U.S. patent application number 17/223858 was filed with the patent office on 2021-10-07 for absolute quantitation of proteins and protein modifications by mass spectrometry with multiplexed internal standards.
This patent application is currently assigned to Pierce Biotechnology, Inc.. The applicant listed for this patent is Pierce Biotechnology, Inc.. Invention is credited to Ryan Daniel Bomgarden, Greg Hermanson, Joel Louette, Bhavinkumar Patel, John Charles Rogers.
Application Number | 20210311072 17/223858 |
Document ID | / |
Family ID | 1000005666033 |
Filed Date | 2021-10-07 |
United States Patent
Application |
20210311072 |
Kind Code |
A1 |
Hermanson; Greg ; et
al. |
October 7, 2021 |
Absolute Quantitation of Proteins and Protein Modifications by Mass
Spectrometry with Multiplexed Internal Standards
Abstract
A method for absolute protein or peptide quantitation by mass
spectroscopy. A sample containing a protein or peptide of interest
is prepared for mass spectroscopy analysis. The sample is subjected
to mass spectroscopy analysis at low resolution whereby a single
additive mass spectroscopy peak is obtained, then is subjected to
high resolution mass spectroscopy analysis whereby a plurality of
mass spectroscopy peaks are obtained. The intensity of each of the
plurality of mass spectroscopy peaks is quantitated either by
comparison to an internal standard set, or by using a standard
curve generated for each isotopologue set. Quantitation using a
standard curve enhances quantitation across a dynamic range of
analyte.
Inventors: |
Hermanson; Greg; (Loves
Park, IL) ; Rogers; John Charles; (Rockton, IL)
; Louette; Joel; (Ulm, DE) ; Bomgarden; Ryan
Daniel; (Winnebago, IL) ; Patel; Bhavinkumar;
(Rockford, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pierce Biotechnology, Inc. |
Rockford |
IL |
US |
|
|
Assignee: |
Pierce Biotechnology, Inc.
Rockford
IL
|
Family ID: |
1000005666033 |
Appl. No.: |
17/223858 |
Filed: |
April 6, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14980155 |
Dec 28, 2015 |
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17223858 |
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14297696 |
Jun 6, 2014 |
9252003 |
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14980155 |
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61832380 |
Jun 7, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6848 20130101;
G01N 33/60 20130101; H01J 49/0027 20130101; H01J 49/0031 20130101;
H01J 49/26 20130101; G01N 2458/15 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; H01J 49/00 20060101 H01J049/00; G01N 33/60 20060101
G01N033/60; H01J 49/26 20060101 H01J049/26 |
Claims
1-20. (canceled)
21. A multiplexed internal standard reference set composition for
peptide quantification comprising at least one first premixed set
of multiplexed internal standard reference peptides comprising a
plurality of isolated peptides labeled with heavy isotopes, wherein
each peptide of the multiplexed internal standard reference set has
the same amino acid sequence and same nominal mass, wherein the
nominal mass of each peptide is increased by at least 4 Daltons
from the naturally occurring peptide, and the peptides within the
set differ by milliDalton mass defects created by incorporating
heavy isotopes on different atoms within the amino acid sequence,
wherein the heavy isotopes are chosen from .sup.13C--, .sup.15N--,
.sup.18O--, .sup.34S-- or .sup.2H--; and at least one second
premixed set of multiplexed internal standard reference peptides
comprising a plurality of isolated peptides labeled with heavy
isotopes having a different amino acid sequence from the first set,
wherein each peptide of the multiplexed internal standard reference
set has the same amino acid sequence and same nominal mass, wherein
the nominal mass of each peptide is increased by at least 4 Daltons
from the naturally occurring peptide, and the peptides within the
set differ by milliDalton mass defects created by incorporating
heavy isotopes on different atoms within the amino acid sequence,
wherein the heavy isotopes are chosen from .sup.13C--, .sup.15N--,
.sup.18O--, .sup.34S-- or .sup.2H--.
22. The multiplexed internal standard reference set composition
according to claim 21, wherein each heavy isotope labeled peptide
of the at least one first premixed set of multiplexed internal
standard reference peptides is present at a different
concentration.
23. The multiplexed internal standard reference set composition
according to claim 22, wherein the different concentrations are
fixed ratios.
24. The multiplexed internal standard reference set composition
according to claim 21, wherein each heavy isotope labeled peptide
of the at least one second premixed set of multiplexed internal
standard reference peptides is present at a different
concentration.
25. The multiplexed internal standard reference set composition
according to claim 24, wherein the different concentrations are
fixed ratios.
26. The multiplexed internal standard reference set composition for
peptide quantification according to claim 21, further comprising a
native sample containing the naturally occurring peptide of the
first premixed set.
27. The multiplexed internal standard reference set composition for
peptide quantification according to claim 21, further comprising a
native sample containing the naturally occurring peptide of the
second set.
28. A method for mass spectrometry (MS) calibration, the method
comprising injecting into a first liquid chromatography (LC) column
coupled to a mass spectroscopy detection system a single peptide
composition, a multiplexed internal standard reference set
comprising at least one first set of a plurality of isolated
peptides labeled with heavy isotopes, wherein each heavy isotope
labeled peptide of the multiplexed internal standard reference set
has the same amino acid sequence and same nominal mass that is
increased by at least 4 Daltons from the naturally occurring
peptide, and further wherein the peptides within the multiplexed
internal standard reference set differs by milliDalton mass defects
created by incorporating heavy isotopes on different atoms within
at least one amino acid molecule, wherein the heavy isotopes are
chosen from .sup.13C--, .sup.15N--, .sup.18O--, .sup.34S-- or
.sup.2H--; analyzing the multiplexed internal standard mix and
corresponding native peptide composition by mass spectroscopy;
generating from the single peptide composition injection into the
LC column at least a first calibration curve from the analysis; and
calibrating the MS equipment based on the results of the at least
first dilution curve for quantitation of the native peptide.
29. The method according to claim 28, wherein each heavy isotope
labeled peptide of the premixed multiplexed internal standard
reference set is present at a different concentration.
30. The method according to claim 28, wherein the calibration curve
is a standard curve.
31. The method according to claim 28, wherein the calibration curve
is a dilution curve.
32. A method for mass spectrometry (MS) calibration, the method
comprising generating a calibration curve for MS analysis based on
milliDalton mass defects using a multiplexed internal standard
reference set composition according to claim 21; separating the
mixed heavy isotope labeled peptides with high resolution MS; and
calibrating the MS equipment based on the results of the
separation.
33. A method for mass spectrometry (MS) calibration, the method
comprising injecting into a first liquid chromatography (LC) column
coupled to a mass spectroscopy detection system a multiplexed
internal standard reference set composition according to claim 21;
analyzing each peptide in the co-eluted peptide composition by mass
spectroscopy; generating from the single peptide composition
injection into the LC column at least a first calibration curve
from the analysis; and calibrating the MS equipment based on the
results of the at least first dilution curve for quantitation of
the native peptide.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/297,696 filed Jun. 6, 2014 and claims
priority to U.S. Application Ser. No. 61/832,380 filed Jun. 7, 2013
each of which is expressly incorporated by reference herein in its
entirety.
[0002] Mass spectroscopy (MS), in conjunction with internal
standard peptides labeled with stable heavy isotopes, provides
fast, accurate, and precise absolute quantitation of peptides,
polypeptides, and proteins in biological and other samples.
[0003] The inventive MS method provides target peptide quantitation
using multiplexed internal standard isotopologues by high
resolution mass spectrometry. The method uses AQUA peptides (WO
03/016861) and/or heavy proteins synthesized with NeuCode amino
acids (Hebert et al., Neutron-encoded mass signatures for
multiplexed proteome quantification, Nature America, Inc. 2013
Advance Online Publication). In one embodiment, the method is used
with TMT assays. In one embodiment, the method is used with
universal reporter assays (WO 2012/005838; WO 2012/006406).
SUMMARY OF THE INVENTION
[0004] One embodiment is a method for absolute protein or peptide
quantitation in a sample by mass spectroscopy (MS) using at least
one set of multiplexed heavy peptide internal standards, where each
peptide within the set contains the same amino acid sequence, but
the peptides within the set differ by a mass defect created by
incorporating heavy isotopes on different atoms within at least one
amino acid molecule. The heavy peptide internal standards within
each set contain the same number of total neutrons within each
peptide, but differ in that the heavy atom distribution within the
amino acids is unique to each peptide. The heavy peptide internal
standards within each set are resolved as a single peak under low
resolution mass spectrometry, and are resolved as multiple peaks
under high resolution mass spectrometry. The heavy peptide internal
standards within each set contain mass differences between each
peptide that are less than 1 Dalton. The sample can be a biological
sample with the method used for a diagnostic assay. The method can
be used in a universal reporter assay. The method can be used for
multi-sample analysis. The method can be used for multi-target
analysis. The method can be used for multi-sample analysis and
multi-target analysis. The heavy peptides are prepared by
synthesizing a mixture of at least two peptides with isotopologues
of heavy amino acids, resulting in heavy peptides having different
mass defects. Isotopologues are prepared by mixing solid phase
immobilized AQUA peptide precursors at a defined ratio. The sample
is prepared by effecting cleavage of the target protein or peptide.
The quantity of the target peptide peak can be obtained by using a
standard curve of known quantities of at least two isotopologue
standard peptides. The method may be used to verify the target
protein or peptide is free of isobaric interference.
[0005] One embodiment is a method for protein or peptide
quantitation by mass spectroscopy by (a) preparing a sample
containing a target protein or peptide of interest for mass
spectroscopy analysis, (b) preparing a plurality of heavy isotope
labeled peptides having at least one subsequence of the target
protein or peptide, (c) mixing the heavy isotope labeled peptides
at known concentrations with the sample, (d) subjecting the mixture
containing the prepared sample and the heavy isotope labeled
peptides to mass spectroscopy analysis at low resolution whereby a
single additive heavy peptide mass spectroscopy peak is obtained
with the corresponding single light peptide mass spectrometry peak,
(e) subjecting the low resolution peaks to high resolution mass
spectroscopy analysis whereby a plurality of mass spectroscopy
peaks are obtained representing the heavy isotopomeric peptides,
(f) generating a light peptide intensity:heavy peptide intensity
ratio, and (g) quantifying the intensity of each of the plurality
of mass spectroscopy peaks based on the intensity of the heavy
isotope labeled peptides to quantify the amount of protein or
peptide in the sample. The heavy peptides are prepared by
synthesizing a mixture of at least two peptides with isotopologues
of heavy amino acids, resulting in heavy peptides having mass
defects. Isotopologues are prepared by mixing solid phase
immobilized AQUA peptide precursors at a defined ratio. The sample
is prepared by effecting cleavage of the target protein or peptide.
The quantity of the target peptide peak can be obtained by using a
standard curve of known quantities of at least two isotopologue
standard peptides. Step (f) can use results from a standard curve
for MS analysis. Step (f) can further comprise preparing a
plurality of isotopologues of an amino acid; using the
isotopologues to generate a set of heavy peptide standards; mixing
the plurality of isotopologue-containing peptides at fixed ratios;
separating the mixed isotopologue-containing peptides at low mass
resolution and high mass resolution; using the low mass resolution
to determine the light:heavy peptide ratio; and using the high mass
resolution separation results to prepare a standard curve.
[0006] One embodiment is a method of peptide isotopologue synthesis
where a plurality of precursor amino acid isotopologues, the amino
acids containing alpha carboxylate groups, are mixed at a defined
ratio with a solid phase peptide synthesis resin containing a
coupling group to immobilize the isotopologues through the alpha
carboxylate groups. After coupling the precursor amino acid
isotopologues, the peptide synthesis resin mixture comprises a
plurality of immobilized amino acid isotopologues at known ratios
coupled to the resin. The resin comprising the amino acid
isotopologues immobilized at known ratios may be further used for
peptide synthesis to create a heavy peptide set containing a
plurality of isotopologues.
[0007] One embodiment is a method of peptide isotopologue synthesis
comprising mixing a plurality of precursor amino acid isotopologues
with a solid phase peptide synthesis resin containing at least one
amino acid already synthesized into a precursor peptide having a
desired sequence where the precursor amino acid isotopologues
become attached to the N-terminal of the peptides using standard
amino acid synthesis procedures. Coupling of the precursor amino
acid isotopologues to the immobilized peptide results in a set of
heavy peptides containing a known ratio of isotopologue mixture.
The resin comprising the amino acid isotopologues immobilized at
known ratios can be further used for peptide synthesis to create a
heavy peptide set containing a plurality of isotopologues.
[0008] One embodiment is a method of protein isotopologue synthesis
comprising using a plurality of precursor amino acid isotopologues
in an in vitro or cell-based protein expression system to
synthesize a protein of interest having a desired sequence where
the precursor amino acid isotopologues become incorporated into the
protein of interest. Incorporation of the precursor amino acid
isotopologues in the expressed protein results in a heavy protein
set containing a plurality of isotopologues.
[0009] One embodiment is a method of targeted peptide quantitation
using multiplexed internal standard peptide isotopologues by high
resolution mass spectrometry.
[0010] One embodiment is a method of generating a standard curve
for mass spectroscopy analysis by preparing a plurality of
isotopologues of an amino acid to generate a heavy peptide
standard, mixing the plurality of isotopologues at fixed ratios,
separating the mixed isotopologues at a low resolution quantitation
or a high resolution quantitation, and using the separation results
to prepare a standard curve.
[0011] One embodiment is a method of targeted peptide quantitation
by high resolution mass spectrometry using one or more peptides
derived by proteolytic digestion from multiplexed internal standard
protein isotopologues.
[0012] One embodiment is a method of generating a standard curve
for mass spectroscopy analysis by preparing a plurality of
isotopologues of an amino acid to generate a heavy protein
standard, mixing the plurality of isotopologues at fixed ratios,
digesting the isotopologue set with a protease, separating the
mixed isotopologues at a low resolution quantitation or a high
resolution quantitation, and using the separation results to
prepare a standard curve with all peptides derived from the protein
isotolopologue set by proteolytic digestion.
[0013] One embodiment is a method of generating a standard curve
for mass spectroscopy analysis by preparing a plurality of
isotopologues of an amino acid to generate a heavy protein
standard, mixing the plurality of isotopologues at fixed ratios,
digesting the isotopologue set with a protease, and adding one or
more distinct isotopically distinct internal standard peptides of
known amount, separating the mixed isotopologues at a low
resolution quantitation or a high resolution quantitation, and
using the separation results to prepare a standard curve with all
peptides derived from the protein isotolopologue set by proteolytic
digestion, and the distinct isotopic peptide or peptides for
absolute quantification of the corresponding peptides of the
identical sequence derived by digestion of the native protein and
the heavy isotopologue protein set.
[0014] One embodiment is a method of synthesizing a single
isotopologue set by premixing solid phase immobilized precursor
amino acids at a defined desired ratio.
[0015] One embodiment is a mass spectrometry analyte quantitation
method by (a) preparing, from a sample containing a peptide analyte
having a known amino acid sequence, a plurality of heavy
isotopologue labeled standards comprising at least part of the
analyte sequence; (b) adding a known quantity of the result of step
(a) to the sample; (c) cleaving the protein in the sample to result
in peptides; (d) quantitating the plurality of heavy isotopologue
labeled standards in the sample; and (e) based on the quantitation
of (d), determining the quantity of analyte in the sample.
Quantitation of the plurality of heavy isotopologue labeled
standards in the sample is by high resolution mass spectroscopy.
Each standard of step (a) differs from other standards by
incorporating heavy atoms of different elements but having the same
total number of neutrons. Incorporating different heavy atoms into
the standards causes a mass defect among the standards. A peak
intensity for the plurality of heavy isotopologue labeled standards
contains multiple resolvable peaks under high-resolution MS, each
of the multiple resolvable peaks representing the differently
labeled standards of the plurality of heavy isotopically labeled
standards. The method generates a standard curve using the multiple
resolvable peaks as a series of known concentrations of the
plurality of heavy isotopologue labeled standards.
[0016] One embodiment is a plurality of heavy isotope labeled
peptide standards having the same amino acid sequence, each
comprising different isotopologues of heavy amino acids, and each
having the same nominal mass and chemical formula but different
permutations of .sup.13C--, .sup.15N--, .sup.18O--, .sup.34S--, or
.sup.2H--, to result in peptide standards with milliDalton mass
defects. The heavy isotopically labeled peptide standards are used
in a mass spectrometry analysis.
[0017] One embodiment is a plurality of heavy isotope labeled
protein standards having the same amino acid sequence, each
comprising different isotopologues of heavy amino acids, and each
having the same nominal mass and chemical formula but different
permutations of .sup.13C--, .sup.15N--, .sup.18O--, .sup.34S--, or
.sup.2H--, to result in peptide standards with milliDalton mass
defects. The heavy isotopically labeled protein standards are used
in a mass spectrometry analysis to quantify every peptide of a
given target protein.
[0018] One embodiment is a kit for quantifying proteins,
polypeptides, or peptides in a sample. The kit comprises a
plurality of heavy isotope labeled protein and/or peptide standards
having the same amino acid sequence, each comprising different
isotopologues of heavy amino acids, and each having the same
nominal mass and chemical formula but different permutations of
.sup.13C--, .sup.15N--, .sup.18O--, .sup.34S--, or .sup.2H--, to
result in peptide standards with milliDalton mass defects, and
instructions for quantifying the proteins, polypeptides, or
peptides in the sample by mass spectroscopy using the kit. The kit
may have instructions for using the standards in a multiplex assay.
The kit may have instructions for using the standards in a
diagnostic assay.
[0019] One embodiment is a method for absolute protein or peptide
quantitation in a sample by mass spectroscopy using multiplexed
heavy peptide internal standards prepared using protected amino
acid isotopologues as heavy isotopologue precursors for peptide
synthesis. The protected amino acid isotopologues may comprise
fluorenylmethyloxycarbonyl (FMOC)-protected heavy isotopologue
precursors for peptide synthesis. The method may further include
mixing a set of heavy isotopologue peptides at a desired ratio for
use as a multiplexed internal standard. The FMOC-protected amino
acid precursors are mixed at a defined ratio, and the peptide
isotopologue mixture is subsequently synthesized in a single
reaction. Mixtures of fluorenylmethyloxycarbonyl (FMOC)-protected
isotopologues may be used at a defined ratio in peptide
synthesis.
[0020] One embodiment is a method for absolute protein or peptide
quantitation in a sample by mass spectroscopy using multiplexed
heavy protein internal standards prepared using amino acid
isotopologues as heavy isotopologue precursors for protein
synthesis. The method may further include mixing a set of heavy
isotopologue proteins at a desired ratio for use as a multiplexed
internal standard. The amino acid precursors are mixed at a defined
ratio, and the protein isotopologue mixture is subsequently
synthesized in a single reaction. Mixtures of amino acid
isotopologues may be used at a defined ratio in protein
synthesis.
[0021] One embodiment is a method to absolutely quantitate a target
protein or peptide in a sample using mass spectroscopy analysis
using a standard curve specific for the target protein or peptide
in the MS analysis. The method resolves multiplexed internal
standard protein and/or peptide isotopologues first with low
resolution and subsequently with high resolution. It may be used
with a universal reporter assay. The protein or peptide may be used
for diagnosis of a medical or physiological condition of a patient
over a full dynamic range.
[0022] One embodiment is a method to absolutely quantitate a target
protein or peptide in a sample using mass spectroscopy analysis and
with a standard curve specific for the target protein or peptide in
the MS analysis, the standard curve generated from a peptide
mixture synthesized with different isotopologues of heavy amino
acids resulting in a mass defect of at least 1 mDa in the protein
or peptide. The method may further comprise obtaining a single low
resolution MS peak, then obtaining a plurality of high resolution
peaks, and quantitating an intensity of each isotopologue to
quantitate the target protein or peptide in the sample using the
standard curve.
[0023] One embodiment is a method to absolutely quantitate a target
protein or peptide in a sample using mass spectroscopy analysis and
with a standard curve specific for the target protein or peptide in
the MS analysis, the standard curve generated from a protein
mixture synthesized with different isotopologues of heavy amino
acids resulting in a mass defect of at least 1 mDa resulting
peptides of that protein set from proteolytic digestion. The method
may further comprise obtaining a single low resolution MS peak,
then obtaining a plurality of high resolution peaks, and
quantitating an intensity of each isotopologue to quantitate the
target protein or peptide in the sample using the standard
curve.
[0024] One embodiment is a method of enhancing accuracy and/or
sensitivity relative to an AQUA protein or peptide quantitation by
mass spectroscopy analysis using more than one peptide isotopologue
to generate an internal standard curve containing multiple
concentrations of each standard for each assay. The method may
further comprise preparing at least three isotopologue sets of
heavy target peptides, separating heavy target peptides from light
target peptides by MS, at low resolution comparing peaks of the
heavy target peptides and light target peptides to determine
concentration of the target peptide, and at high resolution
resolving isotopologues and analyzing each peak representing
different isotopologue concentrations based on the internal
standard curve.
[0025] One embodiment is a method of enhancing accuracy and/or
sensitivity relative to an AQUA protein or peptide quantitation by
mass spectroscopy analysis using more than one protein isotopologue
to generate an internal standard curve containing multiple
concentrations of each standard for each corresponding peptide of
the native protein analyte. The method may further comprise
preparing at least two isotopologue sets of heavy target proteins,
separating heavy target peptides from light target peptides by MS,
at low resolution comparing peaks of the heavy target peptides and
light target peptides to determine concentration of the target
peptides, and at high resolution resolving isotopologues and
analyzing each peak representing different isotopologue
concentrations based on the internal standard curve.
[0026] One embodiment is a method of generating a standard curve
for mass spectroscopy analysis based on mass defects. The method
comprises preparing a plurality of isotopologues of amino acids to
generate a heavy peptide standard, mixing the plurality of
isotopologues at a fixed desired ratio, separating the mixed
isotopologues with low resolution MS or with high resolution MS,
then using results of the separation to prepare a standard curve.
Separation may be by LC-MS and the standard curve may be prepared
with intact ions. Separation may be by LC-MSn and the standard
curve may be prepared with fragment ions. The method may be used to
verify the target protein or peptide is free of isobaric
interference.
[0027] One embodiment is a method of generating a standard curve
for mass spectroscopy analysis based on mass defects. The method
comprises preparing a plurality of isotopologues of amino acids to
generate a heavy peptide standard, mixing the plurality of
isotopologues at a fixed desired ratio, separating the mixed
isotopologues with low resolution MS or with high resolution MS,
then using results of the separation to verify the MS
calibration.
[0028] One embodiment is a method of generating a standard curve
for mass spectroscopy analysis based on mass defects. The method
comprises preparing a plurality of isotopologues of amino acids to
generate a heavy peptide standard, mixing the plurality of
isotopologues at a fixed desired ratio, separating the mixed
isotopologues with low resolution MS or with high resolution MS,
then using results of the separation to verify the mass shift of
the target analyte.
[0029] One embodiment is a mass spectrometry quantitation method
comprising (a) preparing a plurality of protein isotopologues, (b)
preparing a heavy peptide internal standard, (c) spiking (a) and
(b) into a target sample. (d) digesting the target sample (c), (e)
quantifying each corresponding light:heavy peptide derived from the
digestion of the native target protein analyte (c) and heavy
protein isotopologues (a), (f) separating the peptides by MS
analysis, and (g) quantifying the light and heavy peptide
isotopologues with the heavy peptide internal standard (b) to
quantify the target analyte peptide using the internal standard and
all corresponding light: heavy isotopologue peptide pairs from the
target and internal standard protein set.
[0030] One embodiment is a mass spectrometry quantitation method
comprising (a) preparing a plurality of mass tag isotopologues, (b)
preparing a peptide internal standard labeled with the mass tag
isotopologues, (c) spiking (a) and (b) into a target sample, (d)
digesting the target sample (c), (e) labeling the target sample and
resulting universal reporter peptide with the light version of the
tag, (f) separating the peptides by MS analysis, and (g)
quantifying the target analyte using the internal standard.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 shows mass spectrometry (MS) quantitation of a
protein target using an internal standard.
[0032] FIG. 2 shows low resolution MS and high resolution MS of two
isotopomers (chemical isomers with different heavy isotopic atoms
such that the isomers have the same unit mass but differ in
isotopic elements).
[0033] FIG. 3 shows the generation of a standard curve at high MS
resolution with three different isotopomers at different
concentrations.
[0034] FIG. 4 schematically illustrates synthesis of internal
standards using a defined mixture of precursor amino acid
isotopologues in solution or immobilized on solid supports.
[0035] FIG. 5 shows possible heavy isotope combinations and masses
of lysine +8 Da isotopologues, with two commercially available
isotopologues (filled black, from Hebert et al. 2013).
[0036] FIG. 6 shows the 39 possible formulas and masses of lysine
+8 Da isotopologues with seven 6 milliDalton (mDa)-spaced
representative isotopologues (boxed).
[0037] FIGS. 7 A and B show the 37 possible formulas and masses of
arginine +6 Da isotopologues with seven 6 mDa-spaced representative
isotopologues (boxed, FIG. 7 A) and the 86 possible formulas and
masses of arginine +10 Da isotopologues with seven 6 mDa-spaced
representative isotopologues (boxed, FIG. 7 B).
[0038] FIG. 8 shows the chemical structures of nine representative
fluorenylmethyloxycarbonyl (FMOC)-protected amino acids that may be
used as heavy isotopologue precursors for peptide synthesis by
isotopic labeling in the boxed region.
[0039] FIGS. 9 A-D show the chemical structures of triazine (FIG. 9
A) and purine (FIG. 9 B) core molecules and the corresponding
amine-reactive N-hydroxysuccinimide derivatives (FIGS. 9 C and 9 D
respectively).
[0040] FIGS. 10 A and B show the 13 possible isotopologues of the
triazine core (FIG. 10 A) and the 15 possible isotopologues of the
purine core (FIG. 10B) with six 6 mDa-spaced representative
isotopologues (boxed).
[0041] FIG. 11 schematically shows peptides for mass spectroscopy
quantitation.
[0042] FIG. 12 schematically shows a configuration for a peptide to
be quantified linked to a reporter peptide and correlated to a
universal peptide U, each to be labeled with one or more heavy
amino acids or heavy amino acid isotopologues.
[0043] FIG. 13 schematically shows an embodiment for quantitation
of more than one peptide, each peptide linked to a separate
reporter peptide R, in which each peptide and reporter peptide is
labeled with heavy amino acids or heavy amino acid
isotopologues.
[0044] FIG. 14 schematically shows an embodiment with three
concatenated heavy peptide isotopologue sets linked to a single
reporter peptide R.
[0045] FIG. 15 schematically shows an embodiment for simultaneous
assay of more than one analyte in a sample using a single assay
(multiplexing) where three proteotypic peptide isotopologue sets,
each cleavably linked to its own reporter peptide R and correlated
to a single universal reporter peptide Uplex isotopologue set.
[0046] FIG. 16 schematically shows configuration and relationship
among components.
[0047] FIG. 17 schematically shows configuration and relationship
among peptide components.
[0048] FIG. 18 schematically shows a naming convention with SEQ ID
NOS. 22-25.
[0049] FIG. 19 schematically shows a configuration for a native
analyte targeted for quantitation, an internal standard (IS)
peptide corresponding to the targeted analyte linked to a reporter
peptide and correlated to a universal peptide U. The concatenated
IS:reporter peptide and the universal peptide are each to be
labeled with a heavy isotopologue sets of a chemical MS tag, and
the digested sample with spiked internal standard is to be labeled
with a light, or unique isotopologue version, of the same chemical
MS tag.
[0050] FIG. 20 shows the simulated spectra for LVALVR (SEQ ID NO.
22)+10 Da isotopologues (boxed in FIG. 7 B) with a zoomed in
expanded view of the isotopologue peaks (inset).
[0051] FIGS. 21 A-C schematically show the method for synthesis of
a heavy protein using a mixture of heavy amino acid isotopologues
with an in vitro protein translation kit (FIG. 21 A) or with
cultured cells expressing a protein of interest and grown in
standard media supplemented with heavy amino acid isotopologues
(FIG. 21 B) followed by purification of the heavy protein
isotopologues for use as an internal standard for relative
quantitation (FIG. 21 C).
[0052] FIG. 22 shows a configuration for the use of a digested
heavy isotopologue labeled protein internal standard for relative
quantitation of all corresponding peptides of the native protein
target in a mixture. A distinct heavy AQUA version of at least one
peptide can be used for absolute quantification of the heavy
isotopologue standard and the target protein.
[0053] FIG. 23 shows LC-MS results for the quantification of native
peptide SLLSGLLK (SEQ ID NO. 64) from Akt1 in a cell lysate using a
heavy protein isotopologue set (1:4 ratio of
.sup.13C.sub.8.sup.15N.sub.2 Lysine:.sup.2H.sub.8-Lysine used in
the in vitro Akt1 synthesis, #), with 100 fmol of double heavy AQUA
peptide .sup.13C.sub.8.sup.15N-Leucine,
.sup.13C.sub.8.sup.15N.sub.2-Lysine, SLL*SGLLK* (SEQ ID NO. 64)
spiked into the mixture for absolute quantitation.
[0054] Embodiments of the invention follow.
[0055] A method for absolute quantitation of a target protein or
peptide in a sample by mass spectroscopy (MS) by preparing internal
standard isotopologues using at least one set of multiplexed heavy
peptide internal standards, where each peptide within the at least
one set contains the same amino acid sequence, but the peptides
within the set differ by a mass defect created by incorporating
heavy isotopes on different atoms within at least one amino acid
molecule resulting in internal standard isotopologues, and
quantitating at least one target protein or peptide using the
multiplexed internal standard isotopologues. In one embodiment the
heavy peptide internal standards within each set contain the same
number of total neutrons within each peptide, but differ in that
the heavy atom distribution within the amino acids is unique to
each peptide. In one embodiment the heavy peptide internal
standards within each set are resolved as a single peak under low
resolution mass spectrometry, and are resolved as multiple peaks
under high resolution mass spectrometry. In one embodiment the
heavy peptide internal standards within each set contain mass
differences between each peptide that are less than 1 Dalton. In
one embodiment the sample is a biological sample and the method is
used for a diagnostic assay. In one embodiment the method is used
in a universal reporter assay. In one embodiment the method is used
for multi-sample analysis. In one embodiment the method is used for
multi-target analysis. In one embodiment the method is used for
multi-sample analysis and multi-target analysis. In one embodiment
the heavy peptides are prepared by synthesizing a mixture of at
least two peptides with isotopologues of heavy amino acids,
resulting in heavy peptides having mass defects. In this embodiment
the isotopologues are prepared by mixing solid phase immobilized
AQUA peptide precursors at a defined ratio. In this embodiment the
sample is prepared by effecting cleavage of the target protein or
peptide. In one embodiment the method is used to verify the target
protein or peptide is free of isobaric interference.
[0056] A method for protein or peptide quantitation by mass
spectroscopy by (a) preparing a sample containing a target protein
or peptide of interest for mass spectroscopy analysis, (b)
preparing a plurality of heavy isotope labeled peptides having at
least one subsequence of the target protein or peptide, (c) mixing
the heavy isotope labeled peptides at known concentrations with the
sample, (d) subjecting the mixture containing the prepared sample
and the heavy isotope labeled peptides to mass spectroscopy
analysis at low resolution whereby a single additive heavy peptide
mass spectroscopy peak is obtained with the corresponding single
light peptide mass spectrometry peak, (e) subjecting the low
resolution peaks to high resolution mass spectroscopy analysis
whereby a plurality of mass spectroscopy peaks are obtained
representing the heavy isotopomeric peptides. (f) generating a
light peptide intensity:heavy peptide intensity ratio, and (g)
quantifying the intensity of each of the plurality of mass
spectroscopy peaks based on the intensity of the heavy isotope
labeled peptides to quantify the amount of protein or peptide in
the sample. In one embodiment the heavy peptides are prepared by
synthesizing a mixture of at least two peptides with isotopologues
of heavy amino acids; resulting in heavy peptides having mass
defects. In this embodiment the isotopologues are prepared by
mixing solid phase immobilized AQUA peptide precursors at a defined
ratio. In this embodiment the sample is prepared by effecting
cleavage of the target protein or peptide. In one embodiment step
(f) uses results from a standard curve for MS analysis. In this
embodiment the method further comprises preparing a plurality of
isotopologues of an amino acid; using the isotopologues to generate
a set of heavy peptide standards; mixing the plurality of
isotopologue-containing peptides at fixed ratios; separating the
mixed isotopologue-containing peptides at low mass resolution and
high mass resolution; using the low mass resolution to determine
the light:heavy peptide ratio; and using the high mass resolution
separation results to prepare a standard curve. In one embodiment
the quantity of the target peptide peak is obtained by using a
standard curve of known quantities of at least two isotopologue
standard peptides.
[0057] A method of peptide isotopologue synthesis where a plurality
of precursor amino acid isotopologues, the amino acids containing
alpha carboxylate groups, are mixed at a defined ratio with a solid
phase peptide synthesis resin containing a coupling group to
immobilize the isotopologues through the alpha carboxylate groups.
In one embodiment, after coupling the precursor amino acid
isotopologues, the peptide synthesis resin mixture comprises a
plurality of immobilized amino acid isotopologues at known ratios
coupled to the resin. In this embodiment the resin comprising the
amino acid isotopologues immobilized at known ratios is further
used for peptide synthesis to create a heavy peptide set containing
a plurality of isotopologues.
[0058] A method of peptide isotopologue synthesis by mixing a
plurality of precursor amino acid isotopologues with a solid phase
peptide synthesis resin containing at least one amino acid already
synthesized into a precursor peptide having a desired sequence
where the precursor amino acid isotopologues become attached to the
N-terminal of the peptides using standard amino acid synthesis
procedures. In one embodiment coupling of the precursor amino acid
isotopologues to the immobilized peptide results in a set of heavy
peptides containing a known ratio of isotopologue mixture. In one
embodiment the resin comprising the amino acid isotopologues
immobilized at known ratios is further used for peptide synthesis
to create a heavy peptide set containing a plurality of
isotopologues.
[0059] A method of targeted peptide quantitation using multiplexed
internal standard peptide isotopologues by high resolution mass
spectrometry.
[0060] A method of generating a standard curve for mass
spectroscopy (MS) analysis by preparing a plurality of
isotopologues of an amino acid to generate a heavy peptide
standard, mixing the plurality of isotopologues at fixed ratios,
separating the mixed isotopologues at a low resolution quantitation
or a high resolution quantitation, and using the separation results
to prepare a standard curve.
[0061] A method of synthesizing a single isotopologue set by
premixing solid phase immobilized precursor amino acids at a
defined desired ratio.
[0062] A MS analyte quantitation method comprising (a) preparing,
from a sample containing a peptide analyte having a known amino
acid sequence, a plurality of heavy isotopologue labeled standards
comprising at least part of the analyte sequence; (b) adding a
known quantity of the result of step (a) to the sample; (c)
cleaving the protein in the sample to result in peptides; (d)
quantitating the plurality of heavy isotopologue labeled standards
in the sample; and (e) based on the quantitation of (d),
determining the quantity of analyte in the sample. In one
embodiment quantitation of the plurality of heavy isotopologue
labeled standards in the sample is by high resolution mass
spectroscopy. In one embodiment each standard of step (a) differs
from other standards by incorporating heavy atoms of different
elements but having the same total number of neutrons. In this
embodiment incorporating different heavy atoms into the standards
causes a mass defect among the standards. In one embodiment a peak
intensity for the plurality of heavy isotopologue labeled standards
contains multiple resolvable peaks under high-resolution MS, each
of the multiple resolvable peaks representing the differently
labeled standards of the plurality of heavy isotopically labeled
standards. In this embodiment a standard curve is generated using
the multiple resolvable peaks as a series of known concentrations
of the plurality of heavy isotopologue labeled standards.
[0063] A plurality of heavy isotope labeled peptide standards
having the same amino acid sequence, each comprising different
isotopologues of heavy amino acids, and each having the same
nominal mass and chemical formula but different permutations of
.sup.13C--, .sup.15N--, .sup.18O--, .sup.34S--, or .sup.2H--, to
result in peptide standards with milliDalton mass defects. The
plurality of heavy isotopically labeled peptide standards are used
in a mass spectrometry analysis.
[0064] A kit for quantifying proteins, polypeptides, or peptides in
a sample, the kit comprising a plurality of heavy isotope labeled
peptide standards having the same amino acid sequence, each
comprising different isotopologues of heavy amino acids, and each
having the same nominal mass and chemical formula but different
permutations of .sup.13C--, .sup.15N--, .sup.18O--, .sup.34S--, or
.sup.2H--, to result in peptide standards with milliDalton mass
defects, and instructions for quantifying the proteins,
polypeptides, or peptides in the sample by mass spectroscopy using
the kit. In one embodiment the instructions are for using the
standards in a multiplex assay. In one embodiment the instructions
are for using the standards in a diagnostic assay.
[0065] A method for absolute protein or peptide quantitation in a
sample by mass spectroscopy (MS) using multiplexed heavy peptide
internal standards prepared using protected amino acid
isotopologues as heavy isotopologue precursors for peptide
synthesis. In one embodiment the protected amino acid isotopologues
comprise fluorenylmethyloxycarbonyl (FMOC)-protected heavy
isotopologue precursors for peptide synthesis. In one embodiment
the method further comprises mixing a set of heavy isotopologue
peptides at a desired ratio for use as a multiplexed internal
standard. In one embodiment the method further comprises mixing a
plurality of the FMOC-protected amino acid precursors at a defined
ratio, and subsequently synthesizing the peptide isotopologue
mixture in a single reaction. In one embodiment mixtures of
fluorenylmethyloxycarbonyl (FMOC)-protected isotopologues at a
defined ratio are used in peptide synthesis.
[0066] A method to absolutely quantitate a target protein or
peptide in a sample using mass spectroscopy (MS) analysis using a
standard curve specific for the target protein or peptide in the MS
analysis, by resolving multiplexed internal standard peptide
isotopologues first with low resolution and subsequently with high
resolution. In one embodiment the method is used with a universal
reporter assay. In one embodiment the protein or peptide is used
for diagnosis of a medical or physiological condition of a patient
over a full dynamic range.
[0067] A method to absolutely quantitate a target protein or
peptide in a sample using mass spectroscopy (MS) analysis and with
a standard curve specific for the target protein or peptide in the
MS analysis, the standard curve being generated from a peptide
mixture synthesized with different isotopologues of heavy amino
acids resulting in a mass defect of at least 1 mDa in the protein
or peptide. In one embodiment the method further comprises
obtaining a single low resolution MS peak, then obtaining a
plurality of high resolution peaks, and quantitating an intensity
of each isotopologue to quantitate the target protein or peptide in
the sample using the standard curve.
[0068] A method of enhancing at least one of accuracy and/or
sensitivity relative to an AQUA protein or peptide quantitation by
mass spectroscopy (MS) analysis by using more than one peptide
isotopologue to generate an internal standard curve containing
multiple concentrations of each standard for each assay. In one
embodiment the method further comprises preparing at least three
isotopologue sets of heavy target peptides, separating heavy target
peptides from light target peptides by MS, at low resolution
comparing peaks of the heavy target peptides and light target
peptides to determine concentration of the target peptide, at high
resolution resolving isotopologues and analyzing each peak
representing different isotopologue concentrations based on the
internal standard curve.
[0069] A method of generating a standard curve for mass
spectroscopy (MS) analysis based on mass defects by preparing a
plurality of isotopologues of amino acids to generate a heavy
peptide standard, mixing the plurality of isotopologues at a fixed
desired ratio, separating the mixed isotopologues with low
resolution MS or with high resolution MS, then using results of the
separation to prepare a standard curve. In one embodiment
separation is by LC-MS and the standard curve is prepared with
intact ions. In one embodiment separation is by LC-MSn and the
standard curve is prepared with fragment ions. In one embodiment
the method is used to verify the target protein or peptide is free
of isobaric interference.
[0070] A method of generating a standard curve for mass
spectroscopy (MS) analysis based on mass defects by preparing a
plurality of isotopologues of amino acids to generate a heavy
peptide standard, mixing the plurality of isotopologues at a fixed
desired ratio, separating the mixed isotopologues with low
resolution MS or with high resolution MS, then using results of the
separation to verify the MS calibration.
[0071] A method of generating a standard curve for mass
spectroscopy (MS) analysis based on mass defects by preparing a
plurality of isotopologues of amino acids to generate a heavy
peptide standard, mixing the plurality of isotopologues at a fixed
desired ratio, separating the mixed isotopologues with low
resolution MS or with high resolution MS, then using results of the
separation to verify the mass shift of the target analyte.
[0072] A MS quantitation method comprising (a) preparing a
plurality of mass tag isotopologues, (b) preparing a peptide
internal standard labeled with the mass tag isotopologues, (c)
spiking (a) and (b) into a target sample, (d) digesting the target
sample (c), (e) labeling the target sample and resulting universal
reporter peptide with the light version of the tag, (f) separating
the peptides by MS analysis, and (g) quantifying the target analyte
using the internal standard.
[0073] A mass spectrometry (MS) quantitation system comprising a
sample prepared for MS quantitation, a plurality of mass tag
isotopologues, an ion source, a mass analyzer with isotopologue
separation, and a detector with isotopologue peptide internal
standard resolution.
[0074] The method extends the ability of the known AQUA method, and
results in enhanced quantitation accuracy by generation of a
standard curve, which allows both low mass resolution and high mass
resolution (generally >100,000) capability. The method extends
the ability of the known AQUA method, and results in enhanced
quantitation accuracy by generation of a standard curve for all
peptides in a target protein and enabling the identification and
quantitation of one or more peptides of the target protein that are
regulated differentially from the overall protein level, such as
through post-translational regulation or modification. The
inventive method advantageously uses mass defects, disclosed in
Hebert et al., Neutron-encoded mass signatures for multiplexed
proteome quantification, Nature America, Inc. 2013 Advance Online
Publication), which is expressly incorporated herein by reference
in its entirety, to obtain a single mass spectroscopy peak at low
resolution, then subsequently resolves additional peaks at high
resolution to quantify the intensity of each isotopologue that
results when a mixture of two or more peptides is synthesized with
different isotopologues of heavy isotope labeled amino acids as the
standard. The resulting heavy isotope labeled peptides, termed
heavy peptides, have the same nominal mass and chemical formula,
but have different permutations of .sup.13C--, .sup.15N--,
.sup.18O--, .sup.34S--, or .sup.2H--, resulting in peptides with
milliDalton mass defects.
[0075] A single isotopologue peptide set is obtained by premixing
amino acid isotopomer precursors or solid phase immobilized AQUA
peptide amino acid isotopomer precursors at a defined desired
ratio, as subsequently explained. A full response curve can be
generated for either low or high resolution quantitation.
[0076] A single isotopologue protein set is obtained by premixing
amino acid isotopomer precursors at a defined desired ratio, as
subsequently explained. A full response curve for every peptide of
the target protein can be generated for either low or high
resolution quantitation.
[0077] The method extends the ability of the known AQUA method, and
results in enhanced quantitation accuracy by generation of a
standard curve in with high resolution measurement for one or more
peptides of the target protein.
[0078] The inventive process thus results in enhanced accuracy,
which is particularly required for diagnostic mass spectroscopy
assays. The invention also discloses methods of protein and peptide
isotopologue synthesis, the use of proteotypic peptide isotopologue
sets concatenated with a reporter peptide and quantified with a
universal reporter standard peptide, the use of a heavy
isotopologue protein standard set, and methods of targeted peptide
quantitation using multiplexed internal standard peptide
isotopologues by high resolution mass spectrometry.
[0079] Absolute protein quantitation uses one or more spiked
internal standard peptides containing heavy stable isotopes, with
the amino acid sequence of the standard peptides corresponding to a
subsequence or subsequences of the target protein to be assayed.
The absolute quantitation of this target protein depends upon the
accuracy and linearity of responses of the spiked internal
standard.
[0080] The known AQUA method provides quantitative analysis of
proteins during MS analysis using a single labeled peptide as an
internal standard to quantify the amount of the target, i.e., the
amount of the corresponding target unlabeled peptide or protein in
a sample. This is shown in FIG. 1.
[0081] AQUA relies on a spike of one peptide standard, so
quantitation of the target peptide or protein depends on the
accuracy of that one spiked peptide. The quantitation assumes a
response factor of 1, and assumes that the analyte is within the
linear range. Neither assumption may be accurate for any particular
assay and/or particular analyte. The lack of an internal standard
curve with each measurement results in high coefficients of
variation (CV) and a limited dynamic range at both inter-assay and
inter-laboratory levels.
[0082] The inventive method thus extends the capability of AQUA to
provide greater quantitation accuracy by using multiple
concentrations of each standard to form an internal standard curve,
henceforth referred to as AQUAplex. AQUAplex uses isotopically
labeled peptide families, where each member of the family differs
from other members by incorporation of isotopologues, i.e., heavy
atoms of different elements, but having the same total number of
neutrons. AQUAplex heavy peaks represent a family of isotopically
labeled peptides that are separated and quantified by low mass
resolution and high mass resolution. A single composite peak that
is more readily detectable is obtained in low-resolution MS; thus,
AQUAplex allows quantitation at the MS1 level. Multiple AQUAplex
isotopologue component peaks are obtained and quantified in
high-resolution MS. The use of multiple heavy isotopologues
increases assay accuracy by using multiple measurements, e.g.,
light:heavy peptide intensity ratios obtained first with low
resolution, then quantified with a heavy internal standard
isotopologue set with high resolution MS. As shown in FIG. 1, in a
low resolution MS1 analysis, the heavy peak, which contains
multiple isotopologues, is seen as a single peak and the peak
height and area are the sum of the isotopologues making up the
mixture. The sum of the different isotopologue concentrations
appears in the mass spectrum as a more intense signal than the
individual isotopologues, and may be more intense than the signal
of the target light peptide being analyzed. In this way, under low
resolution MS analysis the heavy peak functions as a marker or
"sign post" for the presence of the target peptide peak which
occurs at a lower m/z position in the spectrum. Because it is known
precisely what the mass increase of the heavy peak is, the target
peak position and elution time can be accurately identified during
LC-MS or LC-MS/MSn, even if the target peak is of low intensity and
may be somewhat lost in the noise or background of the MS spectrum.
Thus, the method facilitates the analysis and more accurate
quantitation of peptides of lower concentration that may not be
immediately detected or may not be detected at all by the mass
spectrometer. The low resolution comparison of the heavy and light
peptide peaks can provide an initial determination of the
concentration of the target peptide being analyzed. Next, under
high resolution analysis of the heavy peak, the isotopologues are
resolved and each peak representing the different concentrations of
the isotopologues making up the internal standard curve can be
analyzed (FIG. 2). The relative concentrations of these
isotopologues can be used to create an internal standard curve to
accurately determine the concentration of the target light peptide
peak (FIG. 3). The high resolution required to resolve the
isotopologues also resolves the target analyte from interfering
species, which improves the target signal-to-noise, sensitivity,
and quantitative accuracy. The isotopologue peak pattern and
absolute masses can be used to verify that the correct heavy
peptide internal standard is being used as the internal standard,
and the exact masses of these isotopologues can be used as mass
calibrants to enable absolute measurements of the offsets for the
light target peptide. Thus, the inventive method permits accurate
identification and absolute quantification of target peptides, that
is, it provides greater accuracy and at higher sensitivity than
possible using previous AQUA peptide techniques.
[0083] As shown in FIG. 2, the AQUAplex heavy peaks represent a
family of peptides, with a single peak resulting with low
resolution MS analysis, and multiple peaks resulting with high
resolution analysis (two peaks shown in FIG. 2). The AQUA light and
heavy peptide peaks are separated by the number of heavy isotopes
incorporated into the amino acids (shown in FIG. 2 as x Dalton).
The heavy peak is used as a control to determine the concentration
of the light peptide. In this example, at low resolution a
measurement of the absolute concentration of the target peptide can
be determined with the higher mass internal standard peak, and the
two isotopomers of equal concentration are able to be resolved at
high resolution for a duplicate measurement. These low and high
resolution measurements may be performed with MS1 to quantify the
parent masses, or MSn to verify and quantify the fragment ions at
either low or high resolution.
[0084] On mass spectrometers capable of multiple ion isolation and
high resolution scanning, such as the Q Exactive hybrid mass
spectrometer (Thermo Scientific), multiplexed analysis of peptides
may be performed by isolation and storage of multiple light:heavy
sets or mass ranges of ions prior to high resolution scanning.
Thus, multiplexed quantitation at low and high resolution can be
performed more efficiently with improved duty cycle performance
(Michalski et al. (2011). Mass Spectrometry-based Proteomics Using
Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass
Spectrometer, Mol Cell Proteomics 2011 September; 10(9):
M111.011015).
[0085] The sample is prepared for MS analysis as known in the art,
e.g., subjecting the sample peptide to proteolytic cleavage. The
sample and standard are then analyzed by mass spectroscopy at low
mass resolution whereby a single additive mass spectroscopy peak is
obtained from the standard, then analyzed at high mass resolution
(>100,000) wherein a plurality of mass spectroscopy peaks are
obtained. The quantity of each peak is obtained using an internal
standard, that is, by comparison to a peak intensity of a known
quantity of an isotopologue standard peptide, or with reference to
an external standard using known quantities of at least two
isotopologue standard peptides to generate a standard curve (FIG.
3). Use of a standard curve enhances quantitation across a dynamic
range of analyte.
[0086] One embodiment of the method is generating a standard curve
for MS analysis. The method comprises the following steps:
preparing a plurality of isotopologues of an amino acid to generate
a heavy peptide standard; mixing the plurality of isotopologues at
fixed ratios; then separating the mixed isotopologues at a low mass
resolution or a high mass resolution using LC-MS or LC-MSn; then
using the separation results to prepare a standard curve with the
intact or fragment ions, respectively.
[0087] One embodiment is a method for absolute protein or peptide
quantitation by MS with multiplexed internal standards. Heavy
AQUAplex peptides, used as standards for assay of a sample
containing a target peptide or protein, are prepared by
synthesizing a mixture of at least two peptides with isotopologues
of heavy amino acids, resulting in heavy peptides having mass
defects, as subsequently explained. The heavy peptides have at
least one subsequence of the target or analyte. The set of heavy
isotopologue peptides is then mixed at a desired ratio for use as a
multiplexed internal standard. Alternatively, the set of
isotopologues is prepared by mixing fluorenylmethyloxycarbonyl
(FMOC)-protected amino acid precursors in solution or solid
phase-immobilized AQUA peptide precursors at a defined ratio, and
then synthesizing the peptide isotopologue mixture in a single
reaction (FIG. 4).
[0088] For AQUAplex assays, each member of the family differs from
the other members by incorporation of heavy atoms of different
elements, but having the same total number of neutrons. For
example, there are 39 unique isotopologues of lysine +8 Da spanning
a mass range of 36 mDa. Theoretically each of these can be resolved
by sufficiently high resolution mass spectrometry (FIG. 5). The
solid bars represent two different isotopologues that are
commercially available (Cambridge Isotope Laboratories). One of the
lysine isomers contains six .sup.13C atoms and two .sup.15N atoms
in its structure, while the other one contains eight .sup.2H atoms.
Both amino acids are nominally of the same molecular mass (about
154.1 Da) but they differ in their accurate mass by 36 mDa due to
the mass defect differences between the neutrons associated with
carbon atoms and hydrogen atoms. Under high resolution mass
spectroscopy analysis, peptides made from these two lysine
isotopologues can be resolved into two distinct peaks differing in
mass by the associated mass defects. Other peptides may be designed
to contain heavy atoms at different sites within their structures.
In one embodiment, one peptide may contain one or more
.sup.13C-lysine amino acids, while another peptide may contain an
equal number of .sup.15N--, .sup.18O--, .sup.34S, or .sup.2H-lysine
amino acids. The incorporation of different heavy atoms between the
members of the family causes a mass defect to occur between
peptides, which slightly alters the exact mass by mDa. With
sufficiently high MS resolution, more isotopologues that are closer
in mass can be resolved. In one embodiment, seven isotopologues of
FMOC-protected lysine +8 Da that are separated by at least 6 mDa
can be used to synthesize and be mixed at a defined ratio of
internal standard isotopologues (FIG. 6). This extended mixture can
be used with high resolution MS to create a standard curve across a
dynamic range greater than one order of magnitude for more accurate
quantitation of a target peptide that varies widely in
concentration. Similarly, eight isotopologues of arginine +6 Da
that are separated by greater than 6 mDa and eight isotopologues of
arginine +10 Da that are separated by greater than 6 mDa can be
used to synthesize and create a defined mixture of heavy internal
standard isotopologues using FMOC-protected arginine +6 Da and
FMOC-protected arginine +10 Da (FIGS. 7a-b). Higher resolution MS
or MSn can be used to further increase the number of resolvable
isotopomers that can be combined to create a standard curve for the
heavy internal standard peptide mixture and improve quantitative
accuracy and dynamic range of quantitation. In one embodiment,
isotopologues are from one or more of the set of FMOC-protected
alanine, arginine, isoleucine, leucine, lysine, phenylalanine,
proline, and valine (FIG. 8).
[0089] In one embodiment, distinct lysine +8 Da isotopologues can
be used to synthesize the heavy peptide standards. These individual
peptides are mixed at a defined ratio, resulting in a single
isotopologue set. In one embodiment, a multiplexed AQUAplex peptide
set can be synthesized in one reaction using precursor
FMOC-protected amino acid precursors or solid phase resins
pre-mixed at a designated ratio (FIGS. 4, 8). In one embodiment,
two or more peptide isotopologues may be mixed at a defined ratio
to create a multiplexed internal standard heavy peptide
isotopologue mixture. As an example, low resolution separation is
shown in FIG. 3 for peptides synthesized in one reaction with
AQUAplex lysine (K) isotopologues at a defined ratio of four
.sup.13C.sub.6.sup.15N.sub.2, three
.sup.13C.sub.4.sup.15N.sub.2.sup.2H.sub.2, two
.sup.13C.sub.6.sup.2H.sub.2, and one .sup.2H.sub.8. Peptides and
modified peptides are synthesized as isotopologues using protected
amino acid precursors, e.g., lysine, arginine, histidine, proline,
leucine, tyrosine (FIGS. 10a-b). In one embodiment, synthesis is
accomplished by premixing or combining the heavy peptide standards
at a defined or fixed ratio, enabling replicate measurement or
generation of a standard calibration curve. At low mass resolution,
heavy isotopologues are not resolved, but instead are summed or
additive. This results in a composite peak of higher intensity,
with or without a detectable weighted average mass defect, and the
intensity of each of the isotopologue peaks becomes summed within
the low resolution single peak to provide a greater signal in the
mass spectrum. The higher intensity peak is more easily detected by
mass spectrometry than the target peptide or the individual heavy
peptide isotopologues, allowing triggered ion isolation and MS
enrichment. This invention thus extends the sensitivity of mass
spectroscopy analysis to lower concentrations, because the combined
isotopologue peak is used as the marker or "sign post" for the
presence of the light target peptide peak, which may not have been
noticed by the mass spectrometer without this marker due to the low
intensity of the peak.
[0090] The heavy peptides can be synthesized individually, or they
can be synthesized as a mixture by combining isotopologue amino
acid precursors or solid phase supports that have been preactivated
with isotopologue precursors at predefined ratios. For example, to
prepare a four-isotopologue mixture, four of the lysine derivatives
represented by the bars of FIG. 6 are first attached to a solid
phase peptide synthesis support using standard methods known in the
art. To prepare four otherwise identical peptides containing
different but known amounts of these isotopologue lysine residues,
the four precursor supports then are mixed in a desired ratio, for
example 4:3:2:1 (FIG. 4). The peptide is then synthesized using
this mixture of isotopologue precursors by adding one amino acid at
a time, as is done in typical amino acid synthesis procedures. Once
the peptide synthesis is complete and the peptide is cleaved from
the support and purified, the resultant mixture will contain four
isotopologues of the same peptide, where one of the isotopologues
is at 1/2, 1/3 or 1/4 the concentration of the other isotopologues.
A peptide isotopologue set that contains the heavy isotopologue at
another position can be synthesized by incorporating a defined
mixture of protected amino acid isotopologues. Other mixtures of
isotopologues may be prepared using similar methods, including
those which would contain more than two isotopologues at known
concentrations to create a multi-point standard curve. Use of this
single reaction or "one pot" synthesis provides several advantages:
it reduces the number of reactions required for synthesizing the
multiplexed isotopologue set, it allows the same predefined mixture
of activated supports to be used as a precursor for multiple
peptide reactions, it ensures enhanced synthesis reproducibility of
quantitative multiplexed peptide isotopologue sets, and it permits
the absolute amount of the isotopologue mixture to be quantified in
one amino acid analysis. The result is that synthesis is simplified
and reproducibility is improved, with a lowered manufacturing cost
for the protein or peptide standards.
[0091] The isotopologue set or sets of the internal standard may be
prepared in a form for commercial use, e.g., packaged with
instructions for use as a kit. Such a kit could include
instructions for high resolution mass spectrometry that may or may
not have the capability to isolate and enrich a mass region (e.g.,
by an ion trap or quadrupole/hexapole/octapole mass filter), and/or
the capability to fragment isolated ions with collisional or
chemical fragmentation methods. Methods using such a kit include
high resolution mass spectrometry with or without mass range
isolation and/or multistage fragmentation.
[0092] Incorporation of different heavy atoms among peptide family
members causes a mass defect to occur among peptides, which
slightly alters the real mass by mDa. The heavy peptide peak of an
AQUAplex MS separation contains multiple resolvable peaks under
high resolution MS. These multiple resolvable peaks represent
multiple concentrations of the isotopomeric peptides within a
single peptide family. Because multiple concentrations of the
isotopomeric peptides can be contained within the heavy AQUAplex
peak, a standard curve is contained within the heavy peak as a
series of known concentrations of the mass-defect labeled
peptide.
[0093] An AQUAplex standard quantitation curve is generated as
follows. Multiple concentrations of the isotopomeric peptides, as a
series of known concentration of the mass-defect labeled peptides,
are contained within the heavy AQUAplex peak. The number of points
in the standard curve is governed by the resolution power of the
mass spectrometer used in the assay. In embodiments, two to six
peaks can be resolved. In one embodiment, an AQUAplex internal
standard curve for quantitation is obtained using two or more
different isotopomers at different concentrations (FIG. 3). An
internal standard curve can be generated readily for each target
peptide being measured. The high intensity of the low mass
resolution peak serves as a signpost of the analyte for triggered
acquisition strategies. The use of standard curves improves and
validates diagnostic assays using mass spectrometry analysis.
[0094] In a similar embodiment, amino acid isotopologue sets can be
used for synthesis of heavy protein isotopologue sets. These heavy
protein isotopologues can be made with fixed ratios of the amino
acid precursors, so that after digestion, isotopologue sets of
every peptide of the heavy protein is present at the same
concentration. Peptides derived by proteolysis of the native and
heavy protein set can be used to quantify all peptides of a target
protein. This heavy protein set may or may not be quantified as an
absolute internal standard. An AQUA peptide with a distinct mass
can be included for absolute quantitation of the native and heavy
protein standard set. In this embodiment, digestion efficiency for
each peptide is normalized, and distinct peptides from the target
analyte that are regulated independent of the protein level, such
as by post translational modification, may be identified. The
stoichiometry of native to modified peptides of the target analyte
may have greater diagnostic utility than the overall target protein
concentration.
[0095] The need to maintain inter-assay precision in an AQUAplex
assay is important, as is the need to improve and validate
diagnostic applications of mass spectroscopy. The inventive method
is compatible with existing quantitative protein workflow systems
that use protein digestion and LC-MS for peptide detection. It
combines AQUAplex methods with NeuCode amino acids or mass tags and
can be used, e.g., in a universal reporter assay, to result in
multi-sample and multi-target analysis with absolute protein or
peptide quantitation. The inventive method provides multiple
replicates of the heavy peptide internal standard, without
increasing the complexity of the sample. The inventive method
allows absolute quantitation with high resolution mass spectrometry
at the MS1 or MSn level, with the ability to verify and quantify
sequence fragment ions with multistage MS. The inventive method
allows triggered ion isolation and MS enrichment using the mass of
the low resolution summed peak of the heavy peptide internal
standard isotopologues. The inventive method permits acquisition of
a set of heavy peptide replicates at high resolution, providing
more accurate target peptide quantitation. The inventive method
permits acquisition of a heavy peptide standard curve at high
resolution for more accurate target peptide quantitation across a
broad dynamic range. The inventive method provides the ability to
incorporate the amino acid isotopologues into the internal standard
heavy peptide and internal standard heavy protein. The inventive
method facilitates multiplexed synthesis of the heavy peptide and
protein isotopologues using a premixed set of precursor amino acids
isotolopogues or solid phase precursors. The inventive method
provides the ability to incorporate the amino acid isotopologues
into a universal reporter peptide sequence.
[0096] Current MS-based method for absolute protein quantitation
(e.g. AQUA) utilize a heavy internal standard heavy peptide that is
spiked into an analyte sample, and the ratio of the MS signal
intensity or AUC of the target analyte peptide to the internal
standard heavy peptide is used to calculate the absolute amount of
target analyte in the sample. Ideally, the concentration of this
internal standard is within one order of magnitude of the target
analyte for accurate quantitation. Clinically relevant protein
biomarkers are present across a broad range of concentrations, such
as cardiac myoglobin (Mb) that is present in plasma from normal
subjects at 1 ng/ml to 85 ng/ml, but is increased to 200 ng/ml to
1,100 ng/ml by a myocardial infarction, and up to 3,000 ng/ml by
fibrinolytic therapy to treat the infarct (Anderson and Anderson
(2002) The human plasma proteome: History, character, and
diagnostic prospects. Mol. Cell. Proteomics 1, 845-867). Troponin I
above a cut off of 100 pg/ml is another approved marker of
myocardial infarction, but this cut off is >500-fold lower than
Mb. Additional examples of the broad dynamic range of clinical
biomarkers include multiple interleukins below 1 pg/ml, CEA above
500 pg/ml, prostate specific antigen above 4 ng/ml,
alpha-fetoprotein above 20 ng/ml, and B-type naturietic peptide
(BNP) above 8 pg/ml (Polanski and Anderson (2006) A List of
Candidate Cancer Biomarkers for Targeted Proteomics. Biomarker
Insights 1, 1-48). For these and other examples, the broad dynamic
range of target analytes requires use of multiple internal standard
concentrations in MS assays for accurate analyte quantitation. The
disclosed inventive method addresses this dynamic range requirement
in one assay.
Labeled Peptide Internal Standards (AQUA Peptides) and Heavy
Protein Internal Standards
[0097] AQUA peptide labeled internal standards are disclosed in WO
03/016861 and U.S. Pat. No. 7,501,286, each of which is expressly
incorporated by reference herein in its entirety. Heavy isotope
labeled proteins as internal standards are disclosed in U.S. Pat.
No. 7,939,331, and proteolytic digestion yields the set of heavy
peptide standards. Briefly, and as disclosed in these references,
the peptide is synthesized using one or more labeled amino acids
(i.e., the label is actually part of the peptide) or less
preferably, labels may be attached after synthesis. The label is a
mass-altering label. The mass of the label should preferably be
unique to shift fragment masses produced by MS analysis to regions
of the spectrum with low background. The ion mass signature
component is the portion of the labeling moiety which preferably
exhibits a unique ion mass signature in mass spectrometric
analyses. The sum of the masses of the constituent atoms of the
label is preferably uniquely different than the fragments of all
the possible amino acids. As a result, the labeled amino acids and
peptides are readily distinguished from unlabeled amino acids and
peptides by their ion/mass pattern in the resulting mass spectrum.
In a preferred embodiment, the ion mass signature component imparts
a mass to a protein fragment produced during mass spectrometric
fragmentation that does not match the residue mass for any of the
20 natural amino acids.
[0098] The label should be robust under the fragmentation
conditions of MS and not undergo unfavorable fragmentation.
Labeling chemistry should be efficient under a range of conditions,
particularly denaturing conditions and the labeled tag preferably
remains soluble in the MS buffer system of choice. Preferably, the
label does not suppress the ionization efficiency of the protein.
More preferably, the label does not alter the ionization efficiency
of the protein and is not otherwise chemically reactive.
Alternatively, or additionally, the label contains a mixture of two
or more isotopically distinct species to generate a unique mass
spectrometric pattern at each labeled fragment position.
[0099] Peptide internal standards comprise mass-altering labels
which are stable isotopes, e.g., isotopes of hydrogen, nitrogen,
oxygen, carbon, or sulfur. Suitable isotopes include, but are not
limited to, .sup.2H, .sup.13C, .sup.15N, .sup.17O, .sup.18O, or
.sup.34S. Pairs of peptide internal standards can be provided,
comprising identical peptide portions but distinguishable labels,
e.g., peptides may be labeled at multiple sites to provide
different heavy forms and isotopologues of the peptide. Multiple
labeled amino acids may be incorporated in a peptide during the
synthesis process. The label may be part of a peptide comprising a
modified amino acid residue, such as a phosphorylated residue, a
glycosylated residue, an acetylated residue, a ribosylated residue,
a farnesylated residue, or a methylated residue. In this
embodiment, pairs or larger sets of peptide internal standards
corresponding to modified and unmodified peptides also can be
produced. In one aspect, such a pair/set is differentially
labeled.
[0100] Peptide internal standards are characterized according to
their mass-to-charge ratio (m/z) and preferably, also according to
their retention time on a chromatographic column (e.g., such as an
HPLC column). Internal standards are selected which co-elute within
30 seconds with peptides of identical sequence but which are not
labeled. Isotopologues with .sup.2H have a slightly different
retention on HPLC, so these isotopologues will elute near but not
simultaneously with the isotopologues. More .sup.2H will result in
more retention time shift.
[0101] The peptide internal standard is then analyzed by MS1 or by
fragmenting the peptide with multistage MS (MSn). Fragmentation can
be achieved by inducing ion/molecule collisions by a process known
as collision-induced dissociation (CID) (also known as
collision-activated dissociation (CAD)). Collision-induced
dissociation is accomplished by selecting a peptide ion of interest
with a mass analyzer and introducing that ion into a collision
cell. The selected ion then collides with a collision gas
(typically argon or helium) resulting in fragmentation. Generally,
any method that is capable of fragmenting a peptide is encompassed
within the scope of the present invention. In addition to CID,
other fragmentation methods include, but are not limited to, high
energy collisional dissociation (HCD), surface induced dissociation
(SID), blackbody infrared radiative dissociation (BIRD), electron
capture dissociation (ECD), post-source decay (PSD), LID, and the
like.
[0102] The fragments are then analyzed to obtain a fragment ion
spectrum. One suitable way to do this is by CID in multistage mass
spectrometry (MS.sup.n). Traditionally used to characterize the
structure of a peptide and/or to obtain sequence information, it is
a discovery of the present invention, that high resolution MS1
and/or high resolution MS.sup.n provides enhanced sensitivity in
methods for quantitating absolute amounts of proteins. Thus, in one
aspect, peptide internal standards are generated for low abundance
proteins (e.g., below 2000 copies/cell).
[0103] High resolution, absolute mass MS1 can be used in
combination with retention time information to reliably verify and
quantify a target peptide. In one embodiment, the internal standard
isotopologue cluster at low resolution can serve as a higher
intensity MS1 signal to trigger isolation and enrichment of one or
more target peptides and internal standard pairs. In one
embodiment, the high resolution and accurate mass measurement of
the isotopologue cluster is used as an MS instrument mass
calibration standard to provide confirmation of the targeted
peptide MS1 mass signal.
[0104] In one embodiment, a peptide internal standard is analyzed
by at least two stages of mass spectrometry to determine the
fragmentation pattern of the peptide and to identify a peptide
fragmentation signature for peptide verification. A peptide
signature is obtained in which peptide fragments have significant
differences in m/z ratios to enable peaks corresponding to each
fragment to be well separated. Signatures are desirably unique,
i.e., diagnostic of a peptide being identified and comprising
minimal if any overlap with fragmentation patterns of peptides with
different amino acid sequences. If a suitable fragment signature is
not obtained at the first stage, additional stages of mass
spectrometry are performed until a unique signature is obtained.
This fragmentation signature ensures that peaks of the same exact
mass are not simply rearrangements of the same amino acids in a
different order.
[0105] Fragment ions in the MS/MS and MS.sup.3 spectra are
generally highly specific and diagnostic for peptides of interest.
In contrast to prior art methods, the identification of peptide
diagnostic signatures provides for a way to perform highly
selective analysis of a complex protein mixture, such as a cellular
lysate in which there may be greater than about 100, about 1000,
about 10,000, or even about 100,000 different kinds of proteins.
Thus, while conventional mass spectroscopy would not be able to
distinguish between peptides with different sequences but similar
m/z ratios (which would tend to co-elute with any labeled standard
being analyzed), the use of peptide fragmentation methods and
multistage mass spectrometry in conjunction with LC methods,
provide a way to detect and quantitate target proteins which are
only a small fraction of a complex mixture (e.g., present in less
than 2000 copies per cell or less than about 0.001% of total
cellular protein) through these diagnostic signatures.
[0106] Multiple peptide subsequences of a single protein may be
synthesized, labeled, and fragmented to identify optimal
fragmentation signatures. In one embodiment, at least two different
peptides are used as internal standards to identify/quantify a
single protein, providing an internal redundancy to any
quantitation system. In another embodiment, peptide internal
standards are synthesized which correspond to a single amino acid
subsequence of a target polypeptide but which vary in one or more
amino acids. The peptide internal standards may correspond to known
variants or mutations in the target polypeptide or can be randomly
varied to identify all possible mutations in an amino acid
sequence.
[0107] In one embodiment, peptides corresponding to different
modified forms of a protein are synthesized, providing internal
standards to detect and/or quantitate changes in protein
modifications in different cell states. In one embodiment, peptide
internal standards are generated which correspond to different
proteins in a molecular pathway and/or modified forms of such
proteins (e.g., proteins in a signal transduction pathway, cell
cycle, metabolic pathway, blood clotting pathway, etc.) providing
panels of internal standards to evaluate the regulated expression
of proteins and/or the activity of proteins in a particular
pathway. Combinations of the above-described internal standards can
be used in a given assay.
[0108] Generally, the sample will have at least about 0.01 mg of
protein, at least about 0.05 mg, and usually at least about 1 mg of
protein or 10 mg of protein or more, typically at a concentration
in the range of about 0.1-10 mg/mi. The sample may be adjusted to
the appropriate buffer concentration and pH, if desired.
[0109] In one embodiment, a known amount of a labeled peptide
internal standard corresponding to a target protein to be detected
and/or quantitated, is added to a sample such as a cell lysate. In
one embodiment, about 10 femtomoles is spiked into the sample. The
sample is contacted with a protease activity, e.g., one or more
proteases or appropriate chemical agent(s) are added to the sample,
and the spiked sample is incubated for a suitable period of time to
allow peptide digestion. If the target protein is present in the
sample, the digestion step should liberate a target peptide
identical in sequence to the peptide portion of the internal
standard and the amount of target peptides so liberated from target
proteins in the sample should be proportional to the amount of
target protein in the sample.
[0110] In one embodiment, a separation procedure is performed to
separate a labeled peptide internal standard and corresponding
target peptide from other peptides in the sample. Representative
examples include high-pressure liquid chromatography (HPLC),
reverse phase-high pressure liquid chromatography (RP-HPLC),
electrophoresis (e.g., capillary electrophoresis), anion or cation
exchange chromatography, and open-column chromatography. Internal
standards are selected so that they co-elute with their
corresponding target peptides as pairs of peptides that differ only
in the mass contributed by the mass-altering label.
[0111] Each peptide then is examined by monitoring of high
resolution absolute mass MS1 or a selected reaction in the mass
spectrometer. This involves using the prior knowledge gained by the
characterization of the peptide internal standard and then
requiring the mass spectrometer to continuously monitor a specific
ion in the MS1, MS/MS, or MS.sup.n spectrum for both the peptide of
interest and the internal standard. After elution, the
areas-under-the-curve (AUC) for both the peptide internal standard
and target peptide peaks are calculated. The ratio of the two areas
provides the absolute quantification that can be normalized for the
number of cells used in the analysis and the protein's molecular
weight, to provide the precise number of copies of the protein per
cell.
[0112] The inventive method determines the presence of and/or
quantity of a modification in a target polypeptide. In one
embodiment, the label in the internal standard is attached to a
peptide comprising a modified amino acid residue or to an amino
acid residue that is predicted to be modified in a target
polypeptide. In one embodiment, multiple internal standards
representing different modified forms of a single protein and/or
peptides representing different modified regions of the protein are
added to a sample and corresponding target peptides (bearing the
same modifications) are detected and/or quantified. In one
embodiment, standards representing both modified and unmodified
forms of a protein are provided to compare the amount of modified
protein observed to the total amount of protein in a sample.
[0113] Reagents for performing the method comprise a peptide
isotopologue internal standard labeled with a stable isotope. In
one embodiment, the standard has a unique peptide fragmentation
signature diagnostic of the peptide. The peptide is a subsequence
of a known protein and can be used to identify the presence of
and/or quantify the protein in sample, such as a cell lysate.
[0114] The invention additionally provides kits comprising one or
more peptide internal standards labeled with a stable isotope or
reagents suitable for performing such labeling. In one embodiment,
the method utilizes isotopes of hydrogen, nitrogen, oxygen, carbon,
or sulfur. Suitable isotopes include, but are not limited to,
.sup.2H, .sup.13C, .sup.15N, .sup.17O, .sup.18O or .sup.34S. In one
embodiment, pairs of peptide internal standards are provided,
comprising identical peptide portions but distinguishable labels,
e.g., peptides may be labeled at multiple sites to provide
different heavy or isotopologue forms of the peptide. Pairs of
peptide internal standards corresponding to modified and unmodified
peptides also can be provided.
[0115] In one embodiment, a kit comprises peptide internal
standards comprising different peptide subsequences from a single
known protein. In one embodiment, the kit comprises peptide
internal standards corresponding to different known or predicted
modified forms of a polypeptide. In one embodiment, the kit
comprises peptide internal standards corresponding to sets of
related proteins, e.g., such as proteins involved in a molecular
pathway (a signal transduction pathway, a cell cycle, etc.), or
which are diagnostic of particular disease states, developmental
stages, tissue types, genotypes, etc. Peptide internal standards
corresponding to a set may be provided in separate containers or as
a mixture or "cocktail" of peptide internal standards.
[0116] In one embodiment, the peptide internal standard comprises a
label associated with a modified amino acid residue, such as a
phosphorylated amino acid residue, a glycosylated amino acid
residue, an acetylated amino acid residue, a farnesylated residue,
a ribosylated residue, and the like. In another aspect, a pair of
reagents is provided, a peptide internal standard corresponding to
a modified peptide and a peptide internal standard corresponding to
a peptide, identical in sequence but not modified.
[0117] In one embodiment, one or more control peptide internal
standards are provided. For example, a positive control may be a
peptide internal standard corresponding to a constitutively
expressed protein, while a negative peptide internal standard may
be provided corresponding to a protein known not to be expressed in
a particular cell or species being evaluated. For example, in a kit
comprising peptide internal standards for evaluating a cell state
in a human being, a plant peptide internal standard may be
provided.
[0118] In one embodiment, a kit comprises a labeled peptide
internal standard as described above and software for analyzing
mass spectra (e.g., such as SEQUEST).
[0119] Preferably, the kit also comprises a means for providing
access to a computer memory comprising data files storing
information relating to the diagnostic absolute mass MS1 and MSn
fragmentation signatures of one or more peptide internal standards.
Access may be in the form of a computer readable program product
comprising the memory, or in the form of a URL and/or password for
accessing an internet site for connecting a user to such a memory.
In one embodiment, the kit comprises diagnostic accurate mass MS1
and MSn fragmentation signatures (e.g., such as mass spectral data)
in electronic or written form, and/or comprises data, in electronic
or written form, relating to amounts of target proteins
characteristic of one or more different cell states and
corresponding to peptides that produce the fragmentation
signatures.
Neutron-Encoded Mass Signatures for Multiplexed Proteome
Quantification (NeuCode) Method
[0120] The neutron-encoded protein quantification method is
disclosed in Hebert et al., Neutron-encoded mass signatures for
multiplexed proteome quantification, Nature America, Inc. 2013
Advance Online Publication), which is expressly incorporated by
reference herein in its entirety. As disclosed, it uses subtle mass
differences caused by nuclear binding energy variation in stable
isotopes. These mass differences are synthetically encoded into
amino acids and incorporated in yeast and mouse proteins by
metabolic labeling. Mass spectrometry analysis with high mass
resolution (>100,000) reveals the isotopologue-embedded peptide
signal, permitting quantification.
[0121] The multiplexing capacity of isobaric tandem mass tags has
been expanded from six to eight using the concomitant swapping of a
.sup.12C for a .sup.13C atom and a .sup.15N for a .sup.14N atom to
produce a new tag with a 6-mDa mass difference that can be
distinguished with a mass resolution of 50,000 at a mass-to-charge
ratio (m/z) of 130 (Werner et al. (2012) High-resolution enabled
TMT8-plexing. Anal Chem 84(16):7188-94; McAllister et al. (2012)
Increasing the multiplexing capacity of TMTs using reporter ion
isotopologues with isobaric masses. Anal Chem 84(17):7469-78). Mass
defect, the cause of this subtle mass change, arises from the fact
that nuclear binding energy, the energy required to break down a
nucleus into its component nucleons, is different for each isotope
of every element. The tandem mass tag approach still relies on
MS/MS-based quantification, however, and does not resolve the
accuracy and reproducibility issues of isobaric tagging. It was
suggested that other elements, besides C and N, could encode
neutron mass signatures. Indeed, mass defects can be induced with
many elements and their isotopes: for example, .sup.12C/.sup.13C
(+3.3 mDa), .sup.1H/.sup.2H (+6.3 mDa), .sup.16O/.sup.18O (+4.2
mDa), .sup.14N/.sup.15N (-3.0 mDa) and .sup.32S/.sup.34S (-4.2
mDa). It was hypothesized that calculated incorporation of these
isotopes into proteomes would generate a new MS1-centric
quantification technology that combines the accuracy of SILAC with
the multiplexing capacity of isobaric tagging. This method has been
called neutron encoding (NeuCode).
[0122] In contrast to the use of NeuCode for mass tagging a
proteome, the methods disclosed use the concept of mass defect to
create labeled peptide standards to be used for absolute
quantitation of an analyte.
[0123] Use of these mass defects in the creation of labeled peptide
standards exploits the subtle differences in nuclear binding energy
between isotopes. The approach effectively compresses isotopic
information into a very narrow m/z space (0.0050-0.040) so that it
is easily concealed or revealed by varying mass resolution. Current
Fourier transform MS systems offer ultra-high resolution
(>1,000,000) and will permit the use of mass defect-labeled
peptides separated by as little as -6 mDa. In one embodiment,
synthesis of custom lysine isotopologues that offer 7-plex
quantification: +8 Da at 0, 6, 12, 18, 24, 30, and 36 mDa spacings
are created (FIGS. 6, 7a-b). Further, such >7-plex isotopologue
sets could be generated with 12 additional neutrons, allowing the
combination of isotopologues in multiple isotopic clusters. For
example, each peptide would be present in three isotopic clusters,
just as in a traditional triplex; however, each cluster would
reveal 5-7 distinct peaks upon high-resolution scan analysis. By
combining peptides containing these 19 isotopologues of lysine
(5+7+7), mass defect labeling should facilitate 19-plex
experiments. Similarly, by combining 8-plex isotopologue sets
containing arginine isotopologues with 6 and 10 additional
neutrons, 16-plex multiplexed quantitation of arginine-containing
peptides can be performed as well. By combining mass defect with
mass differential tags for relative and absolute quantification,
highly multiplexed quantitative accuracy and precision was
achieved.
Isotopologue Mass Tag Assays
[0124] Covalent mass tags are an alternative method to create
isotopologue sets of internal standard peptides isotopologues. Mass
tags have greater flexibility in chemical structure and thus these
chemical tags are more easily synthesized over synthesis of
specific chiral amino acid precursors. Isotopologue mass tags may
be used to tag non-protein samples, including nucleotides, glycans,
lipids, and metabolites. In addition, isotopologue mass tags may
incorporate unique features that improve solubility,
chromatographic retention properties, ionization efficiency, and/or
peptide fragmentation behavior. Two example core structures and
their related amine-reactive tags are described (FIG. 9). These
example tags are based upon triazine cyclic and purine heterocyclic
ring structures that are modified with amine-reactive
N-hydroxysuccinimide (NHS). These tags may be synthesized as known
in the art without heavy isotopes or with different combinations of
heavy isotopes. The light and heavy isotopic reagents may have a
minimum mass difference of 4 Da. The possible isotopologues and
masses of the heavy triazine and purine core molecules are shown
(FIGS. 10a-b). These isotopologues may be used to create a
multiplexed reagent set that can be resolved with >100,000
resolution. In one embodiment, the heavy isotopologues are
pre-mixed at a defined ratio. An analyte protein sample is digested
with a protease, such as trypsin or LysC, and the light tags are
covalently attached to the peptides through reactive amines at the
amino-terminus of the peptide and lysine residues. An internal
standard peptide at a known absolute concentration to quantify the
target analyte peptide is labeled with the heavy isotopologue tag
set. At low resolution, the area under the curve (AUC) of the light
targeted analyte and the composite AUC of the heavy isotopologue
set can be used to quantify the target analyte, and at high
resolution the internal standard isotopologues can be used to
define a standard curve for more accurate quantitation of the
target analyte.
Universal Reporter Assays
[0125] The use of a universal reporter is described in WO
2012/005838 and WO 2012/006406. This method results in absolute
quantification of analytes by MS, and enables a simple
concentration calibration of analytes in reference solutions. The
method uses a heavy isotope labeled analyte (internal standard)
that is in equimolar concentration with, and that is cleavably
coupled to, a reporter R (that may or may not be heavy isotope
labeled); and a heavy isotope labeled universal reporter U.
Analytes include, but are not limited to, peptides, polypeptides,
and proteins. Universal reporter U includes, but is not limited to,
peptides (i.e., polymers of amino acids) and other polymers.
[0126] In one embodiment the inventive method resulted in absolute
quantification of peptide, polypeptide, and proteins analytes by
MS. The method used a heavy isotope labeled peptide (proteotypic
peptide, described below; internal standard) that was present in
equimolar concentration with, and was cleavably coupled at a
proteolytic site to, an optionally heavy isotope labeled reporter
peptide R; a heavy labeled universal reporter peptide U analyzed by
amino acid analysis. The heavy isotope labeled peptide need not
undergo amino acid analysis. In one embodiment, several different
proteotypic peptides from a single protein, linked to separate
reporter peptides R, were analyzed. In one embodiment, several
different proteotypic peptides concatenated into one polypeptide,
linked to a single reporter peptide R were analyzed.
[0127] FIG. 11 shows proteotypic peptides A, B, and C from protein
or polypeptide P. A proteotypic peptide is a signature peptide that
fragments into a predictable ion series following MS dissociation
to allow specific identification and quantitation of the parent
protein, whether in a purified form or from a complex mixture. It
has characteristics that render it readily quantified. A signature
peptide is an unambiguous identifier of a specific protein. Any
protein contains between 10 and 100 signature peptides. Any
signature peptide meets most of the following criteria: easily
detected by mass spectroscopy, predictably and stably eluted from a
liquid chromatography (LC) column, enriched by reversed phase high
performance liquid chromatography (RP-HPLC), good ionization, and
good fragmentation. A peptide that is readily quantified meets most
of the following criteria: readily synthesized, ability to be
highly purified (>97%), soluble in .gtoreq.20% acetonitrile, low
non-specific binding, oxidation resistant, post-synthesis
modification resistant, and a hydrophobicity or hydrophobicity
index .gtoreq.10 and .ltoreq.40. The hydrophobicity index is
described in Krokhin, Molecular and Cellular Proteomics 3 (2004)
908, which is expressly incorporated herein by reference. A peptide
having a hydrophobicity index less than 10 will not be reproducibly
resolved by RP-HPLC. A peptide having a hydrophobicity index
greater than 40 will not be reproducibly eluted from a RP-HPLC
column.
[0128] The inventive method uses an internal standard that is the
heavy isotopologue (labeled) form of the analyte to be quantified,
also referred to as a AQUAplex heavy analyte and shown in FIG. 16.
In the embodiment using a proteotypic peptide isotopologue set, the
internal standard is the labeled isotopologue set of the
proteotypic peptide, also referred to as heavy proteotypic peptide
isotopologues, as shown in FIG. 17.
[0129] Isotope dilution mass spectrometry (IDMS) refers to the use
of heavy isotope-labeled peptides as internal standards to
establish the concentration versus MS response relationship and to
perform absolute quantitation of peptide. The heavy isotope labeled
peptide has identical properties as the unlabeled peptide, except
that its mass is shifted by the incorporated isotope(s). As a
result of this mass shift, a known amount of the isotope-labeled
peptide can be used as an internal standard for peptide
quantitation. The IDMS method results in targeted mass spectrometry
(selected ion monitoring (SIM)/selected reaction monitoring
(SRM)/multiple reaction monitoring (MRM)) quantitation of peptides
in complex samples or mixtures. SIM and SRM encompass the MS
acquisition setup to quantify a list of target proteins by the
quantitation of the parent or specific fragment ions from
proteotypic peptides of these target proteins, respectively.
Targeted assay development must be fast, have high throughput, be
sensitive, specific, targeted, robust, reproducible, and cost
effective; it typically uses liquid chromatography-tandem mass
spectrometry (LC-MS/MS, LC/MS.sup.2). However, precise quantitation
of large number of peptides in targeted proteomics experiments
using SRM remains challenging because of specificity and duty cycle
requirements. SRM specificity refers to the choice of
parent/fragment ion combinations (e.g., transitions) that provide a
specific quantitative response for the target and internal standard
peptides with high sensitivity. Duty cycle refers to the limited
number of SRM transitions that can be monitored simultaneously by
MS in a multiplexed SRM assay. To address the duty cycle
limitations, SRM methods are typically scheduled or timed such that
transitions for a given target are only monitored in a retention
time window expected for the given target. This requires complex
SRM method setup prior to data acquisition and reproducible
chromatography to ensure that the target transitions are monitored
completely with sufficient baseline before and after the peak for
accurate AUC determination. SIM monitoring includes the isolation
and enrichment of a targeted peptide and internal standard in order
to improve the sensitivity of the assay. AQUAplex assays acquired
with high resolution MS1 scans do not require scheduling or the
selection of transitions, and all of the quantitative data analysis
can be performed post-acquisition.
[0130] In IDMS, the native proteotypic peptide differs from the
heavy proteotypic peptide only due to incorporation of a heavy
amino acid. A heavy amino acid contains .sup.13C (the heavy isotope
of carbon) and/or .sup.15N (the heavy isotope of nitrogen),
.sup.18O (the heavy isotope of oxygen), and/or .sup.2H (the heavy
isotope of nitrogen), and different combinations of these isotopes
can be combined to produce isotopologue sets containing the same
unit mass but slightly different accurate masses observable with
high resolution mass spectrometry. The insertion of a heavy amino
acid isotopologues results in HeavyPeptide AQUAplex, which differs
from the proteotypic peptide only by the difference in mass. The
purity of the heavy peptide isotopologue mixture is increased to
>97% using preparative high performance liquid chromatography
(HPLC). The precise quantity of HeavyPeptide AQUAplex is determined
by amino acid analysis. The mixture of the peptide to be quantified
and HeavyPeptide AQUAplex as the internal standard yields two peaks
in low resolution mass spectroscopy: the two peaks have the same
elution time, but different masses. With high resolution MS, the
HeavyPeptide AQUAplex set will be resolved as distinct component
masses separated by the mass defect between isotopologues. Peptides
containing .sup.2H may exhibit a slight reduction in retention time
on reversed phase HPLC, but this does not affect quantitative
accuracy because it does not affect the AUC quantitation.
HeavyPeptide AQUAplex is spiked into the sample to be analyzed at a
known quantity, making it possible to use its quantity to calculate
the quantity of the peptide to be analyzed from the peak areas. At
low MS or MSn resolution, the method compares the AUC of the
corresponding combined MS peak from the heavy isotope labeled
peptide isotopologue set, with the peak of the non-labeled peptide
with the exact same sequence originating from the analyte (e.g.,
polymer, protein, peptide, or polypeptide) being quantified. At
high MS or MSn resolution, the method compares the area of the
corresponding MS peaks from each of the heavy isotope labeled
peptide isotopologues, with the peak of the non-labeled peptide
with the exact same sequence originating from the analyte (e.g.,
polymer, protein, peptide, or polypeptide) being quantified. The
quantitation precision is directly correlated to the accuracy of
the amount of the heavy peptide added to the sample.
[0131] The following example, while used specifically with a
protein analyte, illustrates the general method applicable for
analytes, whether protein or non-protein. A sample (e.g.,
biological sample, food sample) containing numerous proteins is
treated with a cleavage agent such as a protease (e.g., trypsin).
Trypsin cleaves at each R amino acid and K amino acid, yielding
numerous fragments, each fragment having about 13 amino acids
(range 6 amino acids to 20 amino acids). Into this
fragment-containing sample to be analyzed is introduced (spiked)
one, two, or three HeavyPeptide AQUAplex internal standards, and
quantitation is performed as described. In embodiments using
proteolytic digestion, the quantitation precision is also directly
correlated to the digestion predictability and efficiency.
[0132] In one embodiment, proteins contain one, two, or three
proteotypic peptide sequences, labeled as heavy or light
(HeavyPeptide AQUAplex). The samples to be analyzed are spiked with
the proteotypic peptides and quantitated by LC-MS/MS.
[0133] In AQUAplex IDMS, the internal standard isotopologue set has
the same sequence as the proteotypic peptide from the protein to be
quantified, but the internal standard is a mixture of heavy
isotopologues that have a different mass from the proteotypic
peptide. The sequence of the internal standard is thus
pre-determined by the protein sequence; it cannot be changed. The
AQUAplex internal standard must be quantified by amino acid
analysis to determine the total peptide concentration and by high
resolution MS to determine the relative proportion and thus
absolute concentration of the component peptide isotopologues.
Because the protein or polypeptide to be quantified differs with
each experiment, the internal standard for this protein or
polypeptide necessarily also differs with each experiment, and
requires that amino acid analysis be performed with each
experiment. Each quantitation requires a dilution curve that
typically encompasses six points, which may be accomplished in one
high resolution MS analysis through the use of AQUAplex internal
standard isotopologues mixed at a define ratio to provide a
standard curve. The costs for amino acid analysis are relatively
high and the procedure is time consuming. Each peptide sequence has
specific solubility, and its non-specific binding constant varies
based upon various factors that may differ with each analysis,
e.g., vessel material, buffer, temperature, etc. Such variability
decreases precision and reproducibility.
[0134] In contrast, with peptides as a non-limiting example, the
AQUAplex inventive method using a modified, optimized, labeled
universal reporter isotopologue mixture Uplex, and one analyte,
more than one analyte, or several concatenated analytes, increased
the analytical precision of quantitation where quality of internal
standards is decisive to ensure precise quantification. Only this
universal reporter Uplex undergoes amino acid analysis, rather than
an internal standard for each peptide to be quantified requiring
amino acid analysis. This universal reporter is a mixture of
isotopologues, permitting the full dilution curve to be acquired in
one MS acquisition. Universal reporter isotopologue Uplex
quantification thus need be performed only once, rather than with
each experiment, and can be used to quantify a reporter peptide
with a standard curve. The universal reporter Uplex can be stocked
and made readily available. In one embodiment, universal reporter
Uplex is labeled with a fluorophore and/or chromophore, and
universal reporter Uplex is quantified by measuring the absorbance
of the fluorophore and/or chromophore, and the relative proportion
of the isotopologues, and thus absolute concentration of the
isotopologues, is determined by high resolution MS. For peptide
analytes, no amino acid analysis is required. In one embodiment,
universal reporter peptide Uplex contains one tryptophan, and
universal reporter peptide Uplex is quantified by measuring
absorbance using the specific extinction factor of the tryptophan
to determine the total peptide concentration with high resolution
MS to determine the component isotopologue concentrations.
[0135] As shown in FIG. 12 using a peptide analyte, in one
embodiment peptide A*, the internal standard, is linked with a
reporter peptide R through a cleavable site (e.g., proteolytic
site) between A* and R. In this embodiment, each of peptide A* and
reporter peptide R contain at least one amino acid labeled with a
heavy isotopologues, known as heavy amino acids. When peptide A* is
labeled with an isotopologue set, the target analyte may be
quantified by AUC comparison to the A* internal standard in low MS
resolution or the target analyte may be quantified by AUC
comparison to the A* isotopologue set with high MS resolution.
Alternatively, when reporter peptide R is labeled with a heavy
isotope, reporter peptide R may be labeled with an isotopologue set
so that it can be quantified by comparison to a universal reporter
peptide U or universal reporter peptide isotopologue set Uplex.
Because the universal reporter peptide isotopologues Uplex must
have a different mass, universal reporter peptide Uplex can be
represented with two heavy amino acids or their isotopologues, and
reporter peptide R one heavy amino acid or its isotopologues. There
are other ways to obtain a difference in atomic mass; e.g., using
different heavy amino acids for reporter peptide R and universal
reporter peptide Uplex to obtain a difference in atomic mass, using
different isotopologues in reporter peptide R and universal
reporter peptide Uplex, or using multiple unique isotopologues in
reporter peptide Rplex and in universal reporter peptide Uplex. In
this manner, the concept of AQUAplex isotopologues may be applied
to improve quantitation of a target peptide with A* isotopologues,
to improve quantitation of reporter peptide R with Rplex
isotopologues, and/or to improve quantitation of reporter peptide R
with Uplex isotopologues.
[0136] Peptide A* has the same sequence as proteotypic peptide A,
but peptide A* has a different mass due to the presence of the
heavy amino acid or heavy amino acid isotopologues as peptide
A*plex. Universal reporter peptide Uplex is a peptide standard
isotopologue set for reporter peptide R. Universal reporter peptide
Uplex isotopologues are not the internal standard used to quantify
the protein or polypeptide. Universal reporter peptide Uplex has
the exact same sequence as reporter peptide R but has a different
atomic mass due to the incorporation of one or more heavy amino
acid isotopologues.
[0137] In the ligation between peptide A* and reporter peptide R,
resulting in a polypeptide, the reporter peptide R can be
C-terminal to A*, i.e., R A*, or the reporter peptide R can be
N-terminal to A*, i.e., A*-R. One or both of A* and R may be an
isotopologue set, A*plex and Rplex, that can be resolved and
quantified by high resolution MS. The nomenclature A'-R is used to
represent either the A*-R polypeptide or the R-A* polypeptide. In
either case when A* is a proteotypic peptide, there must be a
cleavable (e.g., proteolytic) site between peptide A* and reporter
peptide R in the resulting polypeptide.
[0138] The polypeptide isotopologue set A*plex-R is mixed with the
sample that contains the protein or polypeptide P to be quantified.
A known quantity of universal reporter peptide Uplex isotopologues
is added to the sample, i.e., universal reporter peptide Uplex is
spiked into the sample. The sample is digested with a protease
(e.g. trypsin) that cleaves the polypeptide bonds. As a result of
protease action, polypeptide A*-R must be fully digested. In one
embodiment, universal reporter peptide or Uplex is added before
cleavage (e.g., proteolytic digestion). In one embodiment,
universal reporter peptide or Uplex is added after cleavage (e.g.,
proteolytic digestion).
[0139] After digestion the concentration of peptide A*plex and
reporter peptide R in the sample is equimolar. That is, the
quantity of peptide A* is equal to the quantity of reporter peptide
R. Universal reporter peptide Uplex isotopologues are used to
quantify reporter peptide R using high resolution MS quantitation.
The standard curve of peptide A*plex is used to measure the
quantity of peptide A in the sample resulting from the proteolytic
digestion of protein or polypeptide P.
[0140] In the embodiment using a peptide shown in FIGS. 13 and 14,
the same method is applied to proteotypic peptides B and C from
protein P in order to increase the specificity of the quantitation.
Peptide B*plex has the same sequence as proteotypic peptide B but
has a different atomic mass due at low MS resolution and is a
dilution series of peptide B* at high MS resolution to the presence
of the heavy isotopologue labeled amino acids. Peptide C*plex has
the same sequence as proteotypic peptide C but has a different
atomic mass at low MS resolution and is a dilution series of
peptide B* at high MS resolution due to the presence of the heavy
isotopologue labeled amino acids.
[0141] Using a peptide embodiment as an example, A*plex-R includes
a cleavage site (e.g., proteolytic site) between isotopologue set
A*plex and R. Polypeptide isotopologues A*plex-R thus can be used
as a pseudo-surrogate of protein or polypeptide P to monitor
proteolytic digestion in a single experiment, digestion efficiency
among samples and experiments, and in some cases to normalize
results from different samples and/or different experiments.
[0142] In examples using peptides, reporter peptide R can be
optimized for proteolytic digestion. As one example, reporter
peptide R can be selected and/or modified so that it contains a
specific amino acid (e.g., tryptophan) that is easily quantified by
absorption measurements. As shown in FIG. 15, reporter peptide R
may contain more than one heavy isotope labeled amino acid or a
distinct isotopologue for a single heavy amino acid. This
embodiment increases the multiplexing possibilities of the method
by increasing the number of possible atomic masses for the same
reporter peptide R sequence, so that multiple peptides can be
quantified using universal reporter peptide U in a single
experiment.
[0143] In this multiplexing embodiment, reporter peptide R is
synthesized with different atomic masses, using standard methods
known in the art. As shown in FIG. 15, peptides A, B, and C from
protein or polypeptide P are quantified in a single experiment
using heavy peptides A*plex, B*plex, and C*plex, respectively, and
using reporter peptides R1, R2, R3. In the embodiment shown in FIG.
15, reporter peptides R1, R2, R3, and universal reporter peptide U,
have the same sequence but different atomic masses. To maximize the
number of mass combinations available for reporter peptide R, the
sequence may be composed of, but is not limited to, one or more of
the following amino acids: alanine, arginine, isoleucine, leucine,
lysine, phenylalanine, proline, and valine. These amino acids have
a mass shift .gtoreq.4 Da and can be synthesized with different
combinations of heavy isotopes to make isotopologues, such as in
FIGS. 6-9. The minimum mass difference between the proteotypic
peptide (e.g., A), and the internal standard (e.g., A*), should
exceed the sensitivity threshold determination for MS
differentiation. In one embodiment, the minimum mass difference
between the proteotypic peptide and the internal standard is 4 kDa
when 4 kDa is the minimum atomic mass difference that can be
discriminated. In one embodiment, the minimum mass difference
between the internal standard, reporter peptides, and universal
reporter isotopologues is 6 mDa when 6 mDa is the minimum atomic
mass difference that can be discriminated by high resolution MS.
The number of peptides that can be quantified simultaneously using
a universal heavy peptide U is limited only by the number of mass
difference combinations available within the sequence.
[0144] In another example using peptides, reporter peptide R may be
designed with a low hydrophobicity index, which will increase the
aqueous solubility of the polypeptide A*plex-R where peptide A*plex
is a set of isotopologues with a hydrophobicity index.gtoreq.40 or
where peptide A*plex is poorly soluble. One example of a reporter
peptide R having a sequence that renders it highly soluble is
PVVVPR (SEQ ID NO. 1); it has a hydrophobicity index of 13.45. One
example of a reporter peptide R having a sequence that renders it
highly soluble is SSAAPPPPPR (SEQ ID NO. 2) with a hydrophobicity
factor of 7.57. In one example, each of reporter peptide R and
universal reporter peptide Uplex isotopologues contains a
chromophore and/or fluorophore used for quantification by
absorbance measurement. In the embodiment where both universal
reporter peptide U and reporter peptide R include a chromophore
and/o fluorophore, universal reporter peptide Uplex isotopologues
can be quantified by measuring the absorption of the chromophore
and/or fluorophore, and not by amino acid analysis, and the
isotopologues may be quantified by high resolution MS. The process
of protein or polypeptide quantification by absorbance is more
robust than the process of amino acid analysis. Protein or
polypeptide quantification by absorbance is considered more precise
than protein or polypeptide quantification by amino acid analysis.
Examples of a chromophore or fluorophore and methods of assessing
their absorbance are known in the art.
[0145] In the embodiment shown in FIG. 14, the polypeptide contains
three prototypic peptides, each labeled with a heavy amino acid,
concatenated with a single reporter peptide R also labeled with a
heavy isotope amino acid, resulting in C*plex-B*plex-A*plex-R.
Using this polypeptide C*plex-B*plex-A*plex-R guarantees equimolar
quantities of each of peptides A*plex, B*plex and C*plex, and each
isotopologue set provides a standard curve for peptides A, B, and
C, and thus decreases quantitation variability compared to
quantitation using individual peptides. This embodiment increases
the number of peptides that can be quantified with the same
sequence as that of universal reporter peptide U.
[0146] In one embodiment, A*plex-R, or B*plex-A*plex-R, or
C*plex-B*plex-A*plex-R can be cleaved before being introduced into
the sample to be quantified.
[0147] In embodiments using proteotypic peptides, peptides A*plex,
B*plex, C*plex, and reporter peptide R can be randomly arranged, as
long as they are linked through a cleavage site (e.g., proteolytic
site).
[0148] The polypeptide shown in FIG. 14 contains three heavy
isotope labeled peptides (A*plex, B*plex, and C'plex),
corresponding to target peptides A, B, and C, linked to reporter
peptide R, also containing a heavy isotope label. Other embodiments
are possible where R does not contain a heavy isotope label or
contains one or more isotopologues that allow multiple distinct
reporter isotopologues to quantify multiple unique internal
standard peptides in a multiplexed assay. Other embodiments are
possible that contain various numbers (n) of labeled peptides and
their isotopologues corresponding to one or more target peptides,
joined with one or more reporter peptides and isotopologues. The
range for n is governed by, e.g., manufacturing feasibility,
solubility, etc. as known to one skilled in the art. In one
embodiment, a value of n up to 99 is possible. In one embodiment, a
value of n up to 49 is possible. In one embodiment, n=4. In one
embodiment, n=5. In one embodiment, n=6. In one embodiment, n=7. In
one embodiment, n=8. In one embodiment, n=9. In one embodiment,
n=10. In one embodiment, n=11. In one embodiment, n=12. With
isotopologues these limits increase dramatically; one can use a
combination of different heavy amino acids, isotopic shifts, and
isotolopologues.
[0149] Universal reporter peptide U and reporter peptide R can be
designed with different sequences and isotopologues for multiplex
quantitation. The number of mass difference combinations determined
by a peptide sequence is limited. When the number of peptides to be
quantified exceeds the maximum number of mass difference
combinations available for reporter peptide R, one can use
additional isotopologues of universal reporter peptide U, and one
can use additional sequences: e.g., U.sup.1, U.sup.2, . . . U.sup.n
where n is limited only by the number of peptides that can be
simultaneously quantified by an instrument. As one example, the
polypeptide A*-Rplex may have the amino acid sequence
TTVSKTETSQVAPA SEQ ID NO. 3, with peptide A* having the sequence
TETSQVAPA SEQ ID NO. 4, and reporter peptide Rplex having
resolvable lysine isotopologues from FIG. 6 of the sequence TTVSK
SEQ ID NO. 5, as disclosed in WO/2003/046148.
[0150] Because the sequence of universal reporter peptide Uplex
isotopologue set is not restricted or limited, and because
universal reporter peptide Uplex isotopologue set is a product that
can be readily ordered, stocked, maintained, and inventoried, its
use provides flexibility to MS peptide quantitation. In one
embodiment, the sequence of universal reporter peptide Uplex
isotopologue set can be customized to minimize non-specific binding
of the peptide, polypeptide, or protein to, e.g., a vessel, tips,
tubing, etc. by selecting a sequence with a low hydrophobicity
index, e.g., PVVVPR SEQ ID NO. 1 which has a hydrophobicity index
of 13.45 or SSAAPPPPPR (SEQ ID NO. 2), which has a hydrophobicity
index of 7.57, and using multiple arginine isotopologues or
isotopologues of other heavy amino acid precursors (FIGS. 9,
10a-b). In one embodiment, the sequence of universal reporter
peptide Uplex isotopologue set can be customized to maximize
solubility of the polypeptide A*plex-R. For example, because
universal reporter peptide U is used, the polypeptide A*plex-R need
not be quantified precisely prior to MS analysis, and the use of
isotopologues premixed at a defined ration in the synthesis
provides multiple data points across a dilution curve for improved
quantitation. This results in shorter manufacturing time and lower
cost in producing polypeptide A*plex-R. Peptide A*plex is
quantified at very low concentration, at which its solubility is
guaranteed, resulting in enhanced precision and repeatability.
[0151] Because quantitation of peptide A*plex is performed on the
same instrument used fir the quantitation of reporter peptide R and
within the same MS procedure, it always reflects the quantity added
into the sample and is independent of eventual alteration,
degradation, and partial loss of polypeptide A*plex-R during sample
preparation, fractionation, and liquid chromatography separation
prior to MS quantitation. When extending the method described in WO
03/016861, the A*plex isotopologue set is provided in a known
concentration that is too high for use without dilution; thus, it
is typically diluted 1000 to 10,000. If the sequence of A*plex is
relatively hydrophobic and prone to non-specific binding, as is the
case for 3-amyloid peptides, a significant amount of the standard
will be lost during dilution. This decreases the method's
precision. Because the sequence of universal reporter peptide Uplex
isotopologue set can be designed and optimized to decrease
non-specific binding, the dilution of universal reporter peptide U
is not prone to significant non-specific binding. Universal
reporter peptide Uplex isotopologue set is included in the sample
to be quantified, and quantitation of reporter peptide R is
performed in the diluted sample, thus non-specific binding of the
standard (e.g., .beta.-amyloid peptide) will not decrease the
method's precision.
[0152] The polypeptide A*plex R is a pseudo-surrogate of protein P
and can be used to monitor cleavage (e.g., proteolytic digestion).
It can be used to quantify and compare sample-to-sample, and/or
experiment-to-experiment, digestion efficiency, as the dilution
curve provided by a defined isotopologue set provides accurate
quantitation across a broad dynamic range. It can be used to
normalize results from sample-to-sample, and/or from
experiment-to-experiment.
[0153] In one embodiment, the inventive method is adapted to MS
quantitation of analytes, including but not limited to peptides,
polypeptides, and proteins, using a proteotypic peptide that is
coupled, through a cleavable site, to a reporter peptide R or other
moiety. This is shown schematically in FIG. 16 for any analyte, and
in FIG. 17 for a peptide analyte. The heavy proteotypic peptide
isotopologue set contains the same amino acid sequence, but
different atomic masses, as the native proteotypic peptide. Each
heavy proteotypic peptide isotopologue in the set is in equimolar
concentration with the reporter peptide R. In one embodiment,
reporter peptide R is labeled with a heavy isotope or set of
isotopologues. In one embodiment, reporter peptide R is not labeled
with a heavy isotope. Universal reporter peptide U has the same
sequence as reporter peptide R. Universal reporter peptide U has a
different mass than reporter peptide R because it contains a heavy
set of isotopologues. Only universal reporter peptide Uplex is
quantified. After cleavage (e.g., proteolytic digestion), the heavy
proteotypic peptide or isotopologue set and the reporter peptide R
are released at equimolar concentration into the sample. The
quantity of the reporter peptide R is determined using the quantity
of heavy universal reporter peptide Uplex isotopologue set.
[0154] Universal reporter peptide Uplex isotopologue set is
sequence independent and is used as a quantitation standard and a
cleavage standard. Universal reporter peptide Uplex isotopologue
set has a peptide sequence that is identical to reporter peptide R
but is independent from the protein to be assayed. Because the
sequence of universal reporter peptide Uplex isotopologue set and
reporter peptide R is identical, the atomic mass difference between
universal reporter peptide Uplex isotopologue set and reporter
peptide R is obtained using a heavy labeled reporter peptide R and
a heavy labeled universal reporter peptide Uplex isotopologue set.
The atomic mass difference is obtained by using different heavy
labels in reporter peptide R and universal reporter peptide Uplex
isotopologue set, or by using an additional heavy amino acid in
reporter peptide R or universal reporter peptide Uplex isotopologue
set (FIG. 15). Reporter peptide R may have a lower atomic mass or a
higher atomic mass than universal reporter peptide Uplex
isotopologue set.
[0155] As represented in FIG. 18, a convenient convention for
naming components is as follows: proteotypic peptides are named as
letters, e.g., A, B, C; heavy isotope labeled proteotypic peptides
are named as letters with an asterisk indicating a heavy isotope
label and the "plex" suffix to reflect the use of multiple
isotopologues, e.g., A*plex, B*plex, C*plex; R is a reporter; Uplex
is a universal reporter isotopologue set; the amino acid bearing
the heavy isotope label is indicated by a degree symbol and either
conventional amino acid one- or three-letter naming in bold font,
e.g., either R or Arg indicates the amino acid arginine with a
heavy isotope label; the amino acid bearing the set of heavy
isotopologue labels is indicated by an asterisk symbol "*" and
either conventional amino acid one- or three-letter naming in bold
font, e.g., either R* or Arg* indicates the amino acid arginine
with one or more heavy isotopologue labels; one composition of
concatenated peptides and universal reporter, commercially
available under the trademark HeavyPeptide IGNIS.TM., is
A*plexB*plexC*plexR.
[0156] In one embodiment, the sequence of universal reporter
peptide Uplex isotopologue set is optimized and/or customized to be
compatible with the properties of the proteotypic peptide by
optimizing chromatographic ionization and fragmentation properties.
As one example, universal reporter peptide Uplex isotopologue set
is modified to enhance ionization and/or desolvation by introducing
additional charge or hydrophobic properties. As one example,
universal reporter peptide Uplex isotopologue set is modified to
enhance fragmentation by introducing an aspartate-proline (DP)
group that contains a highly scissile bond that fragments in tandem
MS at lower collisions energies than other dipeptide linkages. As
one example, universal reporter peptide Uplex isotopologue set is
modified to have a similar retention time on liquid chromatography
as the proteotypic peptide by choosing a reporter peptide with a
similar hydrophobicity factor to the proteotypic peptide. Thus,
universal reporter peptide Uplex isotopologue set can be optimized
by design. For example, the number of mass combinations and
isotopologues for the identical peptide sequence can be optimized
to increase the multiplexing capacity, yielding up to 500 proteins
capable of being quantified in a single assay. Yet because the
peptide sequences are identical, the full set of isotopologues can
be resolved with high MS resolution at MS1 or MSn levels, and the
peptide sequence can be verified by MS/MS or MSn fragmentation at
low MS resolution, only one dilution curve is required to quantify
universal reporter peptide U. By increasing the number of identical
sequences with different masses and isotopologues, the number of
proteins that can be quantified in a single experiment increases,
without concomitant increase in instrumentation use and
resources.
[0157] In one embodiment, the universal reporter peptide Uplex
isotopologue set was optimized for low specific binding, high
solubility, high MS signal intensity, and/or desired liquid
chromatography retention time. In one embodiment, the universal
reported peptide Uplex isotopologue set peptide sequence was
modified to change its chromatographic retention properties; this
is one example of internal modification. In one embodiment, the
universal reporter peptide Uplex isotopologue set structure was
modified by attaching tags to change its chromatographic retention
properties; this is one example of external modification. In one
embodiment, the universal reporter peptide Uplex isotopologue set
structure was modified by attaching tags that themselves had been
modified to change its chromatographic retention properties; this
is another example of external modification.
[0158] In one embodiment, a universal isotopologue polymer set is
used, where polymer is broadly defined as a joined group of
monomers. The monomers either need not be peptides, or need not be
entirely peptides, and may contain one or more isotopes. In one
embodiment, polysaccharides (i.e., glycan monomers) are used as
universal polymers (U.sup.polymer). A polysaccharide is a
combination of two or more monosaccharides linked by glycosidic
bonds, and the polysaccharide is synthesized from a set of monomers
such as to produce an isotopomer set. Examples of such
polysaccharides include isotopologues of starch, cellulose, and
glycogen. Their structures and synthesis are known in the art. In
one embodiment, deoxyribonucleic acid (DNA) or ribonucleic acid
(RNA) isotopologue sets are used as universal polymers
(U.sup.polymer). Their structures and synthesis are known in the
art. Methods to detect and quantify nucleotides are well
established, e.g.; PCR, quantitative PCR. Nucleotides attached to
an analyte can be used as a unique identifier (i.e., "barcode") of
the analyte and for quantitation purposes using high resolution MS
with isotopic dilution of the isotopologue set.
[0159] In one example, isotopologues of one of the following
peptide sequences shown in the table below, currently used as
retention time calibrator peptides, were used as a universal
reporter peptide Uplex isotopologue set. These peptides exhibited
sufficient ionization and had defined elution properties. The
following table below shows their sequence, hydrophobicity, and
chromatograph behavior on a Hypersil Gold C.sub.18 column.
TABLE-US-00001 SEQ ID Hydrophobicity Retention Peptide No. Factor
Time (min) SSAAPPPPPR 2 7.57 4.77 GISNEGQNASIK 6 15.50 6.62
HVLTSIGEK 7 15.52 7.22 DIPVPKPK 8 17.65 7.67 IGDYAGIK 9 19.15 8.18
TASEFDSAIAQDK 10 25.88 9.01 SAAGAFGPELSR 11 25.24 9.41
ELGQSGVDTYLQTK 12 28.37 9.63 SFANQPLEVVYSK 13 34.96 10.67
GLILVGGYGTR 14 32.18 10.79 GILFVGSGVSGGEEGAR 15 34.52 10.86
LTILEELR 16 37.30 11.87 NGFILDGFPR 17 40.42 12.16 ELASGLSFPVGFK 18
41.19 12.21 LSSEAPALFQFDLK 19 46.66 12.85
[0160] Hydrophobicity was determined using calculations done with
algorithms described in Spicer. et al (2007). Sequence-specific
retention calculator. A family of peptide retention time prediction
algorithms in reversed-phase HPLC: applicability to various
chromatographic conditions and columns. Anal Chem.
79(22):8762-8.
[0161] In one embodiment, the heavy isotopomeric label is
incorporated in the C-terminal amino acid. For example, using the
peptide SSAAPPPPPR SEQ ID NO. 2, this embodiment can be represented
as SSAAPPPPPR*, where the terminal R contains the heavy isotope
label incorporated as a set of isotopologues resolvable with high
resolution MS.
[0162] In one embodiment, the heavy isotope is incorporated in the
peptide at a position other than the C-terminus. One or more of the
following amino acids may be labeled with a heavy isotopologue set:
alanine, arginine, isoleucine, leucine, lysine, phenylalanine,
valine (FIG. 8). These amino acids have a mass shift >4 Da.
Additionally, multiple amino acids within the peptide can be
labeled with isotopologue sets, and the same amino acid may be
labeled with different isotopes, such as arginine +6 Da and
arginine +10 Da isotopologue sets which would introduce a 6 Da and
10 Da mass shift, respectively at low MS resolution, and with
additive multiplexing capability at high resolution MS (FIGS.
7a-b). For example, using the peptide SSAAPPPPPR SEQ ID NO. 2 where
* indicates the amino acid containing the position of the heavy
isotope label, the following positions are possible: SSA*APPPPPR,
SSA*A*PPPPPR, SSA*A*P*PPPPR, SSA*A*P*P*PPPR, SSA*A*P*P*P*PPR,
SSA*A*P*P*P*P*PR, SSA*A*P*P*P*P*P*R, SSA*A*P*P*P*P*P*R*. This
embodiment, where the heavy isotopologue amino acid is located at
one or more positions, permits higher multiplexing with the same
reporter sequence.
[0163] For multiplexed assays, custom peptides are combined
together into complex targeted assays. Each custom peptide has a
different corresponding universal reporter Uplex isotopologue set
that elutes similarly to the custom peptide. This permits many
peptides to be easily multiplexed and quantified across an LC
gradient without cross contamination. For example, a multiplex
analysis array contains any number of different universal peptides
Uplex isotopologue sets having the same amino acid sequence, but a
different atomic mass due to the presence of a heavy isotopologue
or isotopologue set, and a number of reporter peptides R, each
reporter peptide R cleavably linked to a different isotopically
labeled proteotypic peptide to be quantified in a sample. The
universal peptide Uplex isotopologue sets have substantially
similar chromatography retention time as the custom peptide. In one
embodiment, the heavy isotopologue labels in the universal reporter
peptide Uplex isotopologue set is incorporated at multiple
different amino acids. This embodiment permits higher multiplex
arrays using the same universal reporter peptide Uplex amino acid
sequence.
[0164] In one embodiment, the universal reporter peptide Uplex
isotopologue set is customized for a specific mass spectrometer
and/or specific use for identification, characterization, and
quantitation of disease biomarkers (proteomics, metabolomics,
pharmacoproteomics) discovery, confirmation, validation, and early
clinical diagnosis and disease progression monitoring. For example,
a 4-10 amino acid reporter peptide will allow incorporation of
fewer isotopologue precursors and lower multiplexing capability
with high resolution MS. Alternatively, a larger 11-25 amino acid
peptide could allow incorporation of more isotopologue precursors
and lower multiplexing capability with a high resolution mass
spectrometer. Proteomics has advanced from identification
(qualitative proteomics) to quantitation by incorporating an
internal standard in the assay. An internal standard is required
because the resulting peak height or peak surface in mass
spectroscopy results from a complex function of parameters (e.g.,
peptide quantity, peptide ionization, peptide fragmentation, ion
suppression, etc.). There is no algorithm to measure the quantity
of a peptide from the surface of the mass spectroscopy peak. When a
known quantity of the internal standard is added to the peptide to
be analyzed, the quantity of the peptide is determined by comparing
its peak surface with the internal standard peak surface.
[0165] One embodiment is a described peptide modified with a tag.
Such a tag, used to modify the peptide, differs from the heavy
isotope label that is required or is optional to modify universal
reporter U and reporter R, respectively. The tag, however, may be a
heavy isotopologue set, as subsequently described in the first
example.
[0166] One example of such a tag is a heavy isotopologue set. One
example of such a tag is an isotopologue tag. Such tags include
forms of the same chemical structure with each tag having a unique
isotopologue that can be resolved with high resolution MS.
[0167] One example of such a tag is a different isotopologue of the
same peptide. This example uses as a tag an element that naturally
has multiple isotopes, and where the isotopes have a unique mass
defect.
[0168] Use of a mass defect tag shifts the reporter peptide to a
region of the mass chromatogram in which most isotopes are not
observed, sometimes referred to as a mass quiet space. This example
is useful to enhance sensitivity and specificity of detection in a
mass region with many other background ions.
[0169] Stable isotope labeling with amino acids in cell culture
(SILAC) and its variations, known to one skilled in the art, uses
mass spectrometry to quantitate and compare proteins among samples,
and sample normalization and measurement of biological variation
with structural proteins, chaperones, or housekeeping enzymes
allows large numbers of samples to be processed and compared.
Isobaric labeling using either tandem mass tags (TMT) or isobaric
tags for relative and absolute quantitation (iTRAQ) uses mass
spectrometry to quantitate and compare proteins for quantitation of
peptides from proteins in cell and tissue lysates, serum and
plasma, and formalin-fixed paraffin embedded tissue slices. TMT and
iTRAQ have the general structure M-F-N-R where M=mass reporter
region, F=cleavable linker region, N=mass normalization region, and
R=protein reactive group. Isotopes substituted at various positions
in M and N cause each tag to have a different molecular mass in the
M region with a corresponding mass change in the N region, so that
the set of tags have the same overall molecular weight. Only when
the TMT undergo a second or third fragmentation (such as in tandem
mass spectrometry MS/MS, or triple mass spectroscopy MS/MS/MS) are
they distinguishable, with backbone fragmentation yielding sequence
and tag fragmentation yielding mass reporter ions needed to
quantitate the peptides. iTRAQ and TMT covalently label amine
groups in protein digests and a cysteine reactive TMT labels thiols
of cysteines, resulting in individual digests with unique mass
tags. The labeled digests are then pooled and fragmented into
peptide backbone and reporter ions. The peptide backbone ions are
used to identify the protein from which they came. The reporter
ions are used to quantify this protein in each of the combined
samples.
[0170] Label-free SILAC, TMT, iTRAQ, and other mass spectroscopy
methods known to one skilled in the art of protein quantitation are
used in biomarker discovery to generate candidate markers on
instrumentation that includes LTQ Velos (Thermo Scientific), LTQ
Orbitrap Elite (Thermo Scientific), and Q Exactive (Thermo
Scientific) hybrid mass spectrometers. The candidate markers are
then further evaluated and applied in targeted analysis assays
using selected reaction monitoring (SRM) or selected ion monitoring
(SIM) to target quantitation of peptide markers in many samples.
For confirmation and validation, the universal reporter peptide U
is customized for use, as explained below, with the markers that
were previously identified, for absolute quantitation with
synthetic stable-isotope-labeled peptide standards (HeavyPeptide
AQUAplex and its variations, Thermo Scientific) using existing
discovery data to automate the preliminary selection for targeted
analysis (Pinpoint software (Thermo Scientific); TSQ Vantage triple
stage quadrupole mass spectrometer (Thermo Scientific)). The data
are entered into an integrated data management system for clinical
applications.
[0171] One embodiment is a universal reporter peptide Uplex
isotopologue set synthesized to provide it with similar (e.g.,
.+-.10% to 20%) properties (e.g., retention time, ionization,
optimal fragmentation energy, limit of detection, digestion
efficiency, etc.) to a custom peptide. In a method using this
embodiment, the universal reporter peptide Uplex isotopologue set
is used to assess digestion efficiency. In use, this embodiment
permits one to assess the proteotypic peptide and both the
undigested and digested custom peptide and the universal reporter
peptide Uplex isotopologue set. The efficiency of digestion of the
custom and universal peptide to the individual peptides is then
used to correct the level of proteotypic peptide quantified,
allowing more accurate absolute quantitation of the protein of
interest and more accurate quantification between samples by
correcting for digest efficiency between samples.
[0172] One embodiment is a set of universal peptides Uplex
isotopologue set. This set of universal peptides Uplex isotopologue
set co-elutes in a predictable manner. The peptides in the set may
or may not share a common sequence. The peptides in the set have
stable isotopologue sets incorporated at unique positions to enable
specific quantitation of each.
[0173] One embodiment is a universal isotopologue peptide set
compound, composition, formulation, and/or kit. In one embodiment,
the heavy isotopologue proteotypic peptide set-reporter peptide R
can be formulated dry. In one embodiment, the heavy isotopologue
proteotypic peptide set-reporter peptide R can be formulated in
solution. The heavy isotopologue proteotypic peptide set-reporter
peptide R is stabilized and solubilization is facilitated by
formulating it with a non-reducing sugar (e.g., sorbitol, mannitol,
etc.), using methods known to one skilled in the art. In this form,
it is stable at attomole or femtomole quantities. In one
embodiment, the heavy isotopologue proteotypic peptide set-reporter
peptide R is formulated as a tablet that could be transported and
stored at ambient temperatures and would be easily transferred to
vials with the need for liquid measurement. This format eliminates
concerns about peptide binding nonspecifically to a tube wall or
solvent evaporation resulting in changes in peptide concentration.
This embodiment reduces the number of manipulations required and,
hence, decreases error. This embodiment facilitates automation.
EXAMPLE 1
[0174] Stable isotope labeled peptides containing a universal
reporter peptide Uplex isotopologue set at a predetermined ratio of
isotopologues and several peptides concatenated together with a
reporter peptide are applied to detect and quantify protein
biomarkers in clinical samples, with a focus on markers of lung
cancer.
[0175] To assess the recovery of the sample preparation method,
heavy isotopologue labeled synthetic polypeptide standards
(comprising up to three proteotypic peptides and a universal
reporter R) of human plasma proteins (LDH, NSE, and Myo) are spiked
in samples before and after proteolysis. HPLC-MS analyses are
performed on a hybrid mass spectrometer instrument (Q Exactive,
ThermoFisher Scientific) in SIM mode. A set of concatenated
reference peptides is synthesized based on a list of candidates
previously identified. Synthetic polypeptides are obtained from
ThermoFisher Scientific (Ulm Germany).
[0176] The reporter peptide R is designed with a tryptic cleavage
site at the C-terminus. The isotopologue calibration curve
standards for the universal reporter Uplex peptides are established
during peptide synthesis through the use of a mixture of the
isotopologue amino acid precursors at a pre-defined ratio. The
relative response factor of each targeted analyte peptide compared
to the reporter is readily determined after trypsin treatment by
exploiting the 1:1 stoichiometry of the reporter:targeted analyte
concatenated peptide.
[0177] A panel of proteins indicative of lung cancer is selected to
demonstrate proof-of-principle. For precise quantification of
specific proteins, three synthetic concatenated proteotypic
polypeptide isotopologue mixtures, R:A*plex, R:B*plex, and
R:C*plex, are generated and analyzed. Plasma samples from lung
cancer patients and controls are analyzed.
[0178] The concatenated synthetic polypeptides containing a
universal reporter enables precise determination of the amounts of
targeted proteins present in the sample using concomitantly
multiple reference peptides. The absolute amount of the reporter
peptide (R) released during proteolysis is determined with high
resolution MS by comparing the AUC to that of the universal
reporter internal standard Uplex. The absolute amount of the target
analyte peptides A, B, C in the sample is determined with high
resolution MS by comparing the AUC of each target peptide to that
of the isotopologue internal standard peptide A*plex, B*plex, and
C*plex, respectively. This quantification approach is readily
implemented in a large scale targeted proteomics workflow.
EXAMPLE 2
[0179] Stable isotopologue labeled peptides containing a universal
reporter peptide R and several concatenated targeted analyte
peptides are used to detect and quantify protein biomarkers in
clinical samples, with a focus on markers of bladder cancer.
[0180] Exogenous proteins from yeast (Saccharomyces cerevisiae)
(ADH, enolase, and carboxypeptidase) and human (LDH, NSE, Myo) are
added as internal standards in urine samples. The isotopologue
labeled synthetic polypepticle standards, which are proteotypic
peptides of the protein of interest concatenated with a universal
reporter peptide R, are spiked before proteolysis. Urine samples
are prepared by protein precipitation, reduction/alkylation,
trypsin proteolysis, and desalting using C18 cartridges. A second
set of isotopically labeled synthetic peptides is added after
proteolysis.
[0181] LC-MS/MS analyses are performed on RP-HPLC (Dionex) coupled
with a hybrid high resolution MS instrument (Q Exactive,
ThermoFisher Scientific) operated in multiplexed selected ion
monitoring (mxSIM) mode. Synthetic polypeptide heavy isotopologues
are obtained from ThermoFisher Scientific (Ulm Germany).
[0182] To establish the methodology, stable isotope-labeled
dipeptides containing targeted peptide heavy isotopologues and a
universal reporter R are synthesized. The reporter peptide R is
designed with a tryptic cleavage site at the C-terminus. The
calibration curves for the universal reporter peptides Uplex are
established using the predetermined mixture of amino acid precursor
isotopologues in the peptide synthesis. In parallel, the relative
response factor of each peptide compared to the reporter is
determined after trypsin treatment exploiting the 1:1
stoichiometry. LC-MS analyses are performed in multiplexed SIM mode
(mxSIM).
[0183] To evaluate the methodology, precise quantities of reference
polypeptides are spiked into the urine samples, digested with
trypsin, and analyzed by LC-mxSIM to quantify the targeted human
proteins. Preliminary results include analysis of insulin-like
growth factor binding protein 7 in urine samples using two
individual synthetic stable isotope-labeled peptide isotopologue
sets with a universal reporter R: reporter-HEVTGWVLVSPLSK* (SEQ ID
NO. 20) and reporter-ITVVDALHEIPVK* (SEQ ID NO. 21, isotopologue
residue). Dilution curves are generated in pooled urine samples to
precisely determine the quantity of corresponding protein.
EXAMPLE 3
[0184] To further evidence the method's utility, large synthetic
polypeptides are produced, resulting from concatenation of multiple
peptides representing each of the proteins of interest.
[0185] Three proteotypic peptides per protein with adequate mass
spectrometric properties (precursor m/z, ionization efficiency,
retention time, MS/MS spectra) are selected to construct the
concatenated standards. These reference polypeptides, all
containing a universal reporter, allow measurement of the precise
amount of multiple reference peptides in one LC-MS run.
EXAMPLE 4
[0186] The inventive method decreases peptide synthesis cost and
mass spectrometry usage to generate a calibration curve, resulting
in savings in instrument usage, operator time, and processing
efficiency. To further reduce the cost of peptide synthesis, one
internal standard peptide is synthesized with no heavy isotope, and
this peptide is labeled with a set of six amine-reactive
triazine-based mass tag reagent isotopologues premixed at two-fold
step dilution ratios (e.g. 4:2:1:0.5:0.25:0.0125). In preparing a
calibration curve, these different tagged peptide concentrations
are prepared in one labeling step with the pre-mixed isotopologue
reagents and resolved in the LC-MS system with high resolution MS.
A typical calibration curve requires six peptide injections at
different peptide concentrations, but the disclosed AQUAplex tag
approach described here reduces this to one injection. Each
injection is performed in triplicate (three replicates). The total
LC-MS analysis to generate a calibration curve in triplicate is
thus at least three analyses.
[0187] The universal reporter peptide X:LVALVR (SEQ ID NO. 22),
where X is a proteotypic peptide of the target protein of interest
and LVALVR (SEQ ID NO. 22) is a universal reporter peptide, is
synthesized and labeled at the amino terminus with a heavy
isotopologue triazine MS tag set mixed at a pre-defined ratio.
Similarly, a known concentration of universal reporter internal
standard peptide U is labeled at the amino terminus with the heavy
isotopologue triazine MS tag set mixed at a pre-defined ratio. The
labeled universal reporter peptide and universal reporter internal
standard peptide from above are spiked into the target analyte
sample, this mixture is digested with trypsin, and the resulting
peptides are labeled with an inexpensive amine-reactive triazine
tag that contained no heavy isotopes ("light" tag, FIG. 19). The
final digested and labeled mixtures containing the light
tag-labeled target analyte, universal reporter peptide (now cleaved
into the isotopologue labeled target internal standard and light
tag-labeled reporter peptide), and the universal reporter internal
standard are injected into the LC-MS system. Three replicate
injections are sufficient to generate a complete calibration curve
and quantify the target analyte. This method thus further decreases
the time needed to generate the calibration curve and includes a
universal reporter peptide that served both as a digestion and
labeling control.
EXAMPLE 5
Isotopically-Labeled Proteins as Internal Standards
[0188] The inventive method includes synthesis of sets of heavy
protein internal standards using premixed sets of precursor heavy
amino acid isotopologues. A recombinant human Akt1 protein kinase
was expressed with the Pierce Heavy Protein IVT Kit (Thermo
Scientific, Product #88330). For this heavy .sup.13C.sub.8
.sup.15N.sub.2 lysine in the kit was supplemented with heavy
.sup.2H.sub.8-leucine at a 1:4 ratio to synthesize the heavy
protein isotopologue set (FIGS. 21a-c). Native Akt1 is in the HeLa
cell lysate used for the IVT expression system, while heavy Akt1
isotopologues were made according to instructions. Both native and
heavy Akt1 were immune-enriched with an anti-Akt antibody and
Protein A/G-coated magnetic beads. The immune-enriched Akt sample
was reduced, alkylated, digested with trypsin, and then spiked with
100-1000 fmol of the doubly labeled AKT1 AQUA heavy peptide
SL(L)SGLL(K), where the bracketed amino acids were .sup.13C.sub.6
.sup.15N-leucine and .sup.13C.sub.6 .sup.15N.sub.2-lysine,
respectively (FIG. 22). These sample were desalted and analyzed by
LC-MS/MS on a 15 cm long, 75 .mu.m inside diameter Dionex Pepmap
C18 column connected to a Thermo Scientific Orbitrap XL instrument.
For the MS analysis, both 15K and 100K MS resolution settings were
used to quantify a list of targeted Akt peptides. The resulting MS
spectral peaks were analyzed with Thermo Scientific Xcaliber
software to determine the concentration of native and heavy Akt1
protein by relative quantitation, using the SL(L)SGLL(K) heavy
peptide internal standard for absolute quantitation (FIG. 23). Both
normal resolution (15K) and high resolution (100,000K) MS were used
to resolve the heavy peptide isotopomers to quantify Akt1-specific
peptides as well as Akt peptides that are conserved in Akt2 and
Akt3 isoforms. The ratios of light and heavy peptides were further
verified with low resolution on a Thermo Scientific TSQ Vantage
triple quadrupole mass spectrometer. This method enabled the
relative quantitation of all peptides of a protein and its isoforms
with the corresponding heavy protein isotopologues, and it enabled
absolute quantitation using at least one AQUA peptide internal
standard with a unique resolvable mass.
EXAMPLE 6
Isotopically-Labeled Peptides as Internal Standards
[0189] Table 1 lists targeted human urine and yeast proteins, and
three selected proteotypic peptides to design HeavyPeptide
IGNIS.TM. AQUAplex peptides. As shown in Table 1, ten HeavyPeptide
IGNIS.TM. AQUAplex peptides are designed that corresponded to 30
proteotypic peptides of nine proteins (seven human proteins, three
yeast proteins). Stable isotope-labeled amino acid isotopologue
sets of each HeavyPeptide IGNIS.TM. AQUAplex peptides (purity
>95%, lyophilized in sorbitol) are selected to have a mass shift
and mass defects sufficient for MS analysis, with the endogenous
peptides and the corresponding individual synthetic stable isotope
labeled peptides with C-terminal arginine or lysine isotopologues
premixed at a defined ratio (purity, lyophilized, >99 atom %
isotopic enrichment); Table 2 lists the full sequence of the
HeavyPeptide IGNIS.TM. AQUAplex peptides identifying the stable
isotope amino acid heavy isotopologues.
[0190] The proteotypic peptides are selected from proteomics
shotgun experiments. The reported number of observations is used as
a surrogate indicator for the abundance of proteins in a specific
proteome. The uniqueness of the peptide reporter is verified by
blasting the amino acid sequences, LVALVR (SEQ ID NO. 22) and
LVALVK (SEQ ID NO. 26), against the UniProt database; these
sequences are not associated with a protein.
Calibration Curve of the Peptide Reporter
[0191] The calibration curve is performed by mixing the universal
reporter peptide Uplex solution (purity >97%) with various
isotope label (R for heavy arginine +10 Da using the boxed
isotopologues in FIG. 7b) represented by Arg+10 Da mass defects of
+0 mDa (left-most box), +6 m Da, +12 m Da, +18 mDa, +24 mDa, +30
mDa, +36 mDa, and +42 mDa. Five .mu.L of LVALVR+.sup.0mDa (0.5
fmol/.mu.L) (SEQ ID NO. 22), 15 .mu.L LVALVR.sup.+6mDa (0.5
fmol/.mu.L) (SEQ ID NO. 22), 4.5 .mu.L LVALVR.sup.+12mDa (5
fmol/.mu.L), 13.5 .mu.L LVALVR.sup.+18mDa (5 fmol/.mu.L) (SEQ ID
NO. 22), 40.5 .mu.L LVALVR.sup.+24mDa (5 fmol/.mu.L) (SEQ ID NO.
22), 12.2 .mu.L LVALVR.sup.+30mDa (50 fmol/.mu.L) (SEQ ID NO. 22),
36.5 .mu.L LVALVR.sup.+36mDa (50 fmol/.mu.L) (SEQ ID NO. 22), 109.4
.mu.L LVALVR.sup.+48mDa (50 fmol/.mu.L) (SEQ ID NO. 22), and 13.6
.mu.L of 0.1% (v/v) formic acid (in water) are mixed to obtain a
final volume of 250 .mu.L. Concentrations of these peptides in
solution are 10.0 atmol/.mu.L, 30.0 atmol/.mu.L, 90.0 atmol/.mu.L,
270.0 atmol/.mu.L, 810.0 atmol/.mu.L, 2.4 fmol/.mu.L, 7.3
fmol/.mu.L and 21.9 fmol/.mu.L, respectively. The theoretical MS1
peaks for this series of eight isotopologues of LVALVR+10 Da (SEQ
ID NO. 22) shows that even the z=+2 charge state of this peptide
set is resolved at an MS resolution of 240,000 on a Q Exactive MS
platform (FIG. 20). Further, the high intensity of the composite
low resolution MS peak and the unique isotopologue mass defect peak
series with high MS resolution is used to verify the internal
standard and to provide exact mass information in the relevant MS
region. The high resolution and accurate masses of the isotopologue
series are then used to calculate the mass offsets for the specific
target analyte with sub-ppm mass accuracy, allowing real-time mass
calibration data and verification of the specificity of the
quantitation of the target analyte. Analysis of the calibration
curve is performed in triplicate by one LC-SIM run on the Q
Exactive-platform. All of these isotopologue peaks are monitored
for the reporter peptide.
Proteolysis of HeavyPeptide IGNIS.TM. AQUAplex
[0192] Each HeavyPeptide IGNIS.TM. AQUAplex isotopologue peptide
mixture is solubilized with acetonitrile (ACN)/water (15/85)
(vol/vol) to obtain a final protein concentration of 5 pmol/.mu.L,
and then sonicated for 20 minutes. HeavyPeptide IGNIS.TM. AQUAplex
are individually digested by trypsin 1:20 (w/w) (Thermo Scientific,
Rockford Ill.) for 3.5 hr at 38.degree. C. under agitation (1400
rpm). The kinetic digestion was monitored by reaction mixture
extraction every 15 minutes. To stop the digestion, all samples are
diluted in 0.1% v/v formic acid to obtain a final peptide
concentration of 50 fmol/.mu.L for analysis on LC-MS (in SIM
mode).
[0193] For quantitative measurements on a Thermo Scientific Q
Exactive platform, all HeavyPeptide IGNIS.TM. AQUAplex digestion
kinetic points are stoichiometrically supplemented with the
corresponding synthetic stable isotope-labeled and/or stable
isotopologue-labeled peptides and the universal reporter peptide
U=LVALVR (SEQ ID NO. 22). The two most intense MS1 charge state
ions observed from the SIM assay are monitored for all
peptides.
Urine Collection and Sample Treatment
[0194] Spot midstream urine samples are collected from ten
non-smoking healthy volunteers, five females and five males, age
range 30-40 years. There is no history of renal dysfunction in any
of the subjects or drug administration during the sample
collection. Urine is centrifuged at 1 000 g relative centrifuge
force (rcf) per 20 minutes at room temperature (about 19.degree.
C.-22.degree. C.). The supernatants 1 000 g are pooled together,
portioned into aliquots in 50 Falcon.TM. tubes and stored at
-80.degree. C.
[0195] The amount of urinary protein is estimated by a pyrogallol
assay. Samples corresponding to 250 .mu.g of urinary protein are
precipitated with 100% stock solutions of acetonitrile (for HPLC)
at a ratio 1:5 (v/v). Samples are incubated at room temperature
overnight. After precipitation, urine samples are centrifuged at
14,000 g for 30 minutes at 4.degree. C. The pellet is washed once
with the acetonitrile, air-dried, and resuspended with 250 .mu.L 8
M urea and 0.1 M ammonium bicarbonate. The samples are reduced with
20 mM dithiothreitol in 50 mM ammonium bicarbonate at 37.degree.
C., centrifuged at 800 rpm for 30 minutes, then alkylated with 80
mM iodoacetamide in 50 mM ammonium bicarbonate at 37.degree. C. and
centrifuged at 800 rpm for 30 min. Volume samples are adjusted at 2
M urea with 100 mM BA. Samples are then digested with trypsin
(Thermo Scientific, Rockford Ill.) using a ratio of 1:20 (w/w) at
37.degree. C. overnight. Digestion is halted by adding formic acid
to obtain a pH 2-3. Sep-Pak C18 reverse phase cartridges, 100 mg
(Waters, Milford Mass.) are used to clean and desalt the samples
after protein digestion. The peptides are eluted using 1 mL of 50%
acetonitrile and 0.1% formic acid, dried, and stored at -20.degree.
C. until LC-MS analysis.
Calibration Curve of HeavyPeptide IGNIS.TM. AQUAplex in Urine
Samples
[0196] Dilution curves of the heavy proteotypic peptides from
digested HeavyPeptide IGNIS.TM. are performed in a mixture of
digested pooled urine sample (1 ug/mL urine proteins), containing
three digested exogenous yeast proteins (carboxypeptidase Y,
enolase 1, and alcohol dehydrogenase 1) at 100 ng/mL individually.
Each dilution series corresponds to three data points spanning a
concentration ranging from 0.002 fmol/.mu.L to 40 fmol/.mu.L.
Protein levels of spiked digested yeast proteins and human urine
proteins are determined by iSR using the heavy proteotypic peptides
from digested HeavyPeptide IGNIS.TM. AQUAplex.
LC-MS Conditions
[0197] Urinary and yeast tryptic peptides are analyzed on a Q
Exactive Mass Spectrometer (ThermoFisher, San Jose Calif.).
Instruments are equipped with a nanoelectrospray ion source.
Chromatographic separations of peptides are performed on an
Ultimate 3000 (Dionex, Netherlands) high performance liquid
chromatographer operated in the nano-flow mode. Samples are loaded
on a Trap column (Acclaim PepMap C18, 3 .mu.m, 100 .ANG.,
0.075.times.20 mm, Dionex) and separated on an analytical column
(Acclaim PepMap.RTM. RSLC C18, 2 .mu.m, 100 .ANG., 0.075.times.150
mm, Dionex) coupled with a PicoTip.TM. electrospray emitter (30
.mu.m) (New Objective, Woburn Mass.) maintained at 1.2 kV. The
column temperature is fixed at 35.degree. C. Peptides are separated
with a linear gradient of acetonitrile/water, containing 0.1%
formic acid, at a flow rate of 300 nL/min. A gradient from 2% to
35% acetonitrile in 33 minutes is used. One .mu.L of each sample is
injected.
TABLE-US-00002 TABLE 1 Heavy Peptide IGNIS .TM. AQUAplex Swissprot
Selected protetotypic peptides (PI, PII, PIII) Name Protein Name
Organism ED P I P II P III TRFE uromodulin human P07911 DWVSVVTPAR
DSTIQVVENGESSQGR SGSVIDQSR (SEQ ID NO. 23) (SEQ ID NO. 24) (SEQ ID
NO. 25) serotransferrin human P02787 DGAGDVAFVK SASDLTWDNLK
EGYYGYTGAFR (SEQ ID NO. 28) (SEQ ID NO. 29) (SEQ ID NO. 30) LG3BP*
galectin-3- human Q08380 LADGGATNQGR SDLAVPSELALLK ELSEALGQIFDSQR
binding (SEQ ID NO. 32) (SEQ ID NO. 33) (SEQ ID NO. 34) protein
CD44 CD44 antigen human P16070 FAGVFHVEK YGFIEGHVVIPR ALSIGFETCR
(SEQ ID NO. 36) (SEQ ID NO. 37) (SEQ ID NO. 38) CATD cathepsin d
human P07339 LVDQNIFSFYLSR VSTLPAITLK YSQAVPAVTEGPIPEVLK (SEQ ID
NO. 40) (SEQ ID NO. 41) (SEQ ID NO. 42) KNG 1 kininogen-1 human
P01.042 TVGSDTFYSFK YFIDFVAR YNSQNQSNNQFVLYR (SEQ ID NO. 44) (SEQ
ID NO. 45) (SEQ ID NO. 46) ANAG alpha-N-acetyl- human P54802
LLLTSAPSLATSPAFR YDLLDLTR SDVFEAWR alucosaminidase (SEQ ID NO. 48)
(SEQ ID NO. 49) (SEQ ID NO. 50) ENO1 enolase 1 yeast P00924
NVNDVIAPAFVK LGANAILGVSLAASR TAGIQIVADDLTVTNPK (SEQ ID NO. 52) (SEQ
ID NO. 53) (SEQ ID NO. 54) CBPY carboxypeptidase yeast P00729
YDEEFASQK HFTYLR AWTDVLPWK Y (SEQ ID NO. 56) (SEQ ID NO. 57) (SEQ
ID NO. 58) ADH1 alcohol yeast P00330 GVIFYESHGK SIGGEVFIDFTK
VVGLSTLPEIYEK dehydrogenase 1 (SEQ ID NO. 60) (SEQ ID NO. 61) (SEQ
ID NO. 62) *no analysis performed
TABLE-US-00003 TABLE 2 Heavy Peptide IGNIS .TM. AQUAplex Full
sequence of isotopologue labeled name polypeptide + reporter
peptide P I P II PIII UROM DWVSVVTPARDSTIQVVENGESSQGRSGSVIDQSRLVA
DWVSVVTPAR DSTIQVVENGESSQG SGSVIDQSR LVR (SEQ ID NO. 23) R (SEQ ID
NO. 25) (SEQ ID NO. 27) (SEQ ID NO. 24) TRFE
DGAGDVAFVKSASDLTWDNLKEGYYGYTGAFRLVALVR DGAGDVAFVK SASDLTWDNLK
EGYYGYTGAFR (SEQ ID NO. 31) (SEQ ID NO. 28) (SEQ ID NO. 29) (SEQ ID
NO. 30) LG3BP LADGGATNQGRSDLAVPSELALLKELSEALGQIFDSQR LADGGATNQGR
SDLAVPSELALLK ELSEALGQIFDSQR LVALVR (SEQ ID NO. 32) (SEQ ID NO. 33)
(SEQ ID NO. 34) (SEQ ID NO. 35) CD44
FAGVFHVEKYGFIEGHVVIPRALSIGFETCRLVALVR FAGVFHVEK YGFIEGHVVIPR
ALSIGFETCR (SEQ ID NO. 39) (SEQ ID NO. 36) (SEQ ID NO. 37) (SEQ ID
NO. 38) CATD L VDQNIFSFYLSR VSTLPAITLKYSQAVPAVTEGPI LVDQNIFSFYLSR
VSTLPAITLK YSQAVPAVTEGPIPEV PEVLKLVALVR (SEQ ID NO. 40) (SEQ ID NO.
41) LK (SEQ ID NO. 43) (SEQ ID NO. 42) KNG1
TVGSDTFYSFKYFIDFVARYNSQNQSNNQFVLYR L TVGSDITYSFK YFIDFVAR
YNSQNQSNNQFVLYR VALVR (SEQ ID NO. 44) (SEQ ID NO. 45) (SEQ ID NO.
46) (SEQ ID NO. 47) ANAG LLLTSAPSLATSPAFRYDLLDLTRSDVFEAWRLVALVR
LLLTSAPSLATSPAFR YDLLDLTR SDVFEAWR (SEQ ID NO. 51) (SEQ ID NO. 48)
(SEQ ID NO. 49) (SEQ ID NO. 50) EN01
NVNDVIAPAFVKLGANAILGVSLAASRTAGIQIVADDL NVNDVIAPAFVK LGANAILGVSLAASR
TAGIQIVADDLTVTNP TVTNPKLVALVR (SEQ ID NO. 52) (SEQ ID NO. 53) K
(SEQ ID NO. 55) (SEQ ID NO. 54) CBPY YDEEFASQKHFTYLRAWTDVLPWKLVALVR
YDEEFASQK HFTYLR AWTDVLPWK (SEQ ID NO. 59) (SEQ ID NO. 56) (SEQ ID
NO. 57) (SEQ ID NO. 58) ADH1 GVIFYESHGKSIGGEVFIDFTKVVGLSTLPEIYEKLVA
GVIFYESHGK SIGGEVFIDFTK VVGLSTLPEIYEK LVR (SEQ ID NO. 60) (SEQ ID
NO. 61) (SEQ ID NO. 62) (SEQ ID NO. 63)
[0198] Applicants incorporate by reference the material contained
in the accompanying computer readable Sequence Listing identified
as 073988_281.txt, having a file creation date of Jun. 5, 2014,
1:41 p.m., and a file size of 19.4 kilobytes.
[0199] The embodiments shown and described in the specification are
only specific embodiments of the inventor who is skilled in the art
and is not limiting in any way. Therefore, various changes,
modifications, or alterations to those embodiments may be made
without departing from the spirit of the invention in the scope of
the following claims.
[0200] All references are expressly incorporated by reference
herein in their entirety.
Sequence CWU 1
1
6416PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 1Pro Val Val Val Pro Arg1
5210PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 2Ser Ser Ala Ala Pro Pro Pro Pro Pro
Arg1 5 10314PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 3Thr Thr Val Ser Lys Thr Glu
Thr Ser Gln Val Ala Pro Ala1 5 1049PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 4Thr Glu Thr Ser Gln Val Ala Pro Ala1 555PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 5Thr Thr Val Ser Lys1 5612PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 6Gly Ile Ser Asn Glu Gly Gln Asn Ala Ser Ile Lys1 5
1079PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 7His Val Leu Thr Ser Ile Gly Glu Lys1
588PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 8Asp Ile Pro Val Pro Lys Pro Lys1
598PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 9Ile Gly Asp Tyr Ala Gly Ile Lys1
51013PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 10Thr Ala Ser Glu Phe Asp Ser Ala Ile
Ala Gln Asp Lys1 5 101112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 11Ser Ala Ala Gly Ala Phe Gly Pro Glu Leu Ser Arg1 5
101214PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 12Glu Leu Gly Gln Ser Gly Val Asp Thr
Tyr Leu Gln Thr Lys1 5 101313PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 13Ser Phe Ala Asn Gln Pro Leu Glu Val Val Tyr Ser Lys1 5
101411PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 14Gly Leu Ile Leu Val Gly Gly Tyr Gly
Thr Arg1 5 101517PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 15Gly Ile Leu Phe Val Gly
Ser Gly Val Ser Gly Gly Glu Glu Gly Ala1 5 10 15Arg168PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 16Leu Thr Ile Leu Glu Glu Leu Arg1 51710PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 17Asn Gly Phe Ile Leu Asp Gly Phe Pro Arg1 5
101813PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 18Glu Leu Ala Ser Gly Leu Ser Phe Pro
Val Gly Phe Lys1 5 101914PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 19Leu Ser Ser Glu Ala Pro Ala Leu Phe Gln Phe Asp Leu Lys1
5 102014PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 20His Glu Val Thr Gly Trp
Val Leu Val Ser Pro Leu Ser Lys1 5 102113PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 21Ile Thr Val Val Asp Ala Leu His Glu Ile Pro Val Lys1 5
10226PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 22Leu Val Ala Leu Val Arg1
52310PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 23Asp Trp Val Ser Val Val Thr Pro Ala
Arg1 5 102416PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 24Asp Ser Thr Ile Gln Val
Val Glu Asn Gly Glu Ser Ser Gln Gly Arg1 5 10 15259PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 25Ser Gly Ser Val Ile Asp Gln Ser Arg1 5266PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 26Leu Val Ala Leu Val Lys1 52741PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 27Asp Trp Val Ser Val Val Thr Pro Ala Arg Asp Ser Thr
Ile Gln Val1 5 10 15Val Glu Asn Gly Glu Ser Ser Gln Gly Arg Ser Gly
Ser Val Ile Asp 20 25 30Gln Ser Arg Leu Val Ala Leu Val Arg 35
402810PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 28Asp Gly Ala Gly Asp Val Ala Phe Val
Lys1 5 102911PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 29Ser Ala Ser Asp Leu Thr
Trp Asp Asn Leu Lys1 5 103011PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 30Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg1 5
103138PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 31Asp Gly Ala Gly Asp Val Ala Phe
Val Lys Ser Ala Ser Asp Leu Thr1 5 10 15Trp Asp Asn Leu Lys Glu Gly
Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg 20 25 30Leu Val Ala Leu Val Arg
353211PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 32Leu Ala Asp Gly Gly Ala Thr Asn Gln
Gly Arg1 5 103313PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 33Ser Asp Leu Ala Val Pro
Ser Glu Leu Ala Leu Leu Lys1 5 103414PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 34Glu Leu Ser Glu Ala Leu Gly Gln Ile Phe Asp Ser Gln Arg1
5 103544PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 35Leu Ala Asp Gly Gly
Ala Thr Asn Gln Gly Arg Ser Asp Leu Ala Val1 5 10 15Pro Ser Glu Leu
Ala Leu Leu Lys Glu Leu Ser Glu Ala Leu Gly Gln 20 25 30Ile Phe Asp
Ser Gln Arg Leu Val Ala Leu Val Arg 35 40369PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 36Phe Ala Gly Val Phe His Val Glu Lys1 53712PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 37Tyr Gly Phe Ile Glu Gly His Val Val Ile Pro Arg1 5
103810PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 38Ala Leu Ser Ile Gly Phe Glu Thr Cys
Arg1 5 103937PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 39Phe Ala Gly Val Phe
His Val Glu Lys Tyr Gly Phe Ile Glu Gly His1 5 10 15Val Val Ile Pro
Arg Ala Leu Ser Ile Gly Phe Glu Thr Cys Arg Leu 20 25 30Val Ala Leu
Val Arg 354013PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 40Leu Val Asp Gln Asn Ile
Phe Ser Phe Tyr Leu Ser Arg1 5 104110PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 41Val Ser Thr Leu Pro Ala Ile Thr Leu Lys1 5
104218PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 42Tyr Ser Gln Ala Val Pro Ala Val Thr
Glu Gly Pro Ile Pro Glu Val1 5 10 15Leu Lys4347PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 43Leu Val Asp Gln Asn Ile Phe Ser Phe Tyr Leu Ser Arg
Val Ser Thr1 5 10 15Leu Pro Ala Ile Thr Leu Lys Tyr Ser Gln Ala Val
Pro Ala Val Thr 20 25 30Glu Gly Pro Ile Pro Glu Val Leu Lys Leu Val
Ala Leu Val Arg 35 40 454411PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 44Thr Val Gly Ser Asp Thr Phe Tyr Ser Phe Lys1 5
10458PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 45Tyr Phe Ile Asp Phe Val Ala Arg1
54615PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 46Tyr Asn Ser Gln Asn Gln Ser Asn Asn
Gln Phe Val Leu Tyr Arg1 5 10 154740PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 47Thr Val Gly Ser Asp Thr Phe Tyr Ser Phe Lys Tyr Phe
Ile Asp Phe1 5 10 15Val Ala Arg Tyr Asn Ser Gln Asn Gln Ser Asn Asn
Gln Phe Val Leu 20 25 30Tyr Arg Leu Val Ala Leu Val Arg 35
404816PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 48Leu Leu Leu Thr Ser Ala Pro Ser Leu
Ala Thr Ser Pro Ala Phe Arg1 5 10 15498PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 49Tyr Asp Leu Leu Asp Leu Thr Arg1 5508PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 50Ser Asp Val Phe Glu Ala Trp Arg1 55138PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 51Leu Leu Leu Thr Ser Ala Pro Ser Leu Ala Thr Ser Pro
Ala Phe Arg1 5 10 15Tyr Asp Leu Leu Asp Leu Thr Arg Ser Asp Val Phe
Glu Ala Trp Arg 20 25 30Leu Val Ala Leu Val Arg 355212PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 52Asn Val Asn Asp Val Ile Ala Pro Ala Phe Val Lys1 5
105315PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 53Leu Gly Ala Asn Ala Ile Leu Gly Val
Ser Leu Ala Ala Ser Arg1 5 10 155417PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 54Thr Ala Gly Ile Gln Ile Val Ala Asp Asp Leu Thr Val Thr
Asn Pro1 5 10 15Lys5550PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 55Asn Val Asn Asp Val Ile Ala Pro Ala Phe Val Lys Leu
Gly Ala Asn1 5 10 15Ala Ile Leu Gly Val Ser Leu Ala Ala Ser Arg Thr
Ala Gly Ile Gln 20 25 30Ile Val Ala Asp Asp Leu Thr Val Thr Asn Pro
Lys Leu Val Ala Leu 35 40 45Val Arg 50569PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 56Tyr Asp Glu Glu Phe Ala Ser Gln Lys1 5576PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 57His Phe Thr Tyr Leu Arg1 5589PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 58Ala Trp Thr Asp Val Leu Pro Trp Lys1 55930PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 59Tyr Asp Glu Glu Phe Ala Ser Gln Lys His Phe Thr Tyr
Leu Arg Ala1 5 10 15Trp Thr Asp Val Leu Pro Trp Lys Leu Val Ala Leu
Val Arg 20 25 306010PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 60Gly Val Ile Phe Tyr Glu
Ser His Gly Lys1 5 106112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 61Ser Ile Gly Gly Glu Val Phe Ile Asp Phe Thr Lys1 5
106213PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 62Val Val Gly Leu Ser Thr Leu Pro Glu
Ile Tyr Glu Lys1 5 106341PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 63Gly Val Ile Phe Tyr Glu Ser His Gly Lys Ser Ile Gly
Gly Glu Val1 5 10 15Phe Ile Asp Phe Thr Lys Val Val Gly Leu Ser Thr
Leu Pro Glu Ile 20 25 30Tyr Glu Lys Leu Val Ala Leu Val Arg 35
40648PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 64Ser Leu Leu Ser Gly Leu Leu Lys
5
* * * * *