atoms of the alkyl and aryl
groups independently comprise linked hydrogen, deuterium and/or
fluorine atoms. Each carbon of the heterocyclic ring can have the
formula CJ.sub.2, wherein each J is the same or different and is
selected from the group consisting of H, deuterium (D), R.sup.1,
OR.sup.1, SR.sup.1, NHR.sup.1, N(R.sup.1).sub.2, fluorine,
chlorine, bromine and iodine. Each R.sup.1 can be 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.
[0116] In some embodiments, the labeled analytes can be generated
by first reacting the analyte with a support comprising the
labeling reagent, cleavably linked to the support through a
cleavable linker, and then cleaving the labeled analyte from the
support. Accordingly, a sample mixture can comprise one or more
isobarically labeled analytes of the formula: ##STR10## wherein: G'
can be an amino alkyl, hydroxy alkyl or thio 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 and/or deuterium atoms. Each carbon of the heterocyclic
ring can have the formula CJ.sub.2, wherein each J is the same or
different and is selected from the group consisting of: H,
deuterium (D), R.sup.1, OR.sup.1, SR.sup.1, NHR.sup.1,
N(R.sup.1).sub.2, fluorine, chlorine, bromine and iodine. Each
R.sup.1 can be 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. Here the alkyl
amine group, hydroxy alkyl group or thio alkyl group can be the
moiety that was linked to the cleavable linker of the solid
support. The product of each cleavage reaction can be combined to
produce a sample mixture suitable for analysis of labeled analytes
by the methods described herein.
[0117] 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 (Also see the
above section entitled: "Sample Processing"). For example, the
enzyme can be a protease (to degrade proteins and peptides) or a
nuclease (to degrade nucleic acids). 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.
[0118] In some embodiments, methods can further comprise separating
the sample mixture prior to performing the first mass analysis
(Also see the above section entitled: "Separation Of The Sample
Mixture"). 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
and 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.
[0119] In still other embodiments, the methods of the invention can
comprise both an enzyme treatment to degrade sample components and
a separations step.
[0120] 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".
[0121] 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
in the second mass analysis 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. 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 in the second mass analysis.
[0122] 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.
[0123] Alternatively, 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] B. Mixtures
[0129] In some embodiments, this invention pertains to mixtures
(i.e. 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.
[0130] 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, nucleic acids
carbohydrates, lipids, steroids and/or small molecules of less than
1500 daltons.
[0131] In some embodiments, the label of each isobarically labeled
analyte can be a 5, 6 or 7 membered heterocyclic ring comprising a
ring nitrogen atom that is N-alkylated with a substituted or
unsubstituted acetic acid moiety to which the analyte is linked
through the carbonyl carbon of the N-alkyl acetic acid moiety,
wherein each different label comprises one or more heavy atom
isotopes. The heterocyclic ring can be substituted or
unsubstituted. The heterocyclic ring can be aliphatic or aromatic.
Possible substituents of the heterocylic moiety include alkyl,
alkoxy and aryl groups. The substituents can comprise protected or
unprotected groups, such as amine, hydroxyl or thiol groups,
suitable for lining the analyte to a support. The heterocyclic ring
can comprise additional heteroatoms such as one or more nitrogen,
oxygen or sulfur atoms.
[0132] In some embodiments, the labeled analytes of the mixture are
isobars and each comprise the formula: ##STR11## wherein Z, J and W
have been previously described and their characteristics disclosed.
For example, the sample mixture can comprise one or more
isobarically labeled analytes of the formula: ##STR12## wherein
isotopes of carbon 13 and oxygen 18 are used to balance the gross
mass between the morpholine reporter and the carbonyl linker of the
different labeling reagents.
[0133] In some embodiments, the sample mixture can comprise one or
more isobarically labeled analytes of the formula: ##STR13##
wherein isotopes of carbon 13, oxygen 18 and nitrogen 15 are used
to balance the gross mass between the reporter and the carbonyl
linker of the different labeling reagents. In some embodiments, the
sample mixture can comprise one or more isobarically labeled
analytes of the formula: ##STR14## wherein: isotopes of carbon 13,
oxygen 18 and nitrogen 15 are used to balance the gross mass
between the reporter and the carbonyl linker of the different
labeling reagents and wherein; 1) 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; and 2) each K is independently selected as
hydrogen or an amino acid side chain.
[0134] In some embodiments, the labeled analytes of the mixture are
isobars and each comprise the formula: ##STR15## wherein: Z, J and
R' have been previously described and their characteristics
disclosed. For example, the sample mixture can comprise one or more
isobarically labeled analytes of the formula: ##STR16## wherein
isotopes of carbon 13 and oxygen 18 are used to balance the gross
mass between the reporter and the carbonyl linker of the different
labeling reagents.
[0135] In other embodiments, the labeled analytes can be generated
by first reacting the analyte with a support comprising the
labeling reagent, cleavably linked to the support through a
cleavable linker, and then cleaving the labeled analyte from the
support. For example the labeled analytes of the mixture can be one
or more isobars comprising the general formula: ##STR17## wherein:
G' has been previously described and its characteristics
disclosed.
[0136] C. Kits
[0137] In some embodiments, this invention pertains to kits. The
kits can comprise a set of two or more labeling reagents of the
formula: RP-X-LK-Y-RG and one or more reagents, containers,
enzymes, buffers and/or instructions. The atoms, moieties or bonds
X, Y, RP, LK RG have been previously described and their
characteristics disclosed. The labeling reagents of a kit can be
isomeric or isobaric. Other properties of the labeling reagents of
the kits have likewise been disclosed. For example, the kits can be
useful for the multiplex analysis of one or more analytes in the
same sample, or in two or more different samples.
[0138] In some embodiments, the label of each isobarically labeled
analyte can be a 5, 6 or 7 membered heterocyclic ring comprising a
ring nitrogen atom that is N-alkylated with a substituted or
unsubstituted acetic acid moiety to which the analyte is linked
through the carbonyl carbon of the N-alkyl acetic acid moiety,
wherein each different label comprises one or more heavy atom
isotopes. The heterocyclic ring can be substituted or
unsubstituted. The heterocyclic ring can be aliphatic or aromatic.
Possible substituents of the heterocylic moiety include alkyl,
alkoxy and aryl groups. The substituents can comprise protected or
unprotected groups, such as amine, hydroxyl or thiol groups,
suitable for linking the analyte to a support. The heterocyclic
ring can comprise additional heteroatoms such as one or more
nitrogen, oxygen or sulfur atoms.
[0139] In some embodiments, the different reagents of a kit are
isobars and each comprise the formula: ##STR18## wherein RG, Z, J
and W have been previously described and their characteristics
disclosed. For example, the reagents of a kit can comprise one or
more isobarically labeled reagents of the formula: ##STR19##
wherein RG is the reactive group and isotopes of carbon 13 and
oxygen 18 are used to balance the gross mass between the morpholine
reporter and the carbonyl linker of the different labeling
reagents.
[0140] In some embodiments, the kit can comprise one or more
isobarically labeled reagents of the formula: ##STR20## wherein RG
is the reactive group and isotopes of carbon 13, oxygen 18 and
nitrogen 15 are used to balance the gross mass between the reporter
and the carbonyl linker of the different labeling reagents. In some
embodiments, the reagents of a kit can comprise one or more
isobarically labeled reagents of the formula: ##STR21## wherein:
isotopes of carbon 13, oxygen 18 and nitrogen 15 are used to
balance the gross mass between the reporter and the carbonyl linker
of the different labeling reagents and wherein; 1) 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; and 2) each K is independently
selected as hydrogen or an amino acid side chain. In yet other
embodiments, the labeled analytes of the kit are isobars and each
comprises the formula: ##STR22## wherein: RG Z, J and R' have been
previously described and their characteristics disclosed. For
example, the reagents of a kit can comprise one or more
isobarically labeled analytes of the formula: ##STR23## wherein RG
has been previously described and disclosed and isotopes of carbon
13 and oxygen 18 are used to balance the gross mass between the
reporter and the carbonyl linker of the different labeling
reagents.
[0141] In some embodiments, this invention pertains to kits
comprising one or more sets of supports, each support comprising a
different labeling reagent, cleavably linked to the support through
a cleavable linker. For example, the cleavable linker can be
chemically or photolytically cleavable. The supports can be reacted
with different samples thereby labeling the analytes of a sample
with the same reporter/linker, and analytes of different samples
with different reporter/linker combinations. Supports of a set that
can be used in embodiments of this invention have the general
formula: E-F-G-RP-X-LK-Y-RG, wherein E, F, G, RP, X, LK, Y and RG
have been previously defined herein and their characteristics
disclosed. Each different support of the set can comprise a unique
reporter.
[0142] For example the supports of a kit can comprise two or more
of the reagent supports of the formula: ##STR24## wherein: E, F, G
and RG have been previously described and their characteristics
disclosed.
[0143] In some embodiments, the kit comprises a proteolytic enzyme.
The proteolytic enzyme can be trypsin, papain, pepsin, ArgC, LysC,
V8 protease, AspN, pronase, chymotrypsin or carboxypeptidase C. In
some embodiments, the kit can comprise instructions for using the
labeling reagents to differentially label the analytes of different
samples.
[0144] D. Compositions
[0145] In some embodiments, this invention pertains to compositions
that can be used as labeling reagents. The compositions can be
labeling reagents of the formula: RP-X-LK-Y-RG, wherein the atoms,
moieties or bonds X, Y, RP, LK RG have been previously described
and their characteristics disclosed. The labeling reagents can be
isomeric or isobaric. 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.
[0146] The labeling reagents can be 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 is
incorporated into a carbonyl or thiocarbonyl group of the labeling
reagent and at least one other heavy atom isotope is incorporated
into the reporter group of the labeling reagent.
[0147] Each incorporated heavy atom isotope can be present in at
least 80 percent isotopic purity. Each incorporated heavy atom
isotope can be present in at least 93 percent isotopic purity. Each
incorporated heavy atom isotope can be present in at least 96
percent isotopic purity.
[0148] 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 morpholine,
piperidine or piperazine compound. 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. For example,
piperazine containing labeling reagents can be obtained as a
mono-TFA salt, a mono-HCl salt, a bis-TFA salt or a bis-HCl salt.
The number of counterions present in the labeling reagent can
depend in the number of acidic and/or basic groups present in the
labeling reagent.
[0149] 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). In some embodiments, the alcohol (LG)
of the active ester can have the formula: ##STR25## wherein X is O
or S. The active ester can be an N-hydroxysuccinimidyl ester.
[0150] In some embodiments, the active ester compound can be a 5, 6
or 7 membered heterocyclic ring comprising a ring nitrogen atom
that is N-alkylated with a substituted or unsubstituted acetic acid
moiety to which the alcohol moiety of the active ester is linked
through the carbonyl carbon of the N-alkyl acetic acid moiety,
wherein the compound is isotopically enriched with one or more
heavy atom isotopes. The heterocyclic ring of the active ester can
be substituted with one or more substituents. The one or more
substituents can be alkyl, alkoxy or aryl groups. The one or more
substituents can be alkylamine, alkylhydroxy or alkylthio groups.
The one or more substituents can be protected or unprotected amine
groups, hydroxyl groups or thiol groups. The heterocyclic ring can
be aliphatic. The heterocyclic ring can be aromatic. The
heterocyclic ring can comprise one or more additional nitrogen,
oxygen or sulfur atoms.
[0151] In some embodiments, the active ester compound can be an
N-substituted morpholine acetic acid active ester compound of the
formula: ##STR26## or a salt thereof, wherein; LG is the leaving
group of an active ester; X is O or S; each Z is independently
hydrogen, deuterium, fluorine, chlorine, bromine, iodine, an amino
acid side chain or a straight chain or branched C1-C6 alkyl group
that may optionally contain a substituted or unsubstituted aryl
group wherein the carbon atoms of the alkyl and aryl groups each
independently comprise linked hydrogen, deuterium or fluorine
atoms. In some embodiments, Z independently can be hydrogen,
deuterium, fluorine, chlorine, bromine or iodine. In some
embodiments, Z independently can be hydrogen, methyl or methoxy. In
some embodiments, X is .sup.16O or .sup.18O. The nitrogen atom of
the morpholine ring can be .sup.14N or .sup.15N. In some
embodiments, the active ester is a compound comprising the formula:
##STR27## wherein each C* is independently .sup.12C or .sup.13C; LG
is the leaving group of an active ester; X is O or S; and each Z is
independently hydrogen, deuterium, fluorine, chlorine, bromine,
iodine, an amino acid side chain or a straight chain or branched
C1-C6 alkyl group that may optionally contain a substituted or
unsubstituted aryl group wherein the carbon atoms of the alkyl and
aryl groups each independently comprise linked hydrogen, deuterium
or fluorine atoms.
[0152] In some embodiments, the active ester compound can be an
N-substituted piperidine acetic acid active ester compound of the
formula: ##STR28## or a salt thereof, wherein; LG is the leaving
group of an active ester; X is O or S; each Z is independently
hydrogen, deuterium, fluorine, chlorine, bromine, iodine, an amino
acid side chain or a straight chain or branched C1-C6 alkyl group
that may optionally contain a substituted or unsubstituted aryl
group wherein the carbon atoms of the alkyl and aryl groups each
independently comprise linked hydrogen, deuterium or fluorine
atoms. In some embodiments, Z independently can be hydrogen,
deuterium, fluorine, chlorine, bromine or iodine. In some
embodiments, Z independently can be hydrogen, methyl or methoxy. In
some embodiments X is .sup.16O or .sup.18O. The nitrogen atom of
the piperidine ring can be .sup.14N or .sup.15N. In some
embodiments, the active ester is a compound comprising the formula:
##STR29## wherein each C* is independently .sup.12C or .sup.13C; LG
is the leaving group of an active ester; X is O or S; and each Z is
independently hydrogen, deuterium, fluorine, chlorine, bromine,
iodine, an amino acid side chain or a straight chain or branched
C1-C6 alkyl group that may optionally contain a substituted or
unsubstituted aryl group wherein the carbon atoms of the alkyl and
aryl groups each independently comprise linked hydrogen, deuterium
or fluorine atoms.
[0153] In some embodiments, the active ester compound can be an
N-substituted piperidine acetic acid active ester compound of the
formula: ##STR30## or a salt thereof, wherein; LG is the leaving
group of an active ester; X is O or S; Pg is an amine protecting
group; and each Z is independently hydrogen, deuterium, fluorine,
chlorine, bromine, iodine, an amino acid side chain or a straight
chain or branched C1-C6 alkyl group that may optionally contain a
substituted or unsubstituted aryl group wherein the carbon atoms of
the alkyl and aryl groups each independently comprise linked
hydrogen, deuterium or fluorine atoms. In some embodiments, Z
independently can be hydrogen, deuterium, fluorine, chlorine,
bromine or iodine. In some embodiments, Z independently can be
hydrogen, methyl or methoxy. In some embodiments X is .sup.16O or
.sup.18O. In some embodiments, each nitrogen atom of the piperazine
ring is .sup.14N or .sup.15N. In some embodiments, the active ester
is a compound comprising the formula: ##STR31## wherein each C* is
independently .sup.12C or .sup.13C; LG is the leaving group of an
active ester; X is O or S; Pg is an amine protecting group and each
Z is independently hydrogen, deuterium, fluorine, chlorine,
bromine, iodine, an amino acid side chain or a straight chain or
branched C1-C6 alkyl group that may optionally contain a
substituted or unsubstituted aryl group wherein the carbon atoms of
the alkyl and aryl groups each independently comprise linked
hydrogen, deuterium or fluorine atoms.
[0154] Having described embodiments of the invention, it will now
become apparent to one of skill in the art that other embodiments
incorporating the concepts may be used. It is felt, therefore, that
these embodiments should not be limited to disclosed embodiments
but rather should be limited only by the spirit and scope of the
invention.
EXAMPLES
[0155] This invention is now illustrated by the following examples
that are not intended to be limiting in any way.
Example 1
Synthesis of Morpholine Acetic Acid
[0156] Bromoacetic acid (2 g, 14.4 mole) was dissolved in
tetrahydrofuran (50 mL) and added dropwise to a stirred solution of
morpholine (3.76 g, 43.2 mole) in tetrahydrofuran (THF, 20 mL). The
solution was stirred at room temperature for three days. The white
solid (4.17 g) was filtered, washed with THF (100 mL), and
recrystallised from hot ethanol (EtOH), Yield: 2.59 g: IR:1740
cm-1. For the two different isobaric versions of morpholine acetic
acid, either bromoacetic-1-.sup.13C acid (Aldrich PN 27,933-1) or
bromoacetic-2-.sup.13C acid (Aldrich PN 27,935-8) was substituted
for bromoacetic acid.
Example 2
Synthesis of Morpholine Acetic Acid N-Hydroxysuccinimide Ester
[0157] Dimethylformamide (dry, 1.75 g, 0.024M) was dissolved in
tetrahydrofuran (dry, 30 mLs). This solution was added dropwise to
a stirred solution of thionyl chloride (2.85 g, 0.024M) dissolved
in tetrahydrofuran (dry, 20 mLs) and cooled in an ice bath. After
complete addition and 30 minutes on ice, the ice bath was removed
and solid N-hydroxysuccinimide (2 g, 0.017 M) was added (which
completely dissolved) immediately followed by solid pre-powdered
morpholine acetic acid [or -1-.sup.13C or -2-.sup.13C morpholine
acetic acid] (3.64 g, 0.016M). The morpholine acetic acid dissolved
slowly giving a homogeneous solution that rapidly became cloudy.
The reaction was left vigorously stirring over night at room
temperature. The white solid was washed with tetrahydrofuran and
dried under vacuum, weight 3.65 g (67%), IR spectrum 1828.0 cm-1,
1790.0 cm-1, 1736.0 cm-1.
Example 3
Analyte Determination and Relative Quantitation in Two Samples
[0158] 100 pmole amounts of freeze-dried Glu-Fibrinopeptide B
(Sigma) were reacted with 200 .mu.l of freshly-made 2% w/v
solutions of either I or II (See: FIG. 1A for structure and
Examples 1 & 2 for preparation) in ice-cold 0.5M MOPS buffer
(pH 7.8 with NaOH) for 30 minutes on ice. The reaction was
terminated by the addition of TFA to 0.5% v/v final concentration.
The modified peptides were then mixed in various pre-determined
proportions to approximately cover the range 1:10 to 10:1 of the
differentially labeled peptides. Each peptide mixture was
individually purified by reverse-phase de-salting using a Millipore
C18 Zip-Tip. Excess reagent and buffer do not retain on the
reverse-phase packings, and were thus efficiently removed prior to
MS analysis. The mixtures (0.5 .mu.l) were then spotted onto a
MALDI target plate, over-spotted with 0.5 .mu.l of 1% w/v
.alpha.-cyano cinnamic acid in 50% aqueous acetonitrile and each
sample was analyzed using a MALDI source fitted to a QTOF
analyzer.
[0159] FIG. 2 is an expansion plot of the MS spectrum obtained from
the 1:1 mix of Glu-fibrinopeptide as modified with reagents I and
II. The peak at m/z 1699 represents the N-terminally modified mass
of Glu-fibrinopeptide, and as expected, there is no observable
difference in m/z of the two different forms of the peptide (See:
FIGS. 1A(III) and 1A(IV). The modified peptides are isobaric. The
isotopic cluster observed for the peak is exactly as expected for a
single species.
[0160] The singly-charged precursor ion of m/z 1699 was then
selected for fragmentation by low energy CID (collision offset of
approximately -70V), yielding the MS/MS spectrum found in FIG. 3.
As expected, the observed ion series was predominantly of types b-
and y-. All these ions appeared as single species, with no
indication that they comprised a 1:1 mixture of the
differentially-labeled peptide species. For example, an expansion
of the prominent y-ion at m/z 1056.5 is shown in the expansion plot
as FIG. 4 and the prominent b-ion at m/z 886.3 is shown in the
expansion plot as FIG. 5. TABLE-US-00001 TABLE 1 Observed Predicted
0.13 0.125 0.17 0.166 0.2 0.25 0.46 0.5 1.03 1 2.15 2 4.16 4 6.3 6
7.9 8
[0161] Close examination of the spectrum at about 100 m/z (FIG. 6),
however, reveals the presence of both species VII and VIII (FIG.
1B), which are the fragmentation products of species V and VI (FIG.
1B), respectively. No peaks are observed at m/z 128.1, thereby
indicating that species V and VI are not stable enough to be
observed. In this example, therefore, it may be that fragmentation
of the amide bond between the carbonyl group and the amino-terminal
amino acid of the peptide (e.g. bond Y) induced subsequent
fragmentation of the reporter/linker moiety (bond X) and loss of
the carbonyl moiety as neutral CO. Peak integration was performed
using the instrumentation provided with the instrument. Following
compensation for the naturally occurring second C-13 isotopic
contribution of approximately 6 percent, the measured relative
ratio of VIII/VII (101/100) was 1.03 (expected value 1.00). Table 1
shows actual versus observed ratios for additional experimental
mixtures prepared (ratio expressed as intensity m/z 101/m/z 100),
with correction for the naturally occurring second C-13
contribution. This data is also represented graphically in FIG. 7.
There is excellent agreement between observed and predicted values,
with mean error <10%.
Example 4
Proteomic Analysis
[0162] In practice, a representative proteomic analysis can be
performed as follows. Total cellular protein extracts for
comparison (e.g. samples A and B) are separately digested with
trypsin, or another proteolytic enzyme. The resulting peptide
mixtures are separately reacted with different isomeric of isobaric
labeling reagents (for example, compounds I and II) to give
complete modification of N-terminal and lysine amines of the
peptides. For example, sample A can be reacted with compound I and
sample B can be reacted with compound II. Each of the samples
containing modified peptides/proteins are then be mixed together
before chromatographic separation (often using multi-dimensional
HPLC) and analyzed by MS and MS/MS techniques. The labeling can be
performed with a single label treatment (no prior blocking of
lysine groups with a second reagent required) as the groups are
isobaric.
[0163] The mixture of labeled proteins/peptides is then
chromatographically separated and the eluent, or fractions thereof,
analyzed by mass spectrometry as described in Example 3, above.
Effective sensitivity may also be significantly increased using
triple-quadruople or Q-trap mass spectrometers, where the m/z
region of 100 and 101 is monitored in precursor-ion mode. The
relative ratios of the two "signature" peaks are directly
correlated with the ratio of each peptide/protein analyte of
interest in each of samples A and B. As used herein, the
"signature" peaks are the peaks for the reporter.
Example 5
Analyte Determination and Quantitation Relative to an Internal
Standard
[0164] Total cellular protein extracts for comparison (e.g. samples
A and B) are separately digested with trypsin. The resulting
peptide mixtures are separately reacted with X and XI (FIG. 8) to
give substantially complete modification of N-terminal and lysine
amines as described above. For example, sample A peptides are
reacted with X and sample B peptides are reacted with XI. Known
amounts or each of samples A and B, containing substantially
modified peptides, are then mixed together. To the combined mixture
of A and B is now added, in accurately determined amount, a set
(one or more) of synthetic peptide(s) that correspond exactly in
amino acid sequence and/or post-translational modification (e.g.
phosphorylation) to peptide(s) that may be present in the mixture
of samples A and B, and where the synthetic peptide(s) are labeled
with another member of set of isobaric labeling reagents (e.g.
compounds XII or Xm, see: FIG. 8). The combined mixture of peptides
from sample A, sample B and synthetic internal standard peptides
can optionally be subjected to chromatographic separation, for
example by multi-dimensional HPLC, or electrophoretic separation
and then analyzed by MS and MS/MS techniques as described
previously. All equivalent labeled peptides from sample A, B and
synthetic counterparts of identical sequence are isobaric and have
substantially identical chromatographic properties. By
"substantially identical chromatographic properties" we mean that
there is very little, if any, separation of the differentially
labeled but otherwise identical peptides. Following MS/MS analysis,
the absolute concentration of peptides from sample A and B may be
accurately determined by comparison of the relative intensity of
the reporters for X (sample A) and for XI (sample B) with respect
to the intensity of the reporter (the "signature peak") resulting
from the standard peptide labeled with the additional member of the
isobaric set (e.g. XII or XIII).
[0165] Although the foregoing is a description of two samples (i.e.
Samples A and B), this process could be extended in many practical
ways. For example, there may be many samples that are analyzed
simultaneously provided there is a large enough set of labeling
reagents.
[0166] There could be a double (or more where there are more
samples to be analyzed) internal standard (e.g. sample A peptides
may be `spiked` with synthetic peptides labeled with reagent XII
and sample B peptides may be spiked with synthetic peptides labeled
with reagent Xm (of known absolute concentration)). When all are
combined, separated and analyzed as described above, Sample A
peptides can be quantitated relative to the signature peak for
compound XII and sample B peptides can be quantitated relative to
the signature peak for compound XIII.
Example 6
Exemplary Synthesis of Piperidine Acetic Acid N-hydroxysuccinimide
Ester
[0167] Bromoacetic acid is dissolved in tetrahydrofuran (or another
suitable non-nucleophilic solvent) and added dropwise to a stirred
solution containing an excess of piperidine in tetrahydrofuran
(THF, or another suitable non-nucleophilic solvent). The solution
is stirred at room temperature for one to three days. The solid is
filtered, washed with THF (or another suitable non-nucleophilic
solvent), and optionally recrystallised. For the two different
isobaric versions of piperidine acetic acid, either
bromoacetic-1-.sup.13C acid (Aldrich PN 27,933-1) or
bromoacetic-2-.sup.13C acid (Aldrich PN 27,935-8) can be
substituted for bromoacetic acid. Isomer substituted piperidine can
be prepared from suitable starting material or it can be obtained,
on a custom order basis, from sources such as Cambridge Isotope
Laboratories or Isotec.
[0168] To convert the acetic acid derivatives to active esters,
such as an N-hydroxysuccinimidyl ester, dimethylformamide (DMF) is
dissolved in tetrahydrofuran (or another suitable non-nucleophilic
solvent). This solution is added dropwise to a stirred solution of
an equal molar amount of thionyl chloride (based upon the molar
quantity of DMF) dissolved in tetrahydrofuran (or another suitable
non-nucleophilic solvent) and cooled in an ice bath. After complete
addition and 30 minutes on ice, the ice bath is removed and solid
N-hydroxysuccinimide is added immediately followed by piperidine
acetic acid (or -1-.sup.13C or -2-.sup.13C piperidine acetic acid).
The reaction is left vigorously stirring over night at room
temperature. The product piperidine acetic acid
N-hydroxysuccinimide ester is then isolated from the reaction
mixture possibly by mere filtration. Recrystallization and/or
chromatography can optionally be used to purify the crude
product.
Example 7
Exemplary Synthesis of Piperazine Acetic Acid N-hydroxysuccinimide
Ester
[0169] A solution containing two equivalents of piperazine
dissolved in tetrahydrofuran (THF) is added dropwise to a solution
containing one equivalent of bromoacetic acid (as compared with the
amount of piperazine) dissolved in tetrahydrofuran. The two
solutions should be as concentrated as is practical. The resulting
reaction solution is stirred at room temperature for one to three
days. The solid is filtered, washed with THF, and optionally
recrystallised. For the two different isobaric versions of
piperidine acetic acid, either bromoacetic-1-.sup.13C acid (Aldrich
PN 27,933-1) or bromoacetic-2-.sup.13C acid (Aldrich PN 27,935-8)
can be substituted for bromoacetic acid.
[0170] To convert the acetic acid derivatives to active esters,
such as an N-hydroxysuccinimidyl ester, dry dimethylformamide (DMF,
1.75 g, 0.024M) can be dissolved in tetrahydrofuran. This solution
can be added dropwise to a stirred solution of an equal molar
amount of thionyl chloride (based upon the molar quantity of DMF)
dissolved in tetrahydrofuran and cooled in an ice bath. After
complete addition and 30 minutes on ice, the ice bath can be
removed and solid N-hydroxysuccinimide added immediately followed
by piperazine acetic acid (or -1-.sup.13C or -2-.sup.13C piperidine
acetic acid). The reaction can be left vigorously stirring over
night at room temperature. The product piperazine acetic acid
N-hydroxysuccinimide ester can then be isolated from the reaction
mixture possibly by mere filtration. Recrystallization or
chromatography can then be used to purify the crude product.
Example 8
Exemplary Synthesis of N,N'-(2-methoxyethyl)-glycine Active Ester
(Copied From U.S. Pat. No. 6,326,479
[0171] To 1.1 mole of bis(2-methoxyethyl)amine (Aldrich Chemical)
was added dropwise 500 mmol of tert-butyl chloroacetate (Aldrich
Chemical). The reaction was allowed to stir for three days and was
then worked up. To the final reaction contents was added 250 mL of
dichloromethane (DCM) and 200 mL of water. To this stirring
solution was added portionwise, 300 mmol of solid potassium
carbonate (K.sub.2CO.sub.3). After complete mixing, the layers were
separated. The DCM layer was washed once with a volume of water,
dried (Na.sub.2SO.sub.4), filtered and evaporated to yield 66.3 g
of a very thin yellow oil. This crude product was Kugelrohr
distilled at 60.degree. C. (200-500 .mu.M Hg) to yield 58.9 g of a
clear colorless oil (238 mmol; 95%).
[0172] To the purified (stirring)
N,N'-(2-methoxyethyl)-glycine-tert-butyl ester was slowly added
12.1 mL of concentrated hydrochloric acid. The reaction was allowed
to stir overnight and then the byproducts (e.g. water, HCl,
isobutylene) were removed by vacuum evaporation. .sup.1H-MNR
analysis indicated the t-butyl ester was hydrolyzed but it appeared
that there was water and HCl still present. The crude product was
co-evaporated 2.times.from acetonitrile (ACN) but water and HCl
were still present. To eliminate impurities, a 4.4 g aliquot was
removed from the crude product and Kugelrohr distilled at
135-155.degree. C. (100-200 .mu.M Hg with rapidly dropping pressure
after distillation began). Yield 4.2 g (18.4 mmol; 95% recovery of
thick, clear, colorless oil). The distilled product did not contain
any water or HCl.
[0173] The active ester (e.g. N-hydroxysuccinimidyl ester) of any
suitable isotopically labelled substituted or unsubstituted
N,N'-(2-methoxyethyl)-glycine can then be prepared by methods known
in the art, such as those described herein.
Example 9
Exemplary Method for Preparing a Solid Support Comprising
Labelling/Tagging Reagents
[0174] A commercially available peptide synthesis resin comprising
a "sterically hindered cleavable linker" is reacted with at least
two-fold excess of an aminoalkyl piperazine (e.g.
1-(2-aminoethyl)piperazine, Aldrich P/N A5,520-9; isomeric versions
can be made by the process illustrated in FIG. 11 in combination
with the description in the specification). By "sterically hindered
cleavable linker" we mean that the linker comprises a secondary or
tertiary atom that forms the covalent cleavable bond between the
solid support and the atom or group reacted with the cleavable
linker. 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). Excess reagents are then removed by washing the
support. The secondary amine of the support bound piperazine is
then reacted with an excess of bromoacetic acid in the presence of
a tertiary amine such as triethylamine. Excess reagents are then
removed by washing the support. Depending on the method to be used
to make an active ester of the carboxylic acid (e.g. whether or not
a salt of the carboxylic acid is required for the active ester
synthesis), the wash can be selected to have a pH that is adjusted
to protonate the support bound carboxylic acid group of the
bis-alkylated piperazine. The carboxylic acid group of the support
bound piperazine is then converted to an active ester (e.g.
N-hydroxysuccinimidyl ester) using procedures known in the art for
the production of acid esters of a carboxylic acid, such as those
described above. The resulting solid support can thereafter be used
to label analytes of a sample (e.g. peptides) having nucleophilic
functional groups. The labeled analytes can then be released from
the support as described by the manufacturer's product
instructions. The product of each cleavage reaction can then be
combined to form a sample mixture.
* * * * *
References