U.S. patent application number 14/390447 was filed with the patent office on 2015-06-18 for srm methods in alzheimer's disease and neurological disease assays.
The applicant listed for this patent is Integrated Diagnostics, Inc.. Invention is credited to Clive Hayward, Paul Edward Kearney, Xiao-Jun Li.
Application Number | 20150168421 14/390447 |
Document ID | / |
Family ID | 48045730 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150168421 |
Kind Code |
A1 |
Kearney; Paul Edward ; et
al. |
June 18, 2015 |
SRM METHODS IN ALZHEIMER'S DISEASE AND NEUROLOGICAL DISEASE
ASSAYS
Abstract
Provided herein are methods for developing selected reaction
monitoring mass spectrometry (LC-SRM-MS) assays.
Inventors: |
Kearney; Paul Edward;
(Seattle, WA) ; Li; Xiao-Jun; (Bellevue, WA)
; Hayward; Clive; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Integrated Diagnostics, Inc. |
Seattle |
WA |
US |
|
|
Family ID: |
48045730 |
Appl. No.: |
14/390447 |
Filed: |
March 14, 2013 |
PCT Filed: |
March 14, 2013 |
PCT NO: |
PCT/US2013/031520 |
371 Date: |
October 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61620770 |
Apr 5, 2012 |
|
|
|
Current U.S.
Class: |
506/12 |
Current CPC
Class: |
G01N 33/6848 20130101;
G01N 2800/2821 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A multiplexed LC-SRM-MS assay for the measurement of a plurality
of proteins in a single sample comprising: a) generating a set of
optimal peptides and corresponding transitions for each protein
monitored; b) optimizing the collision energy for each transition
such that interference among the transitions monitored is avoided;
c) selecting a set of transitions that have the greatest peak areas
are monitored for each of the proteins, and wherein the selected
transitions do not interfere with the ions in the sample; d)
monitoring the detected set of transitions for each protein in the
sample, there-by measuring a plurality of proteins in the
sample.
2. The assay of claim 1, wherein each monitored peptide (i) has a
monoisotopic mass of 700-5000 Da; and (ii) does not contain a
cysteine or a methionine; and.
3. The assay of claim 1, wherein the transitions for each peptide
(i) have one of the four most intense b or y transition ions; (ii)
has m/z values of at least 30 m/z above or below those of a
precursor ion; (iii) do not interfere with transitions from other
peptides; and (iv) represent transitions due to breakage of peptide
bond at different sites of the protein.
4. The assay according to claim 1, wherein the peptides do not
include any peptide that is bounded by KK, KR, RK or RR, either
upstream of downstream in the corresponding protein sequence.
5. The assay according to claim 1, wherein each peptide of said set
of peptides is unique to the corresponding protein.
6. The assay according to claim 1, wherein the peptides do not
include peptides which were observed in post-translational modified
forms.
7. The assay according to claim 1, wherein each set of peptides is
prioritized according to one or more of the following ordered set
of criteria: (a) unique peptides first, then non-unique; (b)
peptides with no observed post-translational modifications first,
then those observed with post-translational modifications; (c)
peptides within the mass range 800-3500 Da first, then those
outside of 800-3500 Da; and (d) sorted by decreasing number of
variant residues.
8. The assay according to claim 7, wherein each set of peptides is
prioritized according to all of the ordered set of criteria.
9. The assay according to any one of claim 7 or 8, wherein each
prioritized set of peptides contains 1-5 peptides.
10. The assay according to any one of claims 1-9, wherein the two
best peptides per protein and the two best transitions per peptide
are selected based on experimental data resulting from LC-SRM-MS
analysis of one or more of the following experimental samples: a
biological disease sample, a biological control sample, and a
mixture of synthetic peptides of interest.
11. The assay according to claim 10, wherein the biological disease
and biological control samples are processed using an
immunodepletion method prior to LC-SRM-MS analysis.
12. The assay according to claim 11, wherein the experimental
samples contain internal standard peptides.
13. The assay according to claim 11, wherein the LC-SRM-MS analysis
method specifies a maximum of 7000 transitions, including
transitions of the internal standard peptides and transitions.
14. The assay according to claim 1, wherein the top two transitions
per peptide are selected according to one or more of the following
criteria: (1) the transitions exhibit the largest peak areas
measured in either of the two biological experimental samples; (2)
the transitions are not interfered with by other ions; (3) the
transitions do not exhibit an elution profile that visually differs
from those of other transitions of the same peptide; (4) the
transitions are not beyond the detection limit of both of the two
biological experimental samples; and (5) the transitions do not
exhibit interferences.
15. The assay according to claim 1, wherein the top two peptides
per protein are selected according to one or more of the following
criteria: (1) one or more peptides exhibit two transitions
according to claim 12 and represent the largest combined peak areas
of the two transitions according to claims 12; and (2) one or more
peptides exhibit one transition according to claim 12 and represent
the largest combined peak areas of the two transitions according to
claim 12.
16. An assay developed according to the method of claim 1.
Description
RELATED APPLICATIONS
[0001] This application claims priority and benefit of U.S.
Provisional Application No. 61/620,770, filed Apr. 5, 2012, the
contents of which is incorporated by reference in it's
entirety.
BACKGROUND
[0002] Liquid Chromatography Selected Reaction Monitoring Mass
Spectrometry (LC-SRM-MS) has emerged as an alternative technology
to immunoassays for quantification of target proteins in biological
samples. LC-SRM-MS methods are highly desirable because LC-SRM-MS
methods provide both absolute structural specificity for the target
protein and relative or absolute measurement of the target protein
concentration when suitable internal standards are utilized. In
contrast to immunoassays, LC-SRM-MS does not involve the
manufacturing of biologics. LC-SRM-MS protein assays can be rapidly
and inexpensively developed in contrast to the development of
immunoassays. LC-SRM-MS are highly multiplexed, with simultaneous
assays for hundreds of proteins performed in a single sample
analysis. Using LC-SRM-MS in contrast to other proteomic
technologies allows for complex assays for the identification
diagnostic proteins in complex diseases such as cancer, autoimmune,
and metabolic disease. In particular, the development of a highly
multiplexed LC-SRM-MS assay that reproducibly identifies a specific
set of proteins relevant to a clinical disease presents diagnostic
advantages and efficiencies. To date, proteomic techniques have not
enabled such assays to exist where hundreds of proteins can be
accurately quantified within a single sample. The present
disclosure provides accurate measurement of hundreds of Alzheimer's
disease associated proteins within a single sample using
multiplexed techniques.
SUMMARY
[0003] The present disclosure provides a LC-SRM-MS assay for the
measurement of at least 357 Alzheimer's disease associated proteins
in a single sample and in a single LC-SRM-MS assay. The assay was
optimized for protein quantification and minimal interference among
proteins in the assay. This LC-SRM-MS assay is novel because
measurement of a large number of proteins in a single sample
specifically associated with Alzheimer's disease has not been
accomplished. Simultaneous measurement of such a large number of
proteins without interference among the proteins requires specific
techniques to distinguish among the proteins. The current
disclosure provides clinical utility as this assay was used for
development of Alzheimer's disease diagnostic tests for the early
detection of Alzheimer's disease, managing disease treatment, as
well as testing for disease recurrence.
[0004] The object of the present disclosure is to provide improved
methods for the use of LC-SRM-MS in the development of assays.
Accordingly, provided herein is a method for developing peptides
and transitions for a plurality of at least 200 proteins for a
single sample selected reaction monitoring mass spectrometry
(LC-SRM-MS) assay, including the steps of providing a set of 200 or
more proteins; generating transitions for each protein; determining
the Mascot score for SRM-triggered tandem mass spectrometry (MS/MS)
spectra; performing collision energy optimization on the
transitions; selecting peptides with transitions showing the
greatest peak areas of their transitions; selecting a set of
transitions for each peptide, wherein the transitions for each
peptide have one of the four most intense b or y transition ions;
the transitions for each peptide have m/z values of at least 30 m/z
above or below those of the precursor ion; the transitions for each
peptide do not interfere with transitions from other peptides; and
the transitions represent transitions due to breakage of peptide
bond at different sites of the protein.
[0005] In one embodiment of the method, each selected peptide in
the set of peptides has a monoisotopic mass of 700-5000 Da; and
does not contain a cysteine or a methionine or does not contain
cysteine or methionine. In other embodiments, each selected peptide
contains cysteine or methionine. In another embodiment, the
transitions for each peptide have one of the four most intense b or
y transition ions; have m/z values of at least 30 m/z above or
below those of a precursor ion; do not interfere with transitions
from other peptides; and represent transitions due to breakage of
peptide bond at different sites of the protein.
[0006] In another embodiment of the method, the peptides do not
include any peptide that is bounded by KK, KR, RK or RR (either
upstream or downstream) in the corresponding protein sequence.
Specifically, the amino acid is charged at pH 7.0. In another
embodiment, each peptide of said set of peptides is unique to the
corresponding protein. In yet another embodiment, the peptides do
not include peptides which were observed in post-translational
modified forms. In still another embodiment, each set of peptides
is prioritized according to one or more of the following ordered
set of criteria: unique peptides first, then non-unique; peptides
with no observed post-translational modifications first, then those
observed with post-translational modifications; peptides within the
mass range 800-3500 Da first, then those outside of 800-3500 Da;
and sorted by decreasing number of variant residues. In certain
embodiments, the peptides are unique in that they only appear once
among the peptides run in a single assay.
[0007] In one embodiment, each set of peptides is prioritized
according to all of the ordered set of criteria. In another
embodiment, each prioritized set of peptides contains 1-5
peptides.
[0008] In certain embodiments of the preceding methods, the two
best peptides per protein and the two best transitions per peptide
are selected based on experimental data resulting from LC-SRM-MS
analysis of one or more of the following experimental samples: a
biological disease sample, a biological control sample, and a
mixture of synthetic peptides of interest. In a particular
embodiment, the biological disease and biological control samples
are processed using an immunodepletion method prior to LC-SRM-MS
analysis. In another embodiment, the experimental samples contain
internal standard peptides. In yet another embodiment, the
LC-SRM-MS analysis method specifies a maximum of 7000 transitions,
including transitions of the internal standard peptides and
transitions. In other embodiments the method specifies a maximum of
between 1000-7000, 2000-6000, 3000-5000 and about 3500
transitions.
[0009] In one embodiment of the method, the top two transitions per
peptide are selected according to one or more of the following
criteria the transitions exhibit the largest peak areas measured in
either of the two biological experimental samples; the transitions
are not interfered with by other ions; the transitions do not
exhibit an elution profile that visually differs from those of
other transitions of the same peptide; or the transitions are not
beyond the detection limit of both of the two biological
experimental samples.
[0010] In another embodiment of the method, the top two peptides
per protein are selected according to one or more of the following
criteria: one or more peptides exhibit two transitions and
represent the largest combined peak areas of the two transitions;
or one or more peptides exhibit one transition and represent the
largest combined peak areas of the two transitions.
[0011] In another aspect, provided herein is an assay developed
according to the foregoing method, and embodiments thereof.
[0012] In yet another aspect provided herein is the use of an assay
developed according to the foregoing method, and embodiments
thereof, to detect a plurality of at least 200 proteins in a single
biological sample.
[0013] In another aspect, provided herein is an assay developed
according to the foregoing method, and embodiments thereof.
[0014] The disclosure provides a use of a composition, as described
above, for the development of an assay to detect a disease,
disorder or condition in a mammal.
[0015] The disclosure provides a method comprising analyzing a
composition, as described above, using mass spectrometry. The
method can use selected reaction monitoring mass spectrometry.
DETAILED DESCRIPTION
[0016] The present disclosure relates to methods for developing
peptides and transitions for a single sample selected reaction
monitoring mass spectrometry (LC-SRM-MS) assay, generally
comprising the steps of providing a set of proteins; identifying
representative proteolytic peptides for each protein according to a
set of criteria; identifying representative transitions for each
peptide according to another set of criteria; and selecting the
optimum peptides per protein and the optimum transitions per
peptide.
[0017] Selected reaction monitoring mass spectrometry is capable of
highly sensitive and accurate protein quantification based on the
quantification of proteolytic peptides. In terms of clinical
utility, mass spectrometry-based assays are often compared to
immunoassays (e.g., Enzyme-Linked Immunosorbent Assay, or ELISA),
which have the ability to quantify specific analytes in large
sample sets (e.g., 96 or 384 samples in parallel microtitre
plate-based format). Until recently, mass spectrometry-based
protein assays were not able to match these sample sizes or
quantitative accuracy. Considerable time and expense is required to
generate and characterize antibodies required for immunoassays.
Increasingly efficient LC-SRM-MS assays, therefore, may surpass
immunoassays such as ELISA in the rapid development of clinically
useful, multiplexed protein assays.
[0018] LC-SRM-MS is a highly selective method of tandem mass
spectrometry which has the potential to effectively filter out all
molecules and contaminants except the desired analyte(s). This is
particularly beneficial if the analysis sample is a complex mixture
which may comprise several isobaric species within a defined
analytical window. LC-SRM-MS methods may utilize a triple
quadrupole mass spectrometer which, as is known in the art,
includes three quadrupole rod sets. A first stage of mass selection
is performed in the first quadrupole rod set, and the selectively
transmitted ions are fragmented in the second quadrupole rod set.
The resultant transition (product) ions are conveyed to the third
quadrupole rod set, which performs a second stage of mass
selection. The product ions transmitted through the third
quadrupole rod set are measured by a detector, which generates a
signal representative of the numbers of selectively transmitted
product ions. The RF and DC potentials applied to the first and
third quadrupoles are tuned to select (respectively) precursor and
product ions that have m/z values lying within narrow specified
ranges. By specifying the appropriate transitions (m/z values of
precursor and product ions), a peptide corresponding to a targeted
protein may be measured with high degrees of sensitivity and
selectivity. Signal-to-noise ratio in LC_SRM_MS is often superior
to conventional tandem mass spectrometry (MS/MS) experiments, that
do not selectively target (filter) particular analytes but rather
aim to survey all analytes in the sample.
[0019] Accordingly, provided herein is a method for developing
peptides and transitions for a plurality of proteins for use in
selected reaction monitoring mass spectrometry (LC-SRM-MS) assay.
In a preferred embodiment, the assay involves the analysis of a
single sample containing all analytes of interest (e.g., a
proteolytic digest of plasma proteins). As to the selection of the
protease(s) used, trypsin, which cleaves exclusively C-terminal to
arginine and lysine residues, is a preferred choice to generate
peptides because the masses of generated peptides are compatible
with the detection ability of most mass spectrometers (up to 2000
m/z), the number and average length of generated peptides, and also
the availability of efficient algorithms for the generation of
databases of theoretical trypsin-generated peptides. High cleavage
specificity, availability, and cost are other advantages of
trypsin. Other suitable proteases will be known to those of skill
in the art. Miscleavage is a factor for failure or ambiguous
protein identification. A miscleavage can be defined as partial
enzymatic protein cleavages generating peptides with internal
missed cleavage sites reflecting the allowed number of sites
(targeted amino acids) per peptide that were not cut. The presence
of post-translational modifications (PTMs) is also a potential
contributor to the problem of miscleavages.
[0020] LC-SRM-MS mass spectrometry involves the fragmentation of
gas phase ions and occurs between the different stages of mass
analysis. There are many methods used to fragment the ions and
these can result in different types of fragmentation and thus
different information about the structure and composition of the
molecule. The transition ions observed in an LC-SRM-MS spectrum
result from several different factors, which include, but are not
limited to, the primary sequence, the amount of internal energy,
the means of introducing the energy, and charge state. Transitions
must carry at least one charge to be detected. An ion is
categorized as either a, b or c if the charge is on a transition
comprising the original N terminus of the peptide, whereas the ion
is categorized as either x, y or z if the charge is on a transition
comprising the original C terminus of the peptide. A subscript
indicates the number of residues in the transition (e.g., one
peptide residue in x.sub.1, two peptide residues in y.sub.2, and
three peptide residues in z.sub.3, etc.).
[0021] In a generic peptide repeat unit represented --N--C(O)--C--,
an x ion and an a ion resulting from cleavage of the
carbonyl-carbon bond (i.e., C(O)--C). The x ion is an acylium ion,
and the a ion is an iminium ion. A y ion and a b ion result from
cleavage of the carbonyl-nitrogen bond (i.e., C(O)--N, also known
as the amide bond). In this case, the y ion is an ammonium ion and
the b ion is an acylium ion. Finally, a z ion and a c ion result
from cleavage of the nitrogen-carbon (i.e., C--N) bond. The z ion
is a carbocation and the c ion is an ammonium ion.
[0022] Superscripts are sometimes used to indicate neutral losses
in addition to the backbone fragmentation, for example, * for loss
of ammonia and .degree. for loss of water. In addition to protons,
c ions and y ions may abstract an additional proton from the
precursor peptide. In electrospray ionization, tryptic peptides may
carry more than one charge.
[0023] Internal transitions arise from double backbone cleavage.
These may be formed by a combination of b-type and y-type cleavage
(i.e., cleavage producing b and y ions). Internal cleavage ions may
also be formed by a combination of a-type and y-type cleavage. An
internal transition with a single side chain formed by a
combination of a-type and y-type cleavage is called an iminium ion
(sometimes also referred to as an imonium or immonium ion). These
ions are labeled with the one letter code for the corresponding
amino acid.
[0024] Low energy CID (i.e., collision induced dissociation in a
triple quadrupole or an ion trap) involves the fragmentation of a
peptide carrying a positive charge, primarily along its backbone,
to generate primarily a, b and y ions.
[0025] In one aspect, provided herein is a method for developing
peptides and transitions for a plurality of proteins for a single
sample selected reaction monitoring mass spectrometry (LC-SRM-MS)
assay: (a) providing a panel or plurality of proteins; (b)
identifying a set of peptides for each protein, wherein (i) each
peptide in the set of peptides corresponds to a transition of said
protein; (ii) the peptides have a monoisotopic mass of 700-5000 Da;
and (iii) the peptides do not contain cysteine or does not contain
cysteine or methionine. In other embodiments, each selected peptide
contains cysteine or methionine. ; and; (c) identifying a set of
transitions for each peptide, wherein (i) the transitions for each
peptide have one of the four most intense b or y transition ions;
(ii) the transitions for each peptide have m/z values of at least
30 m/z above or below those of the precursor ion; (iii) the
transitions for each peptide do not interfere with transitions from
other peptides; and (iv) the transitions represent transitions due
to breakage of peptide bond at different sites of the protein; and
(d) selecting the peptides for each protein that best fit the
criteria of step (b) and the transitions per peptide that best fit
the criteria of step (c); thereby developing peptides and
transitions for a LC-SRM-MS assay.
[0026] By plurality of proteins it is meant that at least 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 400,
450, 500 or more proteins. In certain embodiments, the plurality of
proteins can encompass between 2 and 10, 10 and 20, 20 and 50, 50
and 100, 100 and 200 or 200 and 500 proteins. In other embodiments,
the plurality of proteins can encompass between 250 and 450; or 300
and 400 proteins.
[0027] Trypsin-like proteases cleave peptide bonds following a
positively charged amino acid (e.g., lysine (K) or arginine (R)).
This specificity is driven by the residue which lies at the base of
the enzyme's S1 pocket (generally a negatively charged aspartic
acid or glutamic acid). Accordingly, in one embodiment of the
method, the peptides do not include any peptide that is bounded by
KK, KR, RK or RR, either upstream of downstream in the
corresponding protein sequence. In another embodiment, each peptide
of said set of peptides is unique to the corresponding protein.
[0028] Post-translational modification (PTM) is the chemical
modification of a protein after its translation. It can include any
modification following translation, including cleavage. It is one
of the later steps in protein biosynthesis, and thus gene
expression, for many proteins. It is desirable to avoid such
peptides for the purpose of protein identification. Thus, in
another embodiment, the peptides do not include peptides which were
observed in post-translational modified forms.
[0029] In still another embodiment, each set of peptides is
prioritized according to one or more of the following ordered set
of criteria: (a) unique peptides first, then non-unique; (b)
peptides with no observed post-translational modifications first,
then those observed with post-translational modifications; (c)
peptides within the mass range 800-3500 Da first, then those
outside of 800-3500 Da; and (d) sorted by decreasing number of
variant residues. In one embodiment, each set of peptides is
prioritized according to all of the ordered set of criteria. In
another embodiment, each prioritized set of peptides contains 1-5
peptides.
[0030] In certain embodiments, one or more liquid chromatography
(LC) purification steps are performed prior to a subsequent
LC-SRM-MS analysis step. Traditional LC analysis relies on the
chemical interactions between sample components and column packing
materials, where laminar flow of the sample through the column is
the basis for separation of the analyte of interest from the test
sample. The skilled artisan will understand that separation in such
columns is a diffusional process. A variety of column packing
materials are available for chromatographic separation of samples,
and selection of an appropriate separation protocol is an empirical
process that depends on the sample characteristics, the analyte of
interest, the interfering substances present and their
characteristics, etc. Various packing chemistries can be used
depending on the needs (e.g., structure, polarity, and solubility
of compounds being purified). In various embodiments the columns
are polar, ion exchange (both cation and anion), hydrophobic
interaction, phenyl, C-2, C-8, C-18 columns, polar coating on
porous polymer, or others that are commercially available. During
chromatography, the separation of materials is effected by
variables such as choice of eluant (also known as a "mobile
phase"), choice of gradient elution and the gradient conditions,
temperature, etc. In certain embodiments, an analyte may be
purified by applying a sample to a column under conditions where
the analyte of interest is reversibly retained by the column
packing material, while one or more other materials are not
retained. In these embodiments, a first mobile phase condition can
be employed where the analyte of interest is retained by the
column, and a second mobile phase condition can subsequently be
employed to remove retained material from the column, once the
non-retained materials are washed through. Alternatively, an
analyte may be purified by applying a sample to a column under
mobile phase conditions where the analyte of interest elutes at a
differential rate in comparison to one or more other materials. As
discussed above, such procedures may enrich the amount of one or
more analytes of interest relative to one or more other components
of the sample.
[0031] The following parameters are used to specify an LC-SRM-MS
assay of a protein under a particular LC-SRM-MS system: (1) a
tryptic peptide of the protein; (2) the retention time (RT) of the
peptide; (3) the m/z value of the peptide precursor ion; (4) the
declustering potential used to ionize the precursor ion; (5) m/z
value of a fragment ion generated from the peptide precursor ion;
and (6) the collision energy (CE) used to fragment the peptide
precursor ion that is optimized for the particular peptide.
[0032] In certain embodiments of the preceding methods, the two
best peptides per protein and the two best transitions per peptide
are selected based on experimental data resulting from LC-SRM-MS
analysis of one or more of the following experimental samples: a
biological disease sample, a biological control sample, and a
mixture of synthetic peptides of interest. Biological samples
include body fluids, tissue samples and cell samples. Body fluid
samples can include blood, serum, sputum, genital secretions,
cerebrospinal fluid, sweat or excreta such as urine. Body tissue
samples can include lung, skin, brain, spine, bone, muscle,
epithelial, liver, kidney, pancreas, gastrointestinal tract,
cardiovascular tissue, heart or nervous tissue. Biological disease
samples can include cancer, benign tumors, infected tissue and
tissue subject to trauma. In a particular embodiment, the
biological disease and biological control samples are processed
using an immunodepletion method prior to LC-SRM-MS analysis.
Immunodepletion involves removal of one or more proteins through
the use of antibodies. Numerous immunodepletion techniques are
known to those of skill in the art. In another embodiment, the
biological disease and biological control samples are processed
using an immunocapture method prior to LC-SRM-MS analysis.
Immunocapture involves selection of one or more proteins through
the use of antibodies. Numerous immunocapture techniques are known
to those of skill in the art.
[0033] To facilitate accurate quantification of the peptide
transitions by the methods disclosed herein, a set of
isotopically-labeled synthetic versions of the peptides of interest
may be added in known amounts to the sample for use as internal
standards. Since the isotopically-labeled peptides have physical
and chemical properties identical to the corresponding surrogate
peptide, they co-elute from the chromatographic column and are
easily identifiable on the resultant mass spectrum. The addition of
the labeled standards may occur before or after proteolytic
digestion. Methods of synthesizing isotopically-labeled peptides
will be known to those of skill in the art. Thus, in another
embodiment, the experimental samples contain internal standard
peptides. Other embodiments may utilize external standards or other
expedients for peptide quantification.
[0034] In yet another embodiment, the LC-SRM-MS analysis method
specifies a maximum of 7000 transitions, including transitions of
the internal standard peptides and transitions. As used herein, the
term "transition" refers to the specific pair of m/z
(mass-to-charge) values associated with the precursor and
transition ions corresponding to a specific peptide and, therefore,
to a specific protein.
[0035] In one embodiment of the method, the top two transitions per
peptide are selected according to one or more of the following
criteria (A): (1) the transitions exhibit the largest peak areas
measured in either of the two biological experimental samples; (2)
the transitions are not interfered with by other ions; (3) the
transitions do not exhibit an elution profile that visually differs
from those of other transitions of the same peptide; (4) the
transitions are not beyond the detection limit of both of the two
biological experimental samples; (5) the transitions do not exhibit
interferences.
[0036] For the mass spectrometric analysis of a particular peptide,
the quantities of the peptide transitions in the sample may be
determined by integration of the relevant mass spectral peak areas,
as known in the prior art. When isotopically-labeled internal
standards are used, as described above, the quantities of the
peptide transitions of interest are established via an
empirically-derived or predicted relationship between peptide
transition quantity (which may be expressed as concentration) and
the area ratio of the peptide transition and internal standard
peaks at specified transitions.
[0037] In another embodiment of the method, the top two peptides
per protein are selected according to one or more of the following
criteria (B): (1) one or more peptides exhibit two transitions
according to criteria (A) and represent the largest combined peak
areas of the two transitions according to criteria (A); and (2) one
or more peptides exhibit one transition according to criteria (A)
and represent the largest combined peak areas of the two
transitions according to criteria (A).
Assays
[0038] The methods of the present disclosure allow the
quantification of high abundance and low abundance plasma proteins
that serve as detectable markers for various health states
(including diseases and disorders), thus forming the basis for
assays that can be used to determine the differences between normal
levels of detectable markers and changes of such detectable markers
that are indicative of changes in health status. In one aspect of
the disclosure, provided herein is an assay developed according to
the foregoing method, and embodiments thereof. In another aspect,
provided herein is the use of an assay developed according to the
foregoing method, and embodiments thereof, to detect a plurality of
at least 200, 300 or more proteins in a single sample.
Definitions
[0039] As used herein, "transition" refers to a pair of m/z values
associated with a peptide. Normally, labeled synthetic peptides are
used as quality controls in SRM assays. However, for very large SRM
assays, labeled peptides are not feasible. However, correlation
techniques (Keary, Butler et al. 2008) were used to confirm the
identity of protein transitions with high confidence. The
correlation between a pair of transitions is obtained from their
expression profile over all samples in the training set study
detailed below. As expected, transitions from the same peptide are
highly correlated. Similarly, transitions from different peptide
fragments of the same protein are also highly correlated. In
contrast, transitions form different proteins are not highly
correlated. This methodology enables a statistical analysis of the
quality of the protein's SRM assay. For example, if the correlation
of the transitions from the two peptides from the same protein is
above 0.5 then there is less than a 5% probability that the assay
is false
[0040] As used herein, a "tryptic peptide" refers to the peptide
that is formed by the treatment of a protein with trypsin.
[0041] As used herein, "RT" refers to "retention time", the elapsed
time between injection and elution of an analyte.
[0042] As used herein, "m/z" indicates the mass-to-charge ratio of
an ion.
[0043] As used herein "DP" refers to the "declustering potential",
a voltage potential to dissolvate and dissociate ion clusters. It
is also known as "fragmentor voltage" or "ion transfer capillary
offset voltage" depending upon the manufacturer.
[0044] As used herein, "CE" refers to "collision energy", the
amount of energy precursor ions receive as they are accelerated
into the collision cell.
[0045] As used herein, "LC-SRM-MS" is an acronym for "selected
reaction monitoring" and may be used interchangeably with
"LC-MRM-MS".
[0046] As used herein, "MS/MS" represents tandem mass spectrometry,
which is a type of mass spectrometry involving multiple stages of
mass analysis with some form of fragmentation occurring in between
the stages.
[0047] As used herein, "ISP" refers to "internal standard
peptides".
[0048] As used herein, "HGS" refers to "human gold standard", which
is comprised of a pool of plasma from healthy individuals.
[0049] As used herein, "MGF" refers to "Mascot generic file".
Mascot is a search engine that uses mass spectrometry data to
identify proteins from primary sequence databases. A Mascot generic
file is a plain text (ASCII) file containing peak list information
and, optionally, search parameters.
[0050] Mascot is a tool for assessing mass spectrometry data
against protein sequences. This data can be acquired from any mass
spectrometry technique including MALDI-TOF and electrospray
ionization MS (including LC-SRM-MS) data. Mascot uses a
`probability-based MOWSE` algorithm to estimate the significance of
a match (i.e., that the observed transitions correspond to a
particular protein). The total score is the absolute probability
that the observed match is a random event. They are reported as
-10.times.LOG10(P), where P is the absolute probability. Lower
probabilities, therefore, are reported as higher scores. For
example, if the absolute probability that an observed match is
random is 1.times.10.sup.-12, Mascot reports it as 120.
[0051] The disclosure also provides compositions. These
compositions can include any of the transition ions described in
Table II. These transition ions exist while peptides derived from
the proteins in Table II are undergoing analysis with LC-SRM-MS. In
one embodiment, the composition includes any of the transition ions
described in Table II. In another embodiment, the composition
includes any two transition ions described in Table II. In other
embodiments, the composition includes, any 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,
300 or 331 transition ions described in Table II.
[0052] In another embodiment, each of the transition ions in the
composition corresponds and/or is derived from a different protein.
In another embodiment, 90% of the transition ions in the
composition correspond with and/or are derived from a protein that
no other transition ion in the composition corresponds. In other
embodiments, 80, 70, 60, 50, 40, 30, 20, 10 or 0% of the transition
ions in the composition correspond and/or are derived from a
protein that no other transition ion in the composition
corresponds.
[0053] The compositions described herein included synthetic
peptides. Synthetic peptides can be used as controls for the
abundance of proteins they are derived from and/or correspond. In
certain embodiments, the abundance of the synthetic peptides is
defined and the results are compared to LC-SRM-MS results from a
peptide found in a sample to the LC-SRM-MS results in the
corresponding synthetic peptide. This allows for the calculation of
the abundance of the peptide in the sample. In certain embodiments,
by knowing the abundance of a peptide in a sample, the abundance of
the protein it corresponded to is determined.
[0054] Synthetic peptides can be generated using any method known
in the art. These methods can include recombinant expression
techniques such as expression in bacteria or in vitro expression in
eukaryotic cell lysate. These methods can also include solid phase
synthesis.
[0055] In one embodiment, the composition includes synthetic
peptides selected from any of the peptides described in Table II.
In another embodiment, the composition included any two peptides
described in Table II. In other embodiments, the composition
included, any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,
220, 230, 240, 250, 260, 270, 280, 290, 300, 331 or more peptides
described in Table II.
[0056] In another embodiment, each of the peptides in the
composition each corresponds with a different protein. In another
embodiment, 90% of the peptides in the composition correspond with
a protein that no other peptide in the composition corresponds
with. In other embodiments, 80, 70, 60, 50, 40, 30, 20, 10 or 0% of
the peptides in the composition correspond with from a protein that
no other peptide in the composition corresponds with.
[0057] The peptides can be isotopically labeled. The isotopes with
which they can be labeled include .sup.13C, .sup.2H, .sup.15N and
.sup.18O. The peptides can also include a polar solvent. Polar
solvents can include water and mixtures of ethanol and water.
[0058] In certain embodiments, the samples described herein are
taken from mammals. These mammals include rats, mice, rabbits,
dogs, non-human primates and humans. Samples can be isolated from
any tissue or organ or from any bodily fluid. Organs from which
samples can be taken include skin, heart, lung, brain, kidney,
liver, pancreas, spleen, testes, ovaries, gall bladder, thymus,
thyroid, eye, ear, nose, mouth, tongue, penis, vagina, bladder or
larynx. Tissues include nervous tissue, vascular tissue, muscle,
bone, gastrointestinal tract, epithelial tissue, fibroblastic
tissue, mucous membranes, hair, skin, reproductive tissue and
connective tissue. Body fluids and excretions include, blood,
serum, saliva, urine, semen, vaginal secretions, excrement, bile,
tears, lymph, ear wax, mucous, shed skin, finger nails, toe nails,
skin oils, sweat and dandruff.
[0059] The relative abundance of one or more of the proteins
represented by the transition ions and synthetic peptides described
above can be used to diagnose, determine likelihood of the presence
of, develop prognoses for and/or stage various diseases and
pathologies. Often the organ, tissue or bodily fluid or excretion
from which the sample is taken is distinct from the organ, tissue
or bodily fluid or excretion involved with the disease or
pathology. For example, the presence of Alzheimer's disease can be
determined from a sample taken from blood. Any type of body fluid
may be used in the assays.
[0060] Diseases and pathologies that status, diagnosis, presence or
prognosis can be found using the transition ions and/or synthetic
peptides described herein include cancer, metabolic diseases,
neurological disorders, infectious diseases and cardiovascular
disorders.
EXAMPLES
I. Exemplary Standard Operating Procedure
Protein Selection
[0061] Proteins known to be over-expressed or under-expressed in
Alzheimer's disease patients were obtained (through literature
searching, experimental data or proprietary databases) as shown in
Table I. The set of proteins was reduced to a set of 357 proteins
(see Table II) by prioritizing those proteins that have been
previously detected my LC-MS/MS in blood (serum or plasma).
[0062] Selected proteins were then identified by their UniProt
protein name and accession, their Entrez gene symbol and gene name,
the isoform accession and their amino acid sequence. The canonical
isoform in UniProt was selected if a protein has more than one
isoform.
Peptide Selection for Synthesis
[0063] The five best peptides per protein for LC-SRM-MS assay were
selected for as follows. Fully tryptic peptides having a
monoisotopic mass of 800-3500 mass units, without miscleavages, not
containing a cysteine (C) or a methionine (M), without having high
miscleavage probability were selected. Further, any peptide that
was bounded by KK, KR, RK or RR (either upstream or downstream) in
the corresponding protein sequence was not selected.
[0064] Peptides were selected that were unique to the protein of
interest. Peptides were only selected that match only one protein
or protein family including analogues of the one protein, when
searched in protein databases. Further, peptides which were
observed in post-translational modified forms were not selected.
Databases were assessed that showed expression of the proteins from
which the peptides were isolated in human blood. Also databases of
good quality MS peptides were searched. Peptides that appeared in
human blood and were good quality MS peptides were favored. If
these methods did not result in a sufficient number of peptides,
rules were relaxed in a step wise manner to allow a greater number
of peptides until a sufficient number was reached. The purity of
the synthesized peptides was >75% and the amount of material was
.gtoreq.25 .mu.g. Peptides did not need to be desalted.
[0065] The four best transitions per peptide are then selected and
optimized based on experimental results from a mixture of synthetic
peptides. LC-SRM-MS-triggered MS/MS spectra was acquired for each
synthetic peptide, using a QTRAP 5500 instrument. One spectrum or
the doubly--and one for the triply--charged precursor ion was
collected for each of the identified peptides (Mascot score
.gtoreq.15), retention time was recorded for the four most intense
b or y transition ions. The selected transition ions possessed m/z
values were at least 30 m/z above or below those of the precursor
ions; they did not interfere with other synthetic peptides; and
they were transition ions due to breakage of peptide bond at
different sites.
[0066] If an insufficient percentage of the synthetic peptides were
acquired, the steps were repeated. In some cases, the second
transition with first with theoretical y+ ions with m/z values at
least 30 m/z above those of the doubly charged precursor ion was
selected if an insufficient percentage was acquired. Peptides that
failed to trigger the acquisition of MS/MS spectrum were
discarded.
II. Exemplary Protein list
[0067] The abundance of the following proteins can be assessed
substantially simultaneously using the MS-LC-SRM-MS system
described herein. Transitions from these proteins can be used to
diagnose diseases including Alzheimer's disease when their
abundance is measured in a biological specimen from a subject to be
diagnosed for Alzheimer's disease. In one embodiment, the
abundances of these proteins are measure in the blood serum of the
subject.
TABLE-US-00001 Lengthy table referenced here
US20150168421A1-20150618-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-00002 Lengthy table referenced here
US20150168421A1-20150618-T00002 Please refer to the end of the
specification for access instructions.
VI. Exemplary Assayed Protein
[0068] The expression of the following 357 proteins were assessed
substantially simultaneously using the MS-LC-SRM-MS system
described herein.
[0069] 41_HUMAN
[0070] 5HT2C_HUMAN
[0071] A1AG1_HUMAN
[0072] A1AG2_HUMAN
[0073] A1BG_HUMAN
[0074] A2MG_HUMAN
[0075] A4_HUMAN
[0076] AACT_HUMAN
[0077] ABCA1_HUMAN
[0078] ACBD7_HUMAN
[0079] ACE2_HUMAN
[0080] ACHA2_HUMAN
[0081] ACHA4_HUMAN
[0082] ACHAS_HUMAN
[0083] ACHB_HUMAN
[0084] ACHB2_HUMAN
[0085] ADA12_HUMAN
[0086] ADA23_HUMAN
[0087] AFAM_HUMAN
[0088] AGAL_HUMAN
[0089] AGAP2_HUMAN
[0090] ALBU_HUMAN
[0091] ALS_HUMAN
[0092] AMFR2_HUMAN
[0093] AMNLS_HUMAN
[0094] AMPB_HUMAN
[0095] ANGP1_HUMAN
[0096] ANGT_HUMAN
[0097] ANO3_HUMAN
[0098] ANT3_HUMAN
[0099] AP1B1_HUMAN
[0100] APC2_HUMAN
[0101] APLP1_HUMAN
[0102] APOA1_HUMAN
[0103] AP0A2_HUMAN
[0104] AP0A4_HUMAN
[0105] APOB_HUMAN
[0106] APOC2_HUMAN
[0107] APOD_HUMAN
[0108] APOE_HUMAN
[0109] APOL4_HUMAN
[0110] APOOL_HUMAN
[0111] ARHG7_HUMAN
[0112] ARP21_HUMAN
[0113] ARSA_HUMAN
[0114] ARSE_HUMAN
[0115] ARTN_HUMAN
[0116] ASGR1_HUMAN
[0117] AT12A_HUMAN
[0118] AT2A2_HUMAN
[0119] AT2B3_HUMAN
[0120] ATS1_HUMAN
[0121] BAX_HUMAN
[0122] BCAS1_HUMAN
[0123] BDNF_HUMAN
[0124] BEST1_HUMAN
[0125] BTNL8_HUMAN
[0126] C1QL2_HUMAN
[0127] C1QT4_HUMAN
[0128] CACB2_HUMAN
[0129] CAD11_HUMAN
[0130] CAD19_HUMAN
[0131] CAD22_HUMAN
[0132] CADH3_HUMAN
[0133] CADH5_HUMAN
[0134] CADH7_HUMAN
[0135] CALL3_HUMAN
[0136] CAMKV_HUMAN
[0137] CAR14_HUMAN
[0138] CATD_HUMAN
[0139] CB080_HUMAN
[0140] CB085_HUMAN
[0141] CBPN_HUMAN
[0142] CD3D_HUMAN
[0143] CD72_HUMAN
[0144] CEL3A_HUMAN
[0145] CEL3B_HUMAN
[0146] CERU_HUMAN
[0147] CETP_HUMAN
[0148] CF072_HUMAN
[0149] CFAB_HUMAN
[0150] CFAH_HUMAN
[0151] CHAD_HUMAN
[0152] CK041_HUMAN
[0153] CLC4M_HUMAN
[0154] CLUS_HUMAN
[0155] CNTN1_HUMAN
[0156] CNTN2_HUMAN
[0157] CO1A2_HUMAN
[0158] CO2_HUMAN
[0159] CO3_HUMAN
[0160] CO4A_HUMAN
[0161] CO4A4_HUMAN
[0162] CO4B_HUMAN
[0163] C06_HUMAN
[0164] CO8A_HUMAN
[0165] CO9A2_HUMAN
[0166] COIA1_HUMAN
[0167] CORT_HUMAN
[0168] CP46A_HUMAN
[0169] CPLX2_HUMAN
[0170] CRLF1_HUMAN
[0171] CRUM1_HUMAN
[0172] CSF1_HUMAN
[0173] CSF1R_HUMAN
[0174] DBC1_HUMAN
[0175] DCBD1_HUMAN
[0176] DCBD2_HUMAN
[0177] DDR2_HUMAN
[0178] DIRA2_HUMAN
[0179] E41L3_HUMAN
[0180] EAA2_HUMAN
[0181] EDNRB_HUMAN
[0182] ELAV3_HUMAN
[0183] EMIL2_HUMAN
[0184] EMIL3_HUMAN
[0185] EPHA8_HUMAN
[0186] ERLN1_HUMAN
[0187] ERMIN_HUMAN
[0188] ERO1B_HUMAN
[0189] F123A_HUMAN
[0190] F13A_HUMAN
[0191] FA20A_HUMAN
[0192] FCGRN_HUMAN
[0193] FETUA_HUMAN
[0194] FEZ1_HUMAN
[0195] FGFR2_HUMAN
[0196] FGFR3_HUMAN
[0197] FGL1_HUMAN
[0198] FIBA_HUMAN
[0199] FIBB _HUMAN
[0200] FIBG_HUMAN
[0201] FINC_HUMAN
[0202] FRS2_HUMAN
[0203] GABR2_HUMAN
[0204] GALR3_HUMAN
[0205] GAS6_HUMAN
[0206] GBRA2_HUMAN
[0207] GBRB2_HUMAN
[0208] GELS_HUMAN
[0209] GFRA2_HUMAN
[0210] GNAQ_HUMAN
[0211] GOLM1_HUMAN
[0212] GOPC_HUMAN
[0213] GP113_HUMAN
[0214] GP125_HUMAN
[0215] GP158_HUMAN
[0216] GP2_HUMAN
[0217] GPC5_HUMAN
[0218] GPC5D_HUMAN
[0219] GPC6_HUMAN
[0220] GPR88_HUMAN
[0221] GRIA2_HUMAN
[0222] GRM5_HUMAN
[0223] GRN_HUMAN
[0224] GT253_HUMAN
[0225] HAS1_HUMAN
[0226] HCN1_HUMAN
[0227] HCN2_HUMAN
[0228] HEMO_HUMAN
[0229] HEP2_HUMAN
[0230] HPCA_HUMAN
[0231] HPT_HUMAN
[0232] HRG_HUMAN
[0233] HS3S5_HUMAN
[0234] I12R1_HUMAN
[0235] IC1_HUMAN
[0236] ICAM3_HUMAN
[0237] IGF1R_HUMAN
[0238] IL12B _HUMAN
[0239] IL1AP_HUMAN
[0240] IL1R2_HUMAN
[0241] INADL_HUMAN
[0242] INHBA_HUMAN
[0243] IPSP_HUMAN
[0244] ITA3_HUMAN
[0245] ITAM_HUMAN
[0246] ITB2_HUMAN
[0247] ITB5_HUMAN
[0248] ITIH1_HUMAN
[0249] ITIH2_HUMAN
[0250] ITIH3_HUMAN
[0251] ITIH4_HUMAN
[0252] JPH3_HUMAN
[0253] KAIN_HUMAN
[0254] KALRN_HUMAN
[0255] KCC1G_HUMAN
[0256] KCC2A_HUMAN
[0257] KCNA1_HUMAN
[0258] KCNA2_HUMAN
[0259] KCNA3_HUMAN
[0260] KCNA5_HUMAN
[0261] KCNQ1_HUMAN
[0262] KCNV2_HUMAN
[0263] KCTD4_HUMAN
[0264] KIF5A_HUMAN
[0265] KIRR2_HUMAN
[0266] KLK3_HUMAN
[0267] KLKB1_HUMAN
[0268] KNG1_HUMAN
[0269] KSYK_HUMAN
[0270] LAMB2_HUMAN
[0271] LAT2_HUMAN
[0272] LAT3_HUMAN
[0273] LCK_HUMAN
[0274] LCN8_HUMAN
[0275] LGI1_HUMAN
[0276] LGMN_HUMAN
[0277] LIPE_HUMAN
[0278] LRMP_HUMAN
[0279] LRP8_HUMAN
[0280] LRTM2_HUMAN
[0281] LSHR_HUMAN
[0282] LTBP1_HUMAN
[0283] LYG2_HUMAN
[0284] MAMC2_HUMAN
[0285] MAP4_HUMAN
[0286] MICA_HUMAN
[0287] MMP1_HUMAN
[0288] MMP16_HUMAN
[0289] MMP17_HUMAN
[0290] MMP2_OHUMAN
[0291] MMP24_HUMAN
[0292] MMP9_HUMAN
[0293] MOT2_HUMAN
[0294] MPDZ_HUMAN
[0295] MTOR_HUMAN
[0296] MYP2_HUMAN
[0297] NCAN_HUMAN
[0298] NCIOC2_HUMAN
[0299] NDF6_HUMAN
[0300] NECP2_HUMAN
[0301] NETO1_HUMAN
[0302] NETR_HUMAN
[0303] NEUG_HUMAN
[0304] NEUM_HUMAN
[0305] NFL_HUMAN
[0306] NKX62_HUMAN
[0307] NMDE1_HUMAN
[0308] NMDE3_HUMAN
[0309] NMDZ1_HUMAN
[0310] NMS_HUMAN
[0311] NOE3_HUMAN
[0312] NPT4_HUMAN
[0313] NPTX1_HUMAN
[0314] NRG3_HUMAN
[0315] NTRK2_HUMAN
[0316] ODP2_HUMAN
[0317] OLFL3_HUMAN
[0318] OLIG1_HUMAN
[0319] OPCM_HUMAN
[0320] OTOAN_HUMAN
[0321] P2RX1_HUMAN
[0322] P4K2A_HUMAN
[0323] PACN1_HUMAN
[0324] PAK3_HUMAN
[0325] PAQR6_HUMAN
[0326] PAR6B_HUMAN
[0327] PARD3_HUMAN
[0328] PARK7_HUMAN
[0329] PCD1S_HUMAN
[0330] PCDA5_HUMAN
[0331] PCDAA_HUMAN
[0332] PCDB6_HUMAN
[0333] PCDB7_HUMAN
[0334] PCDBC_HUMAN
[0335] PCDBF_HUMAN
[0336] PCDGE_HUMAN
[0337] PCDGF_HUMAN
[0338] PCSK1_HUMAN
[0339] PDIA2_HUMAN
[0340] PDYN_HUMAN
[0341] PEDF_HUMAN
[0342] PERL_HUMAN
[0343] PGCB_HUMAN
[0344] PGCP_HUMAN
[0345] PICAL_HUMAN
[0346] PIN1_HUMAN
[0347] PKDRE_HUMAN
[0348] PLCB1_HUMAN
[0349] PON1_HUMAN
[0350] PRIO_HUMAN
[0351] PSMG1_HUMAN
[0352] PTN5_HUMAN
[0353] PTPRB_HUMAN
[0354] PTPRO_HUMAN
[0355] PTPRT_HUMAN
[0356] PVRL1_HUMAN
[0357] PZP_HUMAN
[0358] RCN1_HUMAN
[0359] RELN_HUMAN
[0360] RES18_HUMAN
[0361] RGS11_HUMAN
[0362] RGS2O_HUMAN
[0363] RGS4_HUMAN
[0364] RRAGC_HUMAN
[0365] RUN3A_HUMAN
[0366] S12A5_HUMAN
[0367] S12A6_HUMAN
[0368] S15A2_HUMAN
[0369] S39A4_HUMAN
[0370] SAA4_HUMAN
[0371] SCG1_HUMAN
[0372] SCG3_HUMAN
[0373] SCN2A_HUMAN
[0374] SCNNA_HUMAN
[0375] SCRT1_HUMAN
[0376] SEM4A_HUMAN
[0377] SEMG1_HUMAN
[0378] SEPP1_HUMAN
[0379] SEPT3_HUMAN
[0380] SGCZ_HUMAN
[0381] SHSA7_HUMAN
[0382] SIA8C_HUMAN
[0383] SIG12_HUMAN
[0384] SIX3_HUMAN
[0385] SLIK1_HUMAN
[0386] SLIT1_HUMAN
[0387] SNP25_HUMAN
[0388] SNTB1_HUMAN
[0389] SO1A2_HUMAN
[0390] SO1B3_HUMAN
[0391] SPB5_HUMAN
[0392] SREC_HUMAN
[0393] STH_HUMAN
[0394] SYN2_HUMAN
[0395] SYNPR_HUMAN
[0396] SYTL4_HUMAN
[0397] SYUA_HUMAN
[0398] SYUB_HUMAN
[0399] T151A_HUMAN
[0400] TADBP_HUMAN
[0401] TAU_HUMAN
[0402] TBB2B_HUMAN
[0403] TERA_HUMAN
[0404] TFR2_HUMAN
[0405] TLR7_HUMAN
[0406] TM9S1_HUMAN
[0407] TMPS2_HUMAN
[0408] TNF6B _HUMAN
[0409] TNR19_HUMAN
[0410] TR11B _HUMAN
[0411] TRFR_HUMAN
[0412] TRIM9_HUMAN
[0413] TRPV5_HUMAN
[0414] TYRO_HUMAN
[0415] UGGG2_HUMAN
[0416] UNC5C_HUMAN
[0417] VGFR3_HUMAN
[0418] VTDB_HUMAN
[0419] VTNC_HUMAN
[0420] WNK4_HUMAN
[0421] WNT8B _HUMAN
[0422] XLRS1_HUMAN
[0423] YQ051_HUMAN
[0424] ZIC1_HUMAN
[0425] ZIC2_HUMAN
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150168421A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
* * * * *
References