U.S. patent application number 14/031712 was filed with the patent office on 2014-08-21 for methods of detection of cancer using peptide profiles.
This patent application is currently assigned to Memorial Sloan-Kettering Cancer Center. The applicant listed for this patent is Memorial Sloan-Kettering Cancer Center. Invention is credited to Paul TEMPST, Josep VILLANUEVA.
Application Number | 20140234880 14/031712 |
Document ID | / |
Family ID | 37758335 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234880 |
Kind Code |
A1 |
TEMPST; Paul ; et
al. |
August 21, 2014 |
METHODS OF DETECTION OF CANCER USING PEPTIDE PROFILES
Abstract
The disclosed methods address the identification and monitoring
of cancer in a subject using serum peptide profiles. Such profiles
allow the detection of the differential presence of certain serum
peptide markers in comparison with controls. The profiles can be
determined employing mass spectrometry.
Inventors: |
TEMPST; Paul; (New York,
NY) ; VILLANUEVA; Josep; (Barcelona, ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Memorial Sloan-Kettering Cancer Center |
New York |
NY |
US |
|
|
Assignee: |
Memorial Sloan-Kettering Cancer
Center
New York
NY
|
Family ID: |
37758335 |
Appl. No.: |
14/031712 |
Filed: |
September 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13116956 |
May 26, 2011 |
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14031712 |
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12063968 |
Oct 20, 2008 |
7972770 |
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PCT/US06/31957 |
Aug 16, 2006 |
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13116956 |
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60708676 |
Aug 16, 2005 |
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Current U.S.
Class: |
435/13 ;
250/282 |
Current CPC
Class: |
G01N 33/57484 20130101;
C12Q 1/56 20130101; H01J 49/0027 20130101 |
Class at
Publication: |
435/13 ;
250/282 |
International
Class: |
H01J 49/00 20060101
H01J049/00; C12Q 1/56 20060101 C12Q001/56 |
Goverment Interests
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] This work was funded by NIH grant nos. 1 R21CA1119425, 5 P30
CA08748 and 5 P50 CA 92629. The government may have certain rights
to this invention.
Claims
1-77. (canceled)
78. A method of identifying cancer of the prostate in a subject
comprising detecting prostate cancer in the subject by detecting an
increase in the following peptides: a complement C3f peptide or a
fragment thereof, clusterin, complement C4-alpha, kininogen or
factor XIII peptide fragment, or any combination thereof in a
plasma or serum sample obtained from the subject relative to the
level of those peptides in a healthy control, thereby identifying
cancer of the prostate in the subject.
79. The method of claim 1, further comprising detecting a decrease
in fibrinopeptideA peptide or a fragment thereof, or a
fibrinogen-alpha peptide fragment, or any combination thereof in a
biological sample obtained from the subject.
80. The method of claim 1, further comprising measuring an increase
in an ITIH4 peptide relative to the level in a control.
81. The method of claim 1, wherein the detecting comprises
analyzing the biological sample, or a preparation thereof using
mass spectrometry.
82. The method of claim 81, wherein the mass spectrometry is MALDI
TOF mass spectrometry.
83. A method of identifying cancer of the prostate in a subject
comprising detecting prostate cancer in the subject by detecting a
decrease in a fibrinopeptide A or a fragment thereof and a
fibrinogen-alpha peptide fragment, and an increase in a complement
C3f peptide or a fragment thereof, a ITIH4, clusterin, complement
C4-alpha, kininogen and factor XIII peptide fragment in a plasma or
serum sample obtained from the subject relative to the level of
those peptides in a healthy control, thereby identifying cancer of
the prostate in the subject.
84. The method of claim 83, wherein the fibrinopeptideA peptide
fragment is selected from the group consisting of DSGEGDFLAEGGGVR
(SEQ ID NO. 1), SGEGDFLAEGGGVR (SEQ ID NO. 2), GEGDFLAEGGGVR (SEQ
ID NO. 3), EGDFLAEGGGVR (SEQ ID NO. 4), GDFLAEGGGVR (SEQ ID NO. 5),
DFLAEGGGVR (SEQ ID NO. 6) and LAEGGGVR (SEQ ID NO. 25).
85. The method of claim 83, wherein the complement C3f peptide
fragment is selected from the group consisting of, SSKITHRIHWESASLL
(SEQ ID NO. 8), SKITHRIHWESASLL (SEQ ID NO. 9), KITHRIHWESASLL (SEQ
ID NO. 10), THRIHWESASLL (SEQ ID NO. 11), and IHWESASLL (SEQ ID NO.
28).
86. The method of claim 83, wherein the ITIH4 peptide fragment is
selected from the group consisting of PGVLSSRQLGLPGPPDVPDHAAYHPF
(SEQ ID NO. 13), SRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 15), HAAYHPFR
(SEQ ID NO. 34), QLGLPGPPDVPDHAAYHPFR (SEQ ID NO. 35), HAAYHPF (SEQ
ID NO. 39) NVHSGSTFFKYYLQGAK1PKPEASFSPR (SEQ ID NO. 40) and
NVHSAGAAGSRMNFRPGVLSS (SEQ ID NO. 41).
87. The method of claim 83, wherein the clusterin peptide fragment
is HFFFPKSRIV (SEQ ID NO. 17).
88. The method of claim 83, wherein the complement C4-alpha peptide
fragment is GLEEELQFSLGSKINVKVGGNS (SEQ ID NO. 23).
89. The method of claim 83, wherein the fibrinogen-alpha peptide
fragment is selected from the group consisting of
SSSYSKQFTSSTSYNRGDSTFESKSYKMA (SEQ ID NO. 55) and
SSSYSKQFTSSTSYNRGDSTFESKSYKM (SEQ ID NO. 56).
90. The method of claim 83, wherein the kininogen peptide fragment
is NLGHGHKHERDQGHGHQ (SEQ ID NO. 52).
91. The method of claim 83, wherein the factor XIII peptide
fragment is AVPPNNSNAAEDDLPTVELQGVVPR (SEQ ID NO. 53).
92. A method of identifying cancer of the prostate in a subject
comprising: detecting in a plasma or serum biological sample
obtained from the subject levels of one or more peptides selected
from the group consisting of: a complement C3f peptide or a
fragment thereof, clusterin peptide fragment, complement C4-alpha
peptide fragment, kininogen peptide fragment, and factor XIII
peptide fragment; and identifying cancer of the prostate in the
subject when the levels of the one or more peptides in the subject
are increased relative to the level of the corresponding peptides
in a healthy control.
93. A method of identifying prostate cancer in a subject,
comprising: obtaining a plasma or serum biological sample from the
subject; detecting prostate cancer in the subject by detecting
levels of fibrinopeptideA or a fragment thereof, a fibrinogen-alpha
peptide fragment, complement C3f peptide or a fragment thereof,
ITIH4 peptide fragment, clusterin peptide fragment, complement
C4-alpa peptide fragment, kininogen peptide fragment, and factor
XIII peptide fragment; and identifying cancer of the prostate in
the subject when the levels of the fibrinopeptideA or a fragment
thereof and fibrinogen-alpha peptide fragment in the subject sample
are decreased relative to the level of the corresponding peptides
in a healthy control, and when the levels of complement C3f peptide
or fragment thereof, ITIH4, clusterin, complement C4-alpha,
kininogen and factor XIII peptide fragments are increased relative
to the level of the corresponding peptides in a healthy control.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS/PATENTS & INCORPORATION
BY REFERENCE
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/708,676, filed Aug. 16, 2005, the contents
of which are incorporated herein by reference.
[0003] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the PCT
and foreign applications or patents corresponding to and/or
paragraphing priority from any of these applications and patents,
and each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference. More generally, documents or references are
cited in this text, either in a Reference List before the
paragraphs, or in the text itself; and, each of these documents or
references ("herein-cited references"), as well as each document or
reference cited in each of the herein-cited references (including
any manufacturer's specifications, instructions, etc.), is hereby
expressly incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0004] Serum biomarkers are used for diagnosis of disease and for
predicting and monitoring response to treatment (Sidransky, D.
2002. Nat Rev Cancer 2:210-219; Bidart, J. M., et al. 1999. Clin
Chem 45:1695-1707). Most clinically useful markers, to date, have
been plasma proteins that require individual immunoassays for
quantitation (Jortani, S. A., et al. 2004. Clin Chem 50:265-278;
Watts, N. B. 1999. Clin Chem 45:1359-1368). Human serum also
contains smaller peptides that constitute an entity known as the
serum `peptidome`. Advances in mass spectrometry (MS) now permit
the display of hundreds of small to medium sized peptides from
microliter volumes of serum (Koomen, J. M., et al., 2005. J
Proteome Res 4:972-981; Villanueva, et al., 2004. Anal Chem
76:1560-1570). Several recent reports have advocated the use of
MS-based serum peptide profiling to determine qualitative and
quantitative patterns, or `signatures`, that indicate the
presence/absence of disease such as cancer (Petricoin, E. F., et
al., 2002. Lancet 359:572-577; Adam, B. L., et al., 2002. Cancer
Res 62:3609-3614; Li, J., et al., 2002. Clin Chem 48:1296-1304;
Ebert, M. P., et al., 2004. J Proteome Res 3:1261-1266; Ornstein,
D. K., et al. 2004. J Urol 172:1302-1305; Conrads, T. P., et al.,
2004. Endocr Relat Cancer 11:163-178). To date, it has neither been
accomplished to independently reproduce entire peptidomic patterns,
nor has it been shown that the highly discriminatory peptides have
the same amino acid sequences.
[0005] TOF-MS is the most efficient mass analysis technique in
terms of detection sensitivity and readily achieves high mass
analysis at good mass accuracy (R. J. Cotter, Anal. Chem. 64 (21),
1027 (1992)). It is one of the few analysis techniques that
combines high sensitivity, selectivity and specificity with speed
of analysis. For example, TOF-MS can record a complete mass
spectrum on a microsecond timescale.
[0006] Advances in MS-based serum peptide profiling can have
important implications for cancer diagnostics.
SUMMARY OF THE INVENTION
[0007] It has now been determined that distinctive peptide patterns
that correlate with clinically relevant outcomes can be established
through mass spectrometry (MS). Methods of the present invention
employ serum peptide profiles to identify various types of
cancer.
[0008] The present invention provides peptide markers that are
differentially present in the samples of cancer subjects and in the
samples of control subjects. Measurement of these markers, alone or
in combination, in patient samples provides information correlating
with a probable diagnosis of human cancer or a negative diagnosis
(e.g., normal or disease-free). Accordingly, further disclosed are
methods and kits that employ these markers in diagnosing and
monitoring cancer.
[0009] In one aspect, the present invention provides methods of
diagnosing or monitoring cancer in a subject comprising measuring
at least one peptide marker in a sample from the subject. The
cancer can be cancer of the prostate, bladder, breast or thyroid.
Peptide markers of the invention include but are not limited to
complement C3f, ITIH4, clusterin, complement C4-alpha,
fibrinopeptideA, bradykinin, APO A-I, APOA-IV, APO E, kininogen,
factor XIII, transthyretin and fibrinogenA. Preferably, peptide
markers for ITIH4, clusterin, complement C4-alpha, APO A-I, APO
A-IV, APO E, kininogen, factor XIII, transthyretin and fibrinogenA
are present in the serum as peptide fragments.
[0010] In one embodiment, peptide marker levels are detected in a
combination of two or more of the aforementioned peptide markers.
Thus, the number of individual peptide markers measured in a sample
can range from about 2 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to
30, 30 to 35, 35 to 40, 40 to 45, 45 to 50 and greater than about
50. In specific embodiments, at least about 20 of the peptide
markers are measured.
[0011] In one embodiment, the invention provides a method of
identifying cancer of the prostate in a subject comprising
detecting an increase in a complement C3f peptide or a fragment
thereof, a ITIH4, clusterin, complement C4-alpha, kininogen or
factor XIII peptide fragment, or any combination thereof in a
biological sample obtained from the subject, thereby identifying
cancer of the prostate in the subject. The method can further
comprise detecting a decrease in fibrinopeptideA peptide or a
fragment thereof, or a fibrinogen-alpha peptide fragment, or any
combination thereof in a biological sample obtained from the
subject.
[0012] In another embodiment, the invention provides a method of
identifying cancer of the bladder in a subject comprising detecting
an increase in a complement C3f peptide or a fragment thereof, a
ITIH4, clusterin, complement C4-alpha, fibrinogen-alpha, APO A-I,
APO A-IV, APO E or kininogen peptide fragment, or any combination
thereof in a biological sample obtained from the subject, thereby
identifying cancer of the bladder in the subject. The method can
further comprise detecting a decrease in a fibrinopeptideA peptide,
bradykinin peptide, or a fragment thereof, a C4-alpha, ITIH4, or
fibrinogen-alpha peptide fragment, or any combination thereof in a
biological sample obtained from the subject.
[0013] In yet another embodiment, the invention provides a method
of identifying cancer of the breast in a subject comprising
detecting an increase in a fibrinopeptideA peptide, bradykinin
peptide, or a fragment thereof, a ITIH4, complement C4-alpha,
fibrinogen-alpha, APO A-IV, factor XIII or transthyretin peptide
fragment, or any combination thereof in a biological sample
obtained from the subject, thereby identifying cancer of the breast
in the subject. The method can further comprise detecting a
decrease in a fibrinopeptideA peptide, complement C3f peptide, or a
fragment thereof, or any combination thereof in a biological sample
obtained from the subject.
[0014] In yet another embodiment, the invention provides a method
of identifying cancer of the prostate in a subject comprising
detecting a decrease in a fibrinopeptideA peptide or a fragment
thereof and a fibrinogen-alpha peptide fragment and an increase in
a complement C3f peptide or a fragment thereof, a ITIH4, clusterin,
complement C4-alpha, kininogen and factor XIII peptide fragment in
a biological sample obtained from the subject, thereby identifying
cancer of the prostate in the subject.
[0015] In yet another embodiment, the invention is provides a
method of identifying cancer of the bladder in a subject comprising
detecting a decrease in a fibrinopeptideA peptide, bradykinin
peptide, or a fragment thereof, a C4-alpha, ITIH4, and
fibrinogen-alpha peptide fragment and an increase in a complement
C3f or a fragment thereof, a ITIH4, dusted % complement C4-alpha,
fibrinogen-alpha, APO A-I, APO A-IV, APO E and kininogen peptide
fragment in a biological sample obtained from the subject, thereby
identifying cancer of the bladder in the subject.
[0016] In yet another embodiment, the invention provides a method
of identifying cancer of the breast in a subject comprising
detecting a decrease in a fibrinopeptideA peptide and complement
C3f peptide, or a fragment thereof, and an increase in a
fibrinopeptideA peptide, bradykinin peptide, or a fragment thereof,
a ITIH4, complement C4-alpha, fibrinogen-alpha, APO A-IV, factor
XIII and transthyretin peptide fragment in a biological sample
obtained from the subject, thereby identifying cancer of the breast
in the subject.
[0017] In specific embodiments of the invention concerning cancer
of the prostate, the fibrinopeptideA peptide fragment that is
decreased includes but is not limited to DSGEGDFLAEGGGVR (SEQ ID
NO. 1), SGEGDFLAEGGGVR (SEQ ID NO. 2), GEGDFLAEGGGVR (SEQ ID NO.
3), EGDFLAEGGGVR (SEQ ID NO. 4), GDFLAEGGGVR (SEQ ID NO. 5),
DFLAEGGGVR (SEQ ID NO. 6) or LAEGGGUR (SEQ ID NO. 25).
[0018] In other specific embodiments of the invention concerning
cancer of the bladder, the fibrinopeptideA peptide fragment that is
decreased includes but is not limited to DSGEGDFLAEGGGVR (SEQ ID
NO. 1), SGEGDFLAEGGGVR (SEQ ID NO. 2), GEGDFLAEGGGVR (SEQ ID NO.
3), EGDFLAEGGGVR (SEQ ID NO. 4), GDFLAEGGGVR (SEQ ID NO. 5),
DFLAEGGGVR (SEQ ID NO. 6), FLAEGGGUR (SEQ ID NO. 24) or LAEGGGUR
(SEQ ID NO. 25).
[0019] In other specific embodiments of the invention concerning
cancer of the breast, the fibrinopeptideA peptide fragment that is
decreased includes but is not limited to SGEGDFLAEGGGVR (SEQ ID NO.
2) or GEGDFLAEGGGVR (SEQ ID NO. 3) and the fibrinopeptideA peptide
fragment that is increased is FLAEGGGUR (SEQ ID NO. 24).
[0020] In other specific embodiments of the invention concerning
cancer of the prostate, the complement C3f peptide fragment that is
increased includes but is not limited to SSKITHRIHWESASLL (SEQ ID
NO. 8), SKITHRIHWESASLL (SEQ ID NO. 9), KITHRIHWESASLL (SEQ ID NO.
10), THRIHWESASLL (SEQ ID NO. 11) or IHWESASLL (SEQ ID NO. 28).
[0021] In other specific embodiments of the invention concerning
cancer of the bladder, the complement C3f peptide fragment that is
increased includes but is not limited to SSKITHRIHWESASLL (SEQ ID
NO. 8), SKITHRIHWESASLL (SEQ ID NO. 9), KITHRIHWESASLL (SEQ ID NO.
10), THRIHWESASLL (SEQ ID NO. 11), HWESASLL (SEQ ID NO. 12),
RIHWESASLL (SEQ ID NO. 27), IHWESASLL (SEQ ID NO. 28) or
SSKITHRIHWESASL (SEQ ID NO. 29).
[0022] In other specific embodiments of the invention concerning
cancer of the breast, the complement C3f peptide fragment that is
decreased includes but is not limited to SSKITHRIHWESASLL (SEQ ID
NO. 8), HWESASLL (SEQ ID NO. 12) or ITHRIHWESASLL (SEQ ID NO.
26).
[0023] In other specific embodiments of the invention concerning
cancer of the prostate, ITIH4 peptide fragment that is increased
includes but is not limited to PGVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID
NO. 13), SRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 15), HAAYHPFR (SEQ ID
NO. 34), QLGLPGPPDVPDHAAYHPFR (SEQ ID NO. 35), HAAYHPF (SEQ ID NO.
39), NVHSGSTFFKYYLQGAKIPKPEASFSPR (SEQ ID NO. 40) or
NVHSAGAAGSRMNFRPGVLSS (SEQ ID NO. 41).
[0024] In other specific embodiments of the invention concerning
cancer of the bladder, the ITIH4 peptide fragment that is increased
includes but is not limited to PGVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID
NO. 13), SRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 15), HAAYHPFR (SEQ ID
NO. 34), QAGAAGSRMNFRPGVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 36),
MNFRPGVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 37),
NVHSGSTFFKYYLQGAKIPKPEASFSPR (SEQ ID NO. 40) or
NVHSAGAAGSRMNFRPGVLSS (SEQ ID NO. 41) and the ITIH4 peptide
fragment that is decreased includes but is not limited to
GVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 14) or HAAYHPF (SEQ ID NO.
39).
[0025] In other specific embodiments of the invention concerning
cancer of the breast, the ITIH4 peptide fragment that is increased
includes but is not limited to GLPGPPDVPDHAAYHPF (SEQ ID NO. 16),
HAAYHPFR (SEQ ID NO. 34), QLGLPGPPDVPDHAAYHPFR (SEQ ID NO. 35),
SSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 38) or NVHSAGAAGSRMNFRPGVLSS
(SEQ ID NO. 41).
[0026] In other specific embodiments of the invention concerning
cancer of the prostate, the clusterin peptide fragment includes but
is not limited to HFFFPKSRIV (SEQ ID NO. 17).
[0027] In other specific embodiments of the invention concerning
cancer of the bladder, the clusterin peptide fragment that is
increased includes but is not limited to HFFFPKSRIV (SEQ ID NO. 17)
or HFFFPK (SEQ ID NO. 18).
[0028] In other specific embodiments of the invention concerning
cancer of the bladder, the bradykinin peptide fragment that is
decreased includes but is not limited to RPPGFSPFR (SEQ ID NO. 19)
or RPPGFSPF (SEQ ID NO. 20).
[0029] In other specific embodiments of the invention concerning
cancer of the breast, the bradykinin peptide fragment that is
increased includes but is not limited to RPPGFSPFR (SEQ ID NO. 19)
or RPPGFSPF (SEQ ID NO. 20).
[0030] In other specific embodiments of the invention concerning
cancer of the prostate, the complement C4-alpha peptide fragment
that is increased includes but is not limited to
GLEEELQFSLGSKINVKVGGNS (SEQ ID NO. 23).
[0031] In other specific embodiments of the invention concerning
cancer of the bladder, the complement C4-alpha peptide fragment
that is increased includes but is not limited to RNGFKSHALQLNNRQI
(SEQ ID NO. 21), GLEEELQFSLGSKINVKVGGNS (SEQ ID NO. 23), or
NGFKSHALQLNNR (SEQ ID NO. 31) and the complement C4-alpha peptide
fragment that is decreased is GLEEELQFSLGSKINV (SEQ ID NO. 33).
[0032] In other specific embodiments of the invention concerning
cancer of the breast, the complement C4-alpha peptide fragment that
is increased includes but is not limited to RNGFKSHALQLNNRQI (SEQ
ID NO. 21), NGFKSHALQLNNRQI (SEQ ID NO. 22), GLEEELQFSLGSKINVKVGGNS
(SEQ ID NO. 23), NGFKSHALQLNNRQ (SEQ ID NO. 30),
GLEEELQFSLGSKINVKVGGNSKGTL (SEQ ID NO. 32) or GLEEELQFSLGSKINV (SEQ
ID NO. 33).
[0033] In other specific embodiments of the invention concerning
cancer of the prostate, the fibrinogen-alpha peptide fragment that
is decreased includes but is not limited to
SSSYSKQFTSSTSYNRGDSTFESKSYKMA (SEQ ID NO. 55) or
SSSYSKQFTSSTSYNRGDSTFESKSYKM (SEQ ID NO. 56).
[0034] In other specific embodiments of the invention concerning
cancer of the bladder, the fibrinogen-alpha peptide fragment that
is increased includes but is not limited to
SSSYSKQFTSSTSYNRGDSTFESKSYKMA (SEQ ID NO. 55),
SSSYSKQFTSSTSYNRGDSTFESKSYKM (SEQ ID NO. 56),
SSSYSKQFTSSTSYNRGDSTFESKSY (SEQ ID NO. 57),
SSSYSKQFTSSTSYNRGDSTFESKS (SEQ ID NO. 58), or SSYSKQFTSSTSYNRGDSTFE
(SEQ ID NO. 60), and the fibrinogen-alpha peptide fragment that is
decreased is GSESGIFTNTKESSSHHPGIAEFPSRG (SEQ ID NO. 61).
[0035] In other specific embodiments of the invention concerning
cancer of the breast, the fibrinogen-alpha peptide fragment that is
increased includes but is not limited to SSYSKQFTSSTSYNRGDSTFE (SEQ
ID NO. 60) or DEAGSEADHEGTHSTKRGHAKSRPV (SEQ ID NO. 62).
[0036] In other specific embodiments of the invention concerning
cancer of the prostate, the kininogen peptide fragment is
NLGHGHKHERDQGHGHQ (SEQ ID NO. 52).
[0037] In other specific embodiments of the invention concerning
cancer of the bladder, the kininogen peptide fragment that is
increased includes but is not limited to KHNLGHGHKHERDQGHGHQ (SEQ
ID NO. 51) or NLGHGHKHERDQGHGHQ (SEQ ID NO. 52).
[0038] In other specific embodiments of the invention concerning
cancer of the bladder, the APO A-I peptide fragment that is
increased includes but is not limited to
QGLLPVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO. 42), VSFLSALEEYTKKLNTQ
(SEQ ID NO. 43) or ATEHLSTLSEKAKPALEDL (SEQ ID NO. 44).
[0039] In other specific embodiments of the invention concerning
cancer of the bladder, the APO A-IV peptide fragment that is
increased includes but is not limited to GNTEGLQKSLAELGGHLDQQVEEFR
(SEQ ID NO. 46), SLAELGGHLDQQVEEFR (SEQ ID NO. 47) or
SLAELGGHLDQQVEEF (SEQ ID NO. 48).
[0040] In other specific embodiments of the invention concerning
cancer of the breast, the APO A-IV peptide fragment that is
increased is ISASAEELRQRLAPLAEDVRGNL (SEQ ID NO. 45).
[0041] In other specific embodiments of the invention concerning
cancer of the bladder, the APO E peptide fragment that is increased
includes but is not limited to AATVGSLAGQPLQERAQAWGERLR (SEQ ID NO.
49) or AATVGSLAGQPLQERAQAWGERL (SEQ ID NO. 50).
[0042] In other specific embodiments of the invention concerning
cancer of the prostate, the factor XIII peptide fragment that is
increased is AVPPNNSNAAEDDLPTVELQGVVPR (SEQ ID NO. 53).
[0043] In other specific embodiments of the invention concerning
cancer of the breast, the factor XIII peptide fragment that is
increased is AVPPNNSNAAEDDLPTVELQGVVPR (SEQ ID NO. 53).
[0044] In other specific embodiments of the invention concerning
cancer of the breast, the transthyretin peptide fragment that is
increased is ALGISPFHEHAEVVFTANDSGPR (SEQ ID NO. 54).
[0045] In practicing the methods of the invention, the biological
sample can comprise plasma or serum or a preparation thereof.
Detection can comprise analyzing the biological sample, or a
preparation thereof using mass spectrometry. The mass spectrometry
can be MALDI TOF, Fourier-transform ion cyclotron resonance,
electrospray ionization mass spectrometry, or combinations thereof.
In another aspect, detection can comprise analyzing the biological
sample, or a preparation thereof on a solid support, wherein
peptides in the sample bind to the solid support.
[0046] In another aspect, the invention provides peptide profiles
indicative of cancer of the prostate, bladder, and breast.
[0047] In one embodiment, the invention provides an isolated or
identified peptide profile indicating cancer of the prostate
comprising an increased amount of peptides or peptide fragments of
SSKITHRIHWESASLL (SEQ ID NO. 8), SKITHRIHWESASLL (SEQ ID NO. 9),
KITHRIHWESASLL (SEQ ID NO. 10), THRIHWESASLL (SEQ ID NO. 11),
PGVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 13), SRQLGLPGPPDVPDHAAYHPF
(SEQ. ID NO. 15), HFFFPKSRIV (SEQ ID NO. 17),
GLEEELQFSLGSKINVKVGGNS (SEQ ID NO. 23), IHWESASLL (SEQ ID NO. 28),
HAAYHPFR (SEQ ID NO. 34), QLGLPGPPDVPDHAAYHPFR (SEQ ID NO. 35),
HAAYHPF (SEQ ID NO. 39), NVHSGSTFFKYYLQGAKIPKPEASFSPR (SEQ ID NO.
40), NVHSAGAAGSRMNFRPGVLSS (SEQ ID NO. 41), NLGHGHKHERDQGHGHQ (SEQ
ID NO. 52), AVPPNNSNAAEDDLPTVELQGVVPR (SEQ ID NO. 53), or
combinations thereof. In an additional embodiment, the isolated or
identified peptide profile indicating cancer of the prostate
comprises a decreased amount of peptides or peptide fragments of
DSGEGDFLAEGGGVR (SEQ ID NO. 1), SGEGDFLAEGGGVR (SEQ ID NO. 2),
GEGDFLAEGGGVR (SEQ ID NO. 3), EGDFLAEGGGVR (SEQ ID NO. 4),
GDFLAEGGGVR (SEQ ID NO. 5), DFLAEGGGVR (SEQ ID NO. 6), LAEGGGUR
(SEQ ID NO. 25), SSSYSKQFTSSTSYNRGDSTFESKSYKMA (SEQ ID NO. 55),
SSSYSKQFTSSTSYNRGDSTFESKSYKM (SEQ ID NO. 56), or combinations
thereof.
[0048] In another embodiment, the invention provides an isolated or
identified peptide profile indicating cancer of the bladder
comprising an increased amount of peptides or peptide fragments of
SSKITHRIHWESASLL (SEQ ID NO. 8), SKITHRIHWESASLL (SEQ ID NO. 9),
KITHRIHWESASLL (SEQ ID NO. 10), THRIHWESASLL (SEQ ID NO. 11),
HWESASLL (SEQ ID NO. 12), PGVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO.
13), SRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 15), HFFFPKSRIV (SEQ ID NO.
17), HFFFPK (SEQ ID NO. 18), RNGFKSHALQLNNRQI (SEQ ID NO. 21),
GLEEELQFSLGSKINVKVGGNS (SEQ ID NO. 23), (SEQ ID NO. 27), IHWESASLL
(SEQ ID NO. 28), SSKITHRIHWESASL (SEQ ID NO. 29), NGFKSHALQLNNR
(SEQ II) NO. 31), HAAYHPFR (SEQ ID NO. 34),
QAGAAGSRMNFRPGVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 36),
MNFRPGVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 37),
NVHSGSTFFKYYLQGAKIPKPEASFSPR (SEQ ID NO. 40), NVHSAGAAGSRMNFRPGVLSS
(SEQ ID NO. 41), QGLLPVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO. 42),
VSFLSALEEYTKKLNTQ (SEQ ID NO. 43), ATEHLSTLSEKAKPALEDL (SEQ ID NO.
44), GNTEGLQKSLAELGGHLDQQVEEFR (SEQ ID NO. 46), SLAELGGHLDQQVEEFR
(SEQ ID NO. 47), SLAELGGHLDQQVEEF (SEQ ID NO. 48),
AATVGSLAGQPLQERAQAWGERLR (SEQ ID NO. 49), AATVGSLAGQPLQERAQAWGERL
(SEQ ID NO. 50), KHNLGHGHKHERDQGHGHQ (SEQ ID NO. 51),
NLGHGHKHERDQGHGHQ (SEQ ID NO. 52), GSESGIFTNTKESSSHHPGIAEFPSRG (SEQ
ID NO. 61), or combinations thereof. In an additional embodiment,
the isolated or identified peptide profile indicating cancer of the
bladder comprises a decreased amount of peptides or peptide
fragments of DSGEGDFLAEGGGVR (SEQ ID NO. 1), SGEGDFLAEGGGVR (SEQ ID
NO. 2), GEGDFLAEGGGVR (SEQ ID NO. 3), EGDFLAEGGGVR (SEQ ID NO. 4),
GDFLAEGGGVR (SEQ ID NO. 5), DFLAEGGGVR (SEQ ID NO. 6),
GVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 14), RPPGFSPFR (SEQ ID NO.
19), RPPGFSPF (SEQ ID NO. 20), FLAEGGGUR (SEQ ID NO. 24), LAEGGGUR
(SEQ ID NO. 25), GLEEELQFSLGSKINV (SEQ ID NO. 33), HAAYHPF (SEQ ID
NO. 39), SSSYSKQFTSSTSYNRGDSTFESKSYKMA (SEQ ID NO. 55),
SSSYSKQFTSSTSYNRGDSTFESKSYKM (SEQ ID NO. 56),
SSSYSKQFTSSTSYNRGDSTFESKSY (SEQ ID NO. 57),
SSSYSKQFTSSTSYNRGDSTFESKS (SEQ ID NO. 58), SSYSKQFTSSTSYNRGDSTFE
(SEQ ID NO. 60), or combinations thereof.
[0049] In yet another embodiment, the invention provides an
isolated or identified peptide profile indicating cancer of the
breast comprising an increased amount of peptides or peptide
fragments of GLPGPPDVPDHAAYHPF (SEQ ID NO. 16), RPPGFSPFR (SEQ ID
NO. 19), RPPGFSPF (SEQ ID NO. 20), RNGFKSHALQLNNRQI (SEQ ID NO.
21), NGFKSHALQLNNRQI (SEQ ID NO. 22), GLEEELQFSLGSKINVKVGGNS (SEQ
ID NO. 23), FLAEGGGUR (SEQ ID NO. 24), NGFKSHALQLNNRQ (SEQ ID NO.
30), GLEEELQFSLGSKINVKVGGNSKGTL (SEQ ID NO. 32), GLEEELQFSLGSKINV
(SEQ ID NO. 33), HAAYHPFR (SEQ ID NO. 34), QLGLPGPPDVPDHAAYHPFR
(SEQ ID NO. 35), SSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO. 38),
NVHSAGAAGSRMNFRPGVLSS (SEQ ID NO. 41), ISASAEELRQRLAPLAEDVRGNL (SEQ
ID NO. 45), AVPPNNSNAAEDDLPTVELQGVVPR (SEQ ID NO. 53),
ALGISPFHEHAEVVFTANDSGPR (SEQ ID NO. 54), SSYSKQFTSSTSYNRGDSTFE (SEQ
ID NO. 60), DEAGSEADHEGTHSTKRGHAKSRPV (SEQ ID NO. 62), or
combinations thereof. In an additional embodiment, the isolated or
identified peptide profile indicating cancer of the breast
comprises a decreased amount of peptides or peptide fragments of
SGEGDFLAEGGGVR (SEQ ID NO. 2), GEGDFLAEGGGVR (SEQ ID NO. 3),
SSKITHRIHWESASLL (SEQ ID NO. 8), HWESASLL (SEQ ID NO. 12),
ITHRIHWESASLL (SEQ ID NO. 26), or combinations thereof.
[0050] In one embodiment of the peptide profile of the invention,
the profile is present in an isolated biological sample. In another
embodiment, the identified profile is stored by electronic
means.
[0051] In one aspect, the invention provides a method of generating
a peptide profile of a subject having, or at risk of having, cancer
of the prostate, comprising the steps of:
[0052] i) combining an exogenous peptide including but not limited
to a complement C3f, ITIH4, clusterin, complement C4-alpha,
fibrinopeptide A, kininogen, factor XIII, and fibrinogenA peptide
or a combination thereof with a biological sample from the subject;
and
[0053] ii) proteolytically digesting a peptide of step i),
[0054] thereby generating a peptide profile of the subject.
[0055] In additional embodiments of the invention, the peptide
profile indicates that the subject has or is at risk of having
cancer of the prostate.
[0056] In one aspect, the invention provides a method of generating
a peptide profile of a subject having, or at risk of having, cancer
of the bladder, comprising the steps of:
[0057] i) combining an exogenous peptide including but not limited
to a complement C3f, ITIH4, clusterin, complement C4-alpha,
fibrinopeptide A, bradykinin, APO A-I, APO A-IV, APO E, kininogen,
and fibrinogenA peptide or a combination thereof with a biological
sample from the subject; and
[0058] ii) proteolytically digesting a peptide of step i), thereby
generating a peptide profile of the subject.
[0059] In an additional embodiment of the invention, the peptide
profile indicates that the subject has or is at risk of having
cancer of the bladder.
[0060] In one aspect, the invention provides a method of generating
a peptide profile of a subject having, or at risk of having, cancer
of the breast, comprising the steps of:
[0061] i) combining an exogenous peptide including but not limited
to a ITIH4, bradykinin, complement C4-alpha, fibrinopeptide A,
complement C3f, APO A-IV, factor XIII, transthyretin and
fibrinogenA peptide or a combination thereof with a biological
sample from the subject; and
[0062] ii) proteolytically digesting a peptide of step i), thereby
generating a peptide profile of the subject.
[0063] In an additional embodiment of the invention, the peptide
profile indicates that the subject has or is at risk of having
cancer of the breast.
[0064] In one aspect, the invention is provides a method of
generating a peptide profile of a subject having, or at risk of
having, cancer of the thyroid, comprising the steps of:
[0065] i) combining an exogenous peptide selected from the group
consisting of a fibrinopeptide A, fibrinogenA, complement C3f
peptide and combinations thereof with a biological sample from the
subject, and
[0066] ii) proteolytically digesting a peptide of step i),
thereby generating a peptide profile of the subject.
[0067] In an additional embodiment of the invention, the peptide
profile indicates that the subject has or is at risk of having
cancer of the thyroid.
[0068] In further embodiments of the invention, the exogenous
peptide is labeled with an isotope. In yet further embodiments of
the invention, the biological sample is serum or plasma. In yet
further embodiments of the invention, the exogenous peptide is a
synthetic peptide. In yet further embodiments of the invention, the
exogenous peptide is comprised of D-amino acids. In yet further
embodiments of the invention, the proteolytic digest is analyzed,
for example, using mass spectrometry.
[0069] Methods of the invention can further comprise the step of
obtaining the exogenous peptide.
[0070] In yet another aspect, the invention provides a kit for
generating a peptide profile of a subject having, or at risk of
having, cancer of the bladder, breast, prostate or thyroid
comprising an exogenous peptide or peptide fragment selected from
the group consisting of complement C3f peptide, ITIH4 peptide,
clusterin peptide, complement C4-alpha peptide, fibrinopeptideA
peptide, bradykinin peptide, APO A-I peptide, APOA-IV peptide, APO
E peptide, kininogen peptide, factor XIII peptide, transthyretin
peptide and fibrinogenA peptide and instructions for use and/or a
packaging means thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0071] FIG. 1A shows the color-coding scheme followed in the
representation of data collected for the blood samples from healthy
volunteers (n=33) and from patients with advanced prostate (n=32),
bladder (n=20) and breast (n=21) cancer.
[0072] FIG. 1B shows the results of unsupervised, average-linkage
hierarchical clustering performed using standard correlation as a
distance metrics (`GeneSpring` program), between each cancer group
and the control, in binary format. The entire peak list
(651.times.106) was used. Columns represent samples; rows are
m/z-peaks (i.e., peptides). Dendrogram colors follow the
color-coding scheme of panel A. The heat map scale of normalized
intensities is from 0 (green) to 200 (red), with the midpoint at
100 (yellow).
[0073] FIG. 1C shows the results of hierarchical clustering
performed for the three cancer groups plus control (as in 1B,
above).
[0074] FIG. 1D shows the results of Principal Component Analysis
(PCA) of the three cancer groups plus controls based on the full
peak list. Color-coding is as in panel A. The first three principal
components, accounting for most of the variance in the original
data set are shown.
[0075] FIG. 2A shows pie charts depicting the peak number reduction
in three m/z ranges, which illustrates the impact of each filter on
peptides of different molecular mass.
[0076] FIG. 2B depicts the Venn-diagrams showing the number of
peptides that passed two selection steps. m/z-peaks with higher
intensities in one (or more) of the cancer groups as compared to
controls are shown in the left panel, while those with lower
intensities are shown in the right panel. The numbers shown outside
the diagrams indicate the total number of peptides of a specific
cancer group that were either up or down.
[0077] FIG. 2C shows heat maps comparing the selected features of
the three cancer groups with controls in multi-class and binary
formats. Columns represent samples (as indicated per group); rows
are peptide m/z-peaks (not in numerical order). The number of
peptides used in each binary comparison (i.e., 58, 14, and 14) is
the sum of those that were specifically higher and lower in each
cancer group; the multi-class heat map contains the total,
non-redundant number of peptides (i.e., 68). The `multi-class`,
`bladder` and `breast` heat map scales of normalized intensities
are from 0 (green) to 500 (red), with the midpoint at 250 (yellow);
those of the `prostate` heat map are, respectively, 0, 2,000 and
1,000.
[0078] FIG. 2D depicts overlays of mass spectra obtained from the
three binary comparisons (cancer vs. control). Mono-isotopic masses
are listed for each peak. Two statistically significant differences
in peptide intensities (one higher; one lower) between prostate
cancer (blue) and controls (yellow) are shown, as well as one
higher-intensity peptide for bladder cancer (green) and one for
breast cancer (red).
[0079] FIGS. 3A and 3B show MALDI-TOF mass spectral overlays of
selected peaks derived from serum peptide profiling of three groups
of cancer patients and healthy controls. Each overlay shows a
binary comparison for all spectra from either the bladder cancer
(n=20; green), or prostate cancer (n=32; blue) or breast cancer
patient group (n=21; red) versus the control group (n=33; yellow).
They are arrayed in a way that the same mass range window is shown
for each of the three binary comparisons, in which spectral
intensities were normalized and scaled to the same size, except for
`2021.05`, which is included herein as an example of the vast
majority of peptide-ions with intensities not statistically
different between any two groups. (A) Overlays of mass spectra of
selected peptides of known sequence (see FIG. 3) that showed
statistically significant differences between peak intensities in
one or more of the three binary comparisons. The mono-isotopic mass
(m/z) of the peak is shown for each peptide. (B) Overlays of mass
spectra of some as yet unidentified peptides that also showed
statistically significant differences between peak intensities in
one or more of the three binary comparisons. The bin `name` (a
number that is close to the average isotopic mass) is shown for
each peptide.
[0080] FIG. 4 shows a fragment ion spectrum for MALDI-TOF/TOF MS/MS
identification of serum peptide 2305.20 as a fragment of complement
4a. b''- and y''-fragment ion series are indicated, together with
the limited sequences (above arrows). Note that y''-ions originate
at the C-terminus and that the sequence therefore reads backwards
(see direction of the arrows).
[0081] FIG. 5A lists the groups (`ladders`) of overlapping
sequences of the peptides identified by MALDI-TOF/TOF MS/MS. Taken
together, 61 peptide-ions on the list have clear peptide-ion marker
potential (adjusted p<0.0002; see FIG. 5B, below) for at least
one type of cancer and are color-coded in blue (prostate cancer),
green (bladder cancer) or red (breast cancer). The resulting
`barcodes` for the three cancer types consist of 26 (prostate), 50
(bladder) and 25 (breast) peptide-ions. Color-coded peptides have
either higher (no dot) or lower (black dot) differential ion
intensities in a particular cohort of cancer samples as compared to
controls. Of the 8 non-markers listed here, full-length C3f
(m/z=2021.05) and one member of the fibrinogen-alpha cluster
(m/z=2553.01) gave comparable ion signals in all patient group and
control sera (see FIG. 5B; FIG. 3, `2021`), and, therefore,
represent virtual internal standards (yellow-coded). Six peptides
(pink-coded) in the clusters were randomly observed in samples of
the cancer and control groups and have neither discriminant nor
internal control value. Note that the measured m/z values, as
listed, are mono-isotopic and, therefore, smaller than the
corresponding average isotopic values in FIG. 13a. Amino acids in
brackets were not experimentally observed but are shown to either
indicate putative full-length sequences of the founders, each
resulting from specific proteolyis of precursor proteins, and/or of
the positions of the putative `trypsin-like` cleavage sites
(Arg/Lys-Xaa).
[0082] FIG. 5B depicts a table listing additional details of the
identified peptides as m/z values, MS-ion intensities, and
`barcodes` (blue, green or red--as described above). The actual
barcodes (blue, green or red) are composed of entries that showed
clear peptide-ion marker potential (adjusted p<0.0002) for at
least one type of cancer. Adjusted p-value is the overriding
criterion, leading to final barcodes of 26 (prostate), 50 (bladder)
and 25 (breast) peptide-ions. The second column lists median
intensities of each m/z-peak in the control samples. Peak intensity
ratios (columns 3-5) were calculated by dividing the median values
of each m/z-peak in each cancer group by the median value of the
corresponding peak in the control samples. Ratios (r) for the
peptides that are part of one or more barcodes are shaded; dark
grey when the median signal was of higher intensity in a particular
cancer (r.gtoreq.1.6), lighter grey when it was lower
(r.gtoreq.0.66). The significance levels (p values) of three
different one-way ANOVA Mann-Whitney tests (columns 6-8) and of a
multi-class Kluskal-Wallis test (column 9) are given. C3f (coded
yellow) has virtually no discriminant value.
[0083] FIG. 6 shows, in bar graph form, the median intensity for
each serum peptide in each of the three cancer groups (color-coding
as indicated) plotted as the ratio versus the median intensity of
the counterpart in the control group (r=case/control). Ratios are
plotted on a log scale ranging from 0.1 to 10. Bars pointing to the
left (r<1) or right (r>1) indicate, respectively, lower or
higher median intensities in a cancer group as in the control
group. Peptides that didn't show much difference in median ion
intensity between case and control groups map closely to or onto
the centerline (r=1).
[0084] FIG. 7 shows a flow chart-type diagram delineating the
approach used for development and validation of (i) the
68-peptide-ion signature and (ii) the prostate cancer barcode
consisting of 26 serum peptides with known sequence (blue-coded in
FIG. 5). Numbers that are encircled indicate total number of
selected peptides at that stage of the study.
[0085] FIG. 8A schematically depicts the independent prostate
cancer serum sample groups identified for the validation of the
established biomarkers.
[0086] FIGS. 8B and 8C show the results of Hierarchical Cluster
(HCA) and Principal Component (PCA) Analyses of all spectra from
the Prostate #1 (blue), Prostate #2 (cyan) and control groups
(yellow). Two limited sets of peptide-ions were used for the
analyses: the 68 combined peptides that had statistically
significant differences in intensity for the three binary
comparisons (FIG. 2B; FIG. 17) (left), and the 26 sequenced
peptides that constitute the prostate cancer barcode (color-coded
blue in FIG. 5) (right). The rest of the .about.650 peptide-ions
were ignored for the cluster analysis. Dendrogram colors follow the
color-coding scheme of panel A. The heat map scale of normalized
ion-intensities is from 0 (green) to 2,000 (red), with the midpoint
at 1,000 (yellow). For the PCA, the first three principal
components, accounting for most of the variance in the original
data set, are shown.
[0087] FIG. 8D shows a table listing the results of class
prediction analysis of the prostate cancer validation set (Prostate
#2) using Support Vector Machine (SVM) and either all 651
m/z-values or the 68-, 26-feature sets described above. Analyses
were done using linear kernel. The proportions of correct
predictions are listed. The binomial confidence intervals (at 95%)
were 87.1-99.9% for 40 correct predictions out of 41, and 91.4-100%
for 41/41. The training sets were either Prostate #1 versus control
(`binary`) or the 3 cancer groups (Prostate #1, bladder and breast
cancer) plus controls (`multi-class`).
[0088] FIG. 9 shows MALDI-TOF MS read-outs of fresh plasma (top
panel), indicating very low levels of small peptides, except for
bradykinin and desArg-bradykinin, of an aliquot withdrawn
immediately (i.e., after 15-20 s) after addition of synthetic C3f
(1 pmole/.mu.L plasma) (middle panel, indicating removal of the
C-terminal Arg, by a carboxypeptidase, in a matter of seconds), and
of an aliquot withdrawn after another 15 minutes at room
temperature (lower panel, indicating that C3f is then further
degraded by the activity of aminopeptidases to result in a type of
sequence ladder as endogenously present in serum).
[0089] FIG. 10 schematically depicts the activity of serum
proteases. Amino acids are color-coded to represent sequence
clusters of C3f (left) or FPA (right), which are just two examples
of all the observed clusters.
[0090] FIG. 11A graphically depicts the distribution of serum
peptides. Number of m/z-peaks are plotted as a function of m/z
range. The first bin, from m/z=0 to 700, is empty, as no data was
collected in that region. No bins are shown in the range >10
kDa.
[0091] FIG. 11B likewise graphically depicts the distribution of
serum peptides. Here, however, number of m/z-peaks are plotted as a
function of normalized intensity. No bins are shown in the region
over 1,000 arbitrary units. The highlighted area indicates the
range above the median peak-intensity threshold, used for selecting
potential biomarkers (FIG. 17).
[0092] FIG. 12 depicts a histogram that shows, starting with a
total of 651 unique m/z-peaks (blue bars) derived from three groups
of cancer patients and healthy controls, the number of peptides in
each mass range that passed two filters applied during feature
selection.
[0093] FIG. 13A shows a table listing averages plus (.+-.) standard
deviations and medians (in brackets) of the intensities of each
m/z-peak (i.e., serum peptide) within a particular data set derived
from each of the three cancer patient groups and of the healthy
controls. Intensities refer to normalized units that were
calculated for each peak by dividing its raw intensity by the total
of all of the intensities in that spectrum (TIC--Total Ion Count).
The resultant values were then multiplied by fixed scaling factor
(1.times.10.sup.7) to convert the data to a `user-friendly` scale
(i.e. most values .gtoreq.1).
[0094] FIG. 13B shows a table listing ratios calculated by dividing
the median normalized intensity of each m/z-peak in each cancer
group by the median of the same m/z-peak in the control group. To
avoid having to divide by zero, any median value of less than was
converted to 1. This was applied to all groups. Data for a second,
independent validation set of prostate cancer samples is also
listed.
[0095] FIG. 13C shows a table listing the false discovery rate
adjusted p-values calculated for each m/z-peak using the
Mann-Whitney rank sum test (for binary comparisons) or the
Kruskal-Wallis test (for multi-class comparisons). The group of 68
m/z-peaks listed were derived from the original peak list,
containing normalized ion intensities (and medians within a group,
case/control ratios and adjusted p-values) for each of the 651
m/z-peaks for each of the 106 samples, by applying p-value and
median intensity cut-off filters (p<0.00001; median intensity
.gtoreq.500 `units`). Entries which passed both filters in one or
more cancer groups are color-coded: prostate cancer (14; blue),
breast cancer (14; red) and bladder cancer (58; green).
[0096] FIG. 14 shows a table listing the total serum peptide
sequences, organized per overlapping cluster; with clusters
organized per precursor protein (NCBI ID nos. are given). Positions
in the precursor proteins are indicated. Residues between brackets
were not observed but are listed in the present table to indicate
the putative primary cleavage sites by endoproteases. Additional
information is given, as for instance the relative position of
adjacently located peptides or peptide clusters, identity of
previously known serum petides (e.g., FPA, C3f), position of
propeptides, and location of C-termini (C-t). Key: Met.sub.ox or
M.sub.ox, oxidized methionine; Pro.sub.hydroxyl, hydroxylated
proline.
[0097] FIG. 15 shows a table listing the locations of sequenced
serum peptides in the precursor proteins. NCBI ID nos. are given,
as well as the positions of known, processed serum proteins,
peptides and propeptides. The peptide sequences obtained herein are
shown in bold and are underlined.
[0098] FIG. 16A shows, in table form, the data set of 651 unique
m/z-peaks derived from MALDI-TOF MS serum peptide profiling of
three groups of cancer patients and healthy controls. Presented are
the averages plus (.+-.) standard deviations and the median values
(in brackets) of the intensities of each m/z-peak (i.e., serum
peptide) within a particular data set derived from each of the
three cancer patient groups and of the healthy controls; a second,
independent validation set of prostate cancer samples is also
listed. Intensities refer to normalized units that were calculated
for each peak by dividing its raw intensity by the total of all the
intensities in that spectrum (TIC--Total Ion Count). The resultant
values were then multiplied by fixed scaling factor
(1.times.10.sup.7) to convert the data to a `user-friendly` scale
(i.e. most values .gtoreq.1).
[0099] FIG. 16B shows, in table form, the data set of 651 unique
m/z-peaks derived from MALDI-TOF MS serum peptide profiling of
three groups of cancer patients and healthy controls.
[0100] FIG. 16C shows, in table form, the data set of 651 unique
m/z-peaks derived from MALDI-TOF MS serum peptide profiling of
three groups of cancer patients and healthy controls.
[0101] FIGS. 17A, 17B, and 17C show, in table form, the data set of
68 putative biomarker m/z-peaks, derived from MALDI-TOF MS serum
peptide profiling of three groups of cancer patients and healthy
controls. The figures contain (i) means plus (.+-.) standard
deviations, and medians (in brackets); (ii) discriminant analysis
false positive rates (p-values); and (iii) ratios of the median
intensities in a group for all 68 m/z-peaks retained after applying
p-value and median intensity cutoff filters (p<0.00001; median
intensity .gtoreq.500 units). All values were extracted from FIGS.
16A-C, above. Entries which passed both filters in one or more
cancer groups are color-coded: prostate cancer (14; blue), breast
cancer (14; red) and bladder cancer (58; green).
[0102] FIG. 18 shows SEQ ID NO:63, GENBANK Accession No. AAH00664,
C3F protein (Homo sapiens), amino acid residues 1-436.
[0103] FIG. 19 shows SEQ ID NO:64, GENBANK Accession No. Q14624,
Inter-alpha-trypsin inhibitor heavy chain H4 precursor (ITI heavy
chain H4) (Homo sapiens), amino acid residues 1 to 930, wherein
29-661="70 kDa inter-alpha-trypsin inhibitor heavy chain H4" and
689-930="35 kDa inter-alpha-trypsin inhibitor heavy chain H4."
[0104] FIG. 20 shows SEQ ID NO:65, GENBANK Accession No. AAP88927,
clusterin (complement lysis inhibitor (Homo sapiens), amino acid
residues 1 to 447.
[0105] FIG. 21 shows SEQ ID NO:66, GENBANK Accession No. AAR89159,
C4A (Homo sapiens), amino acid residues 1 to 534.
[0106] FIG. 22 shows SEQ ID NO:67, GENBANK Accession No.
NP.sub.--068657, fibrinogen, alpha chain isoform alpha
preproprotein (Homo sapiens), amino acid residues 1 to 644, wherein
20-35 product="fibrinopeptide A."
[0107] FIG. 23 shows SEQ ID NO:68, GENBANK Accession No. P01042,
kininogen precursor (Alpha-2-thiol proteinase inhibitor) (Homo
sapiens), amino acid residues 1 to 644, wherein
381-389="Bradykinin."
[0108] FIG. 24 shows SEQ ID NO:69, GENBANK Accession No.
NM.sub.--021871, Homo sapiens fibrinogen alpha chain (FGA),
transcript variant alpha, mRNA.
[0109] FIG. 25 shows SEQ ID NO:70, GENBANK Accession No.
NM.sub.--000039, Homo sapiens apolipoprotein A-I (APOA1), mRNA.
[0110] FIG. 26 shows SEQ ID NO:71, GENBANK Accession No.
NM.sub.--000482, Homo sapiens apolipoprotein A-IV (APOA4),
mRNA.
[0111] FIG. 27 shows SEQ ID NO:72, GENBANK Accession No.
NM.sub.--000041, Homo sapiens apolipoprotein E (APOE), mRNA.
[0112] FIG. 28 shows SEQ ID NO:73, GENBANK Accession No.
NM.sub.--000893, Homo sapiens kininogen (KNG1).
[0113] FIG. 29 shows SEQ ID NO:74, GENBANK Accession No.
NM.sub.--000129, Homo sapiens coagulation factor XIII, A1
polypeptide (F13A1), mRNA.
[0114] FIG. 30 shows SEQ ID NO:75, GENBANK Accession No.
NM.sub.--000371, Homo sapiens transthyretin (prealbumin,
amyloidosis type I)(TTR), mRNA.
[0115] FIG. 31 shows, in table form, 66 reference peptides. All
amino acids are D-stereo-isomers, except for the isotope-containing
(L-isomer). Isotope-labeled amino acids: L, .sup.13C(6)-Leu; F,
.sup.13C(6-ring)-Phe; V, .sup.13C(5)/.sup.15N(1)-Val. (Note:
isotope labels result in a molecular mass increase by 6 Da for each
peptide). Surrogate marker code: P, prostate cancer; B, breast
cancer; BL, bladder cancer; T, thyroid cancer; +, median ion
intensity of this particular peptide in MALDI-TOF MS is higher in
cancer samples than in controls; -, median ion intensity lower in
cancer than controls.
[0116] FIG. 32 shows the MALDI-based, relative quantitation of
serum peptides: A, normalized ion intensities as spectral overlays
and B, as a heat plot. C shows the relative quantitation of
normalized ion intensities in bar graph form.
[0117] FIG. 33 shows, in table form, founder peptides. Total 15
syntheses, including 2 (#7 and 11) or more multi-samplings;
.gtoreq.18 cleavages, purifications, QC and quantitation.
Isotope-labeled amino acids: L, .sup.13C(6)-Leu; F,
.sup.13C(6-ring)-Phe; V, .sup.13C(5)/.sup.15N(1)-Val; A,
.sup.13C(3)/.sup.15N(1)-Ala; resulting in molecular mass increase
of 12 Da per peptide.
[0118] FIG. 34A shows median ion intensities in MALDI spectra taken
of breast cancer sera vs. control sera. FIG. 34B shows selected
views of isotopically resolved or partially resolved peptide-ion
peaks; red, breast cancer; black, controls.
[0119] FIG. 35 shows ten peptide-triplets and plots of the ratios
between exogenously derived peptides and reference peptide
calculated. Inset is a small section of the MALDI spectrum showing
the position of the monoisotopic envelopes for each of the three
iso-peptides.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0120] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. As used herein,
the following terms have the meanings ascribed to them unless
specified otherwise.
[0121] A "subject" is a vertebrate, preferably a mammal, more
preferably a primate and still more preferably a human. Mammals
include, but are not limited to, primates, humans, farm animals,
sport animals, and pets.
[0122] As used herein, "serum" refers to the fluid portion of the
blood obtained after removal of the fibrin clot and blood cells,
distinguished from the plasma in circulating blood. As used herein,
"plasma" refers to the fluid, noncellular portion of the blood,
distinguished from the serum obtained after coagulation.
[0123] As used herein, "sample" or "biological sample" refers to
anything, which may contain an analyte (e.g., peptide) for which an
analyte assay is desired. The sample may be a biological sample,
such as a biological fluid or a biological tissue. Examples of
biological fluids include urine, blood, plasma, serum, saliva,
semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic
fluid or the like. Biological tissues are aggregates of cells,
usually of a particular kind including, for example, connective,
epithelium, muscle and nerve tissues. Examples of biological
tissues also include organs, tumors, lymph nodes, arteries and
individual cell(s).
[0124] The term "isolated" refers to one or more compositions
obtained from and/or contained in a sample apart from the body.
[0125] The term "identified" as in an "identified peptide" or
"peptide profile" refers to one or more compositions or information
relating thereto (e.g., a peptide and its amino acid sequence
information) obtained under conditions of selection. Such
information may optionally be stored by electronic means.
[0126] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules comprising an open reading frame
encoding a marker protein.
[0127] "Gas phase ion spectrometer" refers to an apparatus that
detects gas phase ions. Gas phase ion spectrometers include an ion
source that supplies gas phase ions. Gas phase ion spectrometers
include, for example, mass spectrometers, ion mobility
spectrometers, and total ion current measuring devices. "Gas phase
ion spectrometry" refers to the use of a gas phase ion spectrometer
to detect gas phase ions.
[0128] "Mass spectrometer" refers to a gas phase ion spectrometer
that measures a parameter that can be translated into
mass-to-charge ratios of gas phase ions. Mass spectrometers
generally include an ion source and a mass analyzer. Examples of
mass spectrometers are time-of-flight, magnetic sector, quadrupole
filter, ion trap, ion cyclotron resonance, electrostatic sector
analyzer and hybrids of these. "Mass spectrometry" refers to the
use of a mass spectrometer to detect gas phase ions.
[0129] "Laser desorption mass spectrometer" refers to a mass
spectrometer that uses laser energy as a means to desorb,
volatilize, and ionize an analyte.
[0130] "Tandem mass spectrometer" refers to any mass spectrometer
that is capable of performing two successive stages of m/z-based
discrimination or measurement of ions, including ions in an ion
mixture. The phrase includes mass spectrometers having two mass
analyzers that are capable of performing two successive stages of
m/z-based discrimination or measurement of ions tandem-in-space.
The phrase further includes mass spectrometers having a single mass
analyzer that is capable of performing two successive stages of
m/z-based discrimination or measurement of ions tandem-in-time. The
phrase thus explicitly includes Qq-TOF mass spectrometers, ion trap
mass spectrometers, ion trap-TOF mass spectrometers, TOF-TOF mass
spectrometers, Fourier transform ion cyclotron resonance mass
spectrometers, electrostatic sector--magnetic sector mass
spectrometers, and combinations thereof.
[0131] "Mass analyzer" refers to a sub-assembly of a mass
spectrometer that comprises means for measuring a parameter that
can be translated into mass-to-charge ratios of gas phase ions. In
a time-of-flight mass spectrometer the mass analyzer comprises an
ion optic assembly, a flight tube and an ion detector.
[0132] The term "MALDI" is used herein to refer to Matrix-Assisted
Laser Desorption/Ionization, a process wherein analyte is embedded
in a solid or crystalline "matrix" of light-absorbing molecules
(e.g., nicotinic, sinapinic, or 3-hydroxypicolinic acid), then
desorbed by laser irradiation and ionized from the solid phase into
the gaseous or vapor phase, and accelerated as intact molecular
ions towards a detector. The "matrix" is typically a small organic
acid mixed in solution with the analyte in a 10,000:1 molar ratio
of matrix/analyte. The matrix solution can be adjusted to neutral
pH before use.
[0133] The term "MALDI-TOF MS" is used herein to refer to
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass
spectrometry.
[0134] The term "MALDI ionization surface" is used herein to refer
to a surface for presentation of matrix-embedded analyte into a
mass spectrometer for MALDI. In general, the terms "probe" or
"probe element" are used interchangeably to refer to a device for
presenting analyte into a mass spectrometer for irradiation and
desorption. Metals such as gold, copper and stainless steel are
typically used to form MALDI ionization surfaces. However, other
commercially-available inert materials (e.g., glass, silica, nylon
and other synthetic polymers, agarose and other carbohydrate
polymers, and plastics) can be used where it is desired to use the
surface to actively capture an analyte or as a reaction zone for
chemical modification of the analyte.
[0135] "Solid support" refers to a solid material, which can be
derivatized with, or otherwise attached to, a capture reagent.
Exemplary solid supports include probes, microtiter plates and
chromatographic resins.
[0136] "Eluant" or "wash solution" refers to an agent, typically a
solution, which is used to affect or modify adsorption of an
analyte to an adsorbent surface and/or remove unbound materials
from the surface. The elution characteristics of an eluant can
depend on, for example, pH, ionic strength, hydrophobicity, degree
of chaotropism, detergent strength and temperature.
[0137] "Monitoring" refers to recording changes in a continuously
varying parameter (e.g. monitoring progression of a cancer).
[0138] "Biochip" refers to a solid substrate having a generally
planar surface to which an adsorbent is attached. Frequently, the
surface of the biochip comprises a plurality of addressable
locations, each of which location has the adsorbent bound there.
Biochips can be adapted to engage a probe interface, and therefore,
function as probes.
[0139] "Protein biochip" refers to a biochip adapted for the
capture of polypeptides.
[0140] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an analog or mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. Polypeptides can be modified, e.g., by the
addition of carbohydrate residues to form glycoproteins. The terms
"polypeptide," "peptide" and "protein" include glycoproteins, as
well as non-glycoproteins.
[0141] An "exogenous peptide" is a peptide obtained from a
biological source that is external to the subject's body or by
synthetic means.
[0142] The terms "peptide", "peptide marker", "marker" and
"biomarker" are used interchangeably in the context of the present
invention and refer to a polypeptide which is differentially
present in a sample taken from subjects having human cancer as
compared to a comparable sample taken from control subjects (e.g.,
a person with a negative diagnosis or undetectable cancer, normal
or healthy subject). The markers are identified by molecular mass
in Daltons, and include the masses centered around the identified
molecular masses for each marker.
[0143] The term "detecting" means methods which include identifying
the presence or absence of marker(s) in the sample, quantifying the
amount of marker(s) in the sample, and/or qualifying the type of
biomarker. Detecting includes identifying the presence, absence or
amount of the object to be detected (e.g. a serum peptide
marker).
[0144] "Diagnostic" means identifying the presence or nature of a
pathologic condition, i.e., cancer. While a particular diagnostic
method may not provide a definitive diagnosis of a condition, it
suffices if the method provides a positive indication that aids in
diagnosis.
[0145] As used herein, the term "sensitivity" is the percentage of
marker-detected subjects with a particular disease.
[0146] As used herein, the term "specificity" is the percentage of
subjects correctly identified as having a particular disease i.e.,
normal or healthy subjects. For example, the specificity is
calculated as the number of subjects with a particular disease as
compared to non-cancer subjects (e.g., normal healthy
subjects).
[0147] The phrase "differentially present" refers to differences in
the quantity and/or the frequency of a marker present in a sample
taken from subjects having human cancer as compared to a control
subject. For example, serum peptide markers described herein are
present at an elevated level in samples of subjects compared to
samples from control subjects. In contrast, other markers described
herein are present at a decreased level in samples of cancer
subjects compared to samples from control subjects. Furthermore, a
marker can be a polypeptide, which is detected at a higher
frequency or at a lower frequency in samples of human cancer
subjects compared to samples of control subjects. A marker can be
differentially present in terms of quantity, frequency or both. A
polypeptide is differentially present between two samples if the
amount of the polypeptide in one sample is statistically
significantly different from the amount of the polypeptide in the
other sample. Alternatively or additionally, a polypeptide is
differentially present between two sets of samples if the frequency
of detecting the polypeptide in the cancer subjects' samples is
statistically significantly higher or lower than in the control
samples.
[0148] "Optional" or "optionally" means that the subsequently
described feature or structure may or may not be present in the
analysis system or that the subsequently described event or
circumstance may or may not occur, and that the description
includes instances where said feature or structure is present and
instances where the feature or structure is absent, or instances
where the event or circumstance occurs and instances where it does
not.
[0149] The term "obtaining" as in "obtaining the exogenous peptide"
is intended to include purchasing, synthesizing or otherwise
acquiring the exogenous (or indicated substance or material).
[0150] The terms "comprises", "comprising", and the like are
intended to have the broad meaning ascribed to them in U.S. Patent
Law and can mean "includes", "including" and the like.
[0151] It is to be understood that this invention is not limited to
the particular component parts of a device described or process
steps of the methods described, as such devices and methods may
vary. It is also to be understood that the terminology used herein
is for purposes of describing particular embodiments only, and is
not intended to be limiting. As used in the specification and the
appended claims, the singular forms "a", "an", and "the" include
plural referents unless the context clearly indicates otherwise.
Thus, for example, reference to "an analyte" includes mixtures of
analytes, reference to "a MALDI ionization surface" includes two or
more such ionization surfaces, reference to "a microchannel"
includes more than one such component, and the like. Furthermore,
reference to "cancer" may signify cancer in general (i.e., cancer
of any type) or cancer of a specific type. Accordingly, the
description herein of a subject as having no detectable cancer may
signify a subject in which a specific type of cancer (for example,
bladder) is not detectable. However, such a description may not
necessarily signify that the subject has no type of cancer
whatsoever.
[0152] Other definitions appear in context throughout the
specification.
II. Methods and Peptide Profiles of the Invention
[0153] The present invention provides peptide markers generated
from comparisons of protein profiles from subjects diagnosed with
cancer and from subjects without known neoplastic diseases. In
particular, the invention provides that these markers, used
individually or in combination with other markers, provide a method
of diagnosing and monitoring cancer in a subject having cancer of
the prostate, of the bladder, or of the breast.
[0154] Markers that are differentially present in samples of cancer
subjects and control subjects find application in methods and kits
for determining cancer status. Accordingly, methods are provided
for identifying cancer of the prostate, bladder, or breast in a
subject comprising detecting a differential presence of a biomarker
in subjects with cancer of the prostate, bladder, or breast vs.
without cancer of the prostate, bladder, or breast in a biological
sample obtained from the subject. The amount of one or more
biomarkers found in a test sample compared to a control, or the
presence or absence of one or more markers in the test sample
provides useful information regarding the cancer status of the
patient.
[0155] A. Types of Samples
[0156] The markers can be measured in different types of biological
samples. The sample is preferably a biological fluid sample.
Examples of a biological fluid sample useful in this invention
include blood, blood serum, plasma, vaginal secretions, urine,
tears, saliva, urine, tissue, cells, organs, seminal fluids, bone
marrow, cerebrospinal fluid, nipple aspirate, etc. Blood serum is a
preferred sample source for embodiments of the invention.
[0157] If desired, the sample can be prepared to enhance
detectability of the markers. For example, to increase the
detectability of markers, a blood serum sample from the subject can
be preferably fractionated by, e.g., Cibacron blue agarose
chromatography and single stranded DNA affinity chromatography,
anion exchange chromatography, affinity chromatography (e.g., with
antibodies) and the like. The method of fractionation depends on
the type of detection method used. Any method that enriches for the
protein of interest can be used. Typically, preparation involves
fractionation of the sample and collection of fractions determined
to contain the biomarkers. Methods of pre-fractionation include,
for example, size exclusion chromatography, ion exchange
chromatography, heparin chromatography, affinity chromatography,
sequential extraction, gel electrophoresis and liquid
chromatography. The analytes also may be modified prior to
detection. These methods are useful to simplify the sample for
further analysis. For example, it can be useful to remove high
abundance proteins, such as albumin, from blood before
analysis.
[0158] B. Detection of Serum Peptide Markers
[0159] Serum Peptide Marker Modification
[0160] A marker can be modified before analysis to improve its
resolution or to determine its identity. For example, the markers
may be subject to proteolytic digestion before analysis. Any
protease can be used. Proteases, such as trypsin, that are likely
to cleave the markers into a discrete number of fragments are
particularly useful. The fragments that result from digestion
function as a fingerprint for the markers, thereby enabling their
detection indirectly. This is particularly useful where there are
markers with similar molecular masses that might be confused for
the marker in question. Also, proteolytic fragmentation is useful
for high molecular weight markers because smaller markers are more
easily resolved by mass spectrometry. In specific embodiments, the
proteases occur or naturally exist in the biological sample.
[0161] To improve detection resolution of the markers,
neuraminidase can, for instance, be used to remove terminal sialic
acid residues from glycoproteins to improve binding to an anionic
adsorbent (e.g., cationic exchange ProteinChip.RTM. arrays) and to
improve detection resolution. In another example, the markers can
be modified by the attachment of a tag of particular molecular
weight that specifically bind to molecular markers, further
distinguishing them. Optionally, after detecting such modified
markers, the identity of the markers can be further determined by
matching the physical and chemical characteristics of the modified
markers in a protein database (e.g., SwissProt).
[0162] It has been found that proteins frequently exist in a sample
in a plurality of different forms characterized by a detectably
different mass. These forms can result from either, or both, of
pre- and post-translational modification. Pre-translational
modified forms include allelic variants, slice variants and RNA
editing forms. Post-translationally modified forms include forms
resulting from proteolytic cleavage (e.g., fragments of a parent
protein), glycosylation, phosphorylation, lipidation, oxidation,
methylation, cystinylation, sulphonation and acetylation. Modified
forms of any marker of this invention also may be used, themselves,
as biomarkers. In certain cases the modified forms may exhibit
better discriminatory power in diagnosis than the specific forms
set forth herein.
[0163] Serum Peptide Marker Purification
[0164] For some of the method embodiments of the invention, it may
be helpful to purify the marker detected by the methods disclosed
herein prior to subsequent analysis. Nearly any means known to the
art for the purification and separation of small molecular weight
substances, e.g., anion or cation exchange chromatography, gas
chromatography, liquid chromatography or high pressure liquid
chromatography may be used. Methods of selecting suitable
separation and purification techniques and means of carrying them
out are known in the art (see, e.g., Labadarious et. al., J.
Chromatography (1984) 310:223-231, and references cited therein;
and Shahrokhin and Gehrke, J. Chromatography (1968) 36:31-41, and
Niessen J. Chromatography (1998) 794:407-435).
[0165] In another embodiment of the method of the invention,
purification of the marker comprises fractioning a sample
comprising one or more protein markers by size-exclusion
chromatography and collecting a fraction that includes the one or
more marker; and/or fractioning a sample comprising the one or more
markers by anion exchange chromatography and collecting a fraction
that includes the one or more markers. Fractionation is monitored
for purity on normal phase and immobilized nickel arrays.
Generating data on immobilized marker fractions on an array is
accomplished by subjecting the array to laser ionization and
detecting intensity of signal for mass/charge ratio; and
transforming the data into computer readable form. Preferably,
fractions are subjected to gel electrophoresis and correlated with
data generated by mass spectrometry. In one aspect, gel bands
representative of potential markers are excised and subjected to
enzymatic treatment and are applied to biochip arrays for peptide
mapping.
[0166] Methods of Detection
[0167] Any suitable method can be used to detect one or more of the
markers described herein. Successful practice of the invention can
be achieved with one or a combination of methods that can detect
and, preferably, quantify the markers. These methods include,
without limitation, hybridization-based methods including those
employed in biochip arrays, mass spectrometry (e.g., laser
desorption/ionization mass spectrometry), fluorescence (e.g.
sandwich immunoassay), surface plasmon resonance, ellipsometry and
atomic force microscopy. Methods may further include, by one or
more of electrospray ionization mass spectrometry (ESI-MS),
ESI-MS/MS, ESI-MS/(MS).sup.n, matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF-MS),
surface-enhanced laser desorption/ionization time-of-flight mass
spectrometry (SELDI-TOF-MS), desorption/ionization on silicon
(DIOS), secondary ion mass spectrometry (SIMS), quadrupole
time-of-flight (Q-TOF), atmospheric pressure chemical ionization
mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS).sup.n,
atmospheric pressure photoionization mass spectrometry (APPI-MS),
APPI-MS/MS, and APPI-(MS).sub.n, quadrupole mass spectrometry,
fourier transform mass spectrometry (FTMS), and ion trap mass
spectrometry, where n is an integer greater than zero.
[0168] Biochip-Based Methods
[0169] Detection methods may include use of a biochip array.
Biochip arrays useful in the invention include protein and nucleic
acid arrays. One or more markers are captured on the biochip array
and subjected to laser ionization to detect the molecular weight of
the markers. Analysis of the markers is, for example, by molecular
weight of the one or more markers against a threshold intensity
that is normalized against total ion current.
[0170] The biochip surfaces may, for example, be ionic, anionic,
hydrophobic; comprised of immobilized nickel or copper ions,
comprised of a mixture of positive and negative ions; and/or
comprised of one or more antibodies, single or double stranded
nucleic acids, proteins, peptides or fragments thereof, amino acid
probes, or phage display libraries. Many protein biochips are
described in the art. These include, for example, protein biochips
produced by Ciphergen Biosystems (Fremont, Calif.), Packard
BioScience Company (Meriden Conn.), Zyomyx (Hayward, Calif.) and
Phylos (Lexington, Mass.). Examples of such protein biochips are
described in the following patents or patent applications: U.S.
Pat. No. 6,225,047 (Hutchens and Yip, "Use of retentate
chromatography to generate difference maps," May 1, 2001);
International publication WO 99/51773 (Kuimelis and Wagner,
"Addressable protein arrays," Oct. 14, 1999); U.S. Pat. No.
6,329,209 (Wagner et al., "Arrays of protein-capture agents and
methods of use thereof," Dec. 11, 2001) and International
publication WO 00/56934 (Englert et al., "Continuous porous matrix
arrays," Sep. 28, 2000).
[0171] Markers may be captured with capture reagents immobilized to
a solid support, such as a biochip, a multiwell microtiter plate, a
resin, or nitrocellulose membranes that are subsequently probed for
the presence of proteins. Capture can be on a chromatographic
surface or a biospecific surface. For example, a sample containing
the markers, such as serum, may be placed on the active surface of
a biochip for a sufficient time to allow binding. Then, unbound
molecules are washed from the surface using a suitable eluant, such
as phosphate buffered saline. In general, the more stringent the
eluant, the more tightly the proteins must be bound to be retained
after the wash.
[0172] Upon capture on a biochip, analytes can be detected by a
variety of detection methods selected from, for example, a gas
phase ion spectrometry method, an optical method, an
electrochemical method, atomic force microscopy and a radio
frequency method. Gas phase ion spectrometry methods are described
herein. Of particular interest is the use of mass spectrometry, and
in particular, SELDI. Optical methods include, for example,
detection of fluorescence, luminescence, chemiluminescence,
absorbance, reflectance, transmittance, birefringence or refractive
index (e.g., surface plasmon resonance, ellipsometry, a resonant
mirror method, a grating coupler waveguide method or
interferometry). Optical methods include microscopy (both confocal
and non-confocal), imaging methods and non-imaging methods.
Immunoassays in various formats (e.g., ELISA) are popular methods
for detection of analytes captured on a solid phase.
Electrochemical methods include voltametry and amperometry methods.
Radio frequency methods include multipolar resonance
spectroscopy.
[0173] Mass Spectrometry-Based Methods
[0174] Mass spectrometry (MS) is a well-known tool for analyzing
chemical compounds. Thus, in one embodiment, the methods of the
present invention comprise performing quantitative MS to measure
the serum peptide marker. The method may be performed in an
automated (Villanueva, et al., Nature Protocols (2006)
1(2):880-891) or semi-automated format. This can be accomplished,
for example with MS operably linked to a liquid chromatography
device (LC-MS/MS or LC-MS) or gas chromatography device (GC-MS or
GC-MS/MS). Methods for performing MS are known in the field and
have been disclosed, for example, in US Patent Application
Publication Nos: 20050023454; 20050035286; U.S. Pat. No. 5,800,979
and references disclosed therein.
[0175] The protein fragments, whether they are peptides derived
from the main chain of the protein or are residues of a side-chain,
are collected on the collection layer. They may then be analyzed by
a spectroscopic method based on matrix-assisted laser
desorption/ionization (MALDI) or electrospray ionization (ESI). The
preferred procedure is MALDI with time of flight (TOF) analysis,
known as MALDI-TOF MS. This involves forming a matrix on the
membrane, e.g. as described in the literature, with an agent which
absorbs the incident light strongly at the particular wavelength
employed. The sample is excited by UV, or IR laser light into the
vapour phase in the MALDI mass spectrometer. Ions are generated by
the vaporization and form an ion plume. The ions are accelerated in
an electric field and separated according to their time of travel
along a given distance, giving a mass/charge (m/z) reading which is
very accurate and sensitive. MALDI spectrometers are commercially
available from PerSeptive Biosystems, Inc. (Frazingham, Mass., USA)
and are described in the literature, e.g. M. Kussmann and P.
Roepstorff, cited above.
[0176] Magnetic-based serum processing can be combined with
traditional MALDI-TOF. Through this approach, improved peptide
capture is achieved prior to matrix mixture and deposition of the
sample on MALDI target plates. Accordingly, methods of peptide
capture are enhanced through the use of derivatized magnetic bead
based sample processing.
[0177] MALDI-TOF MS allows scanning of the fragments of many
proteins at once. Thus, many proteins can be run simultaneously on
a polyacrylamide gel, subjected to a method of the invention to
produce an array of spots on the collecting membrane, and the array
may be analyzed. Subsequently, automated output of the results is
provided by using the ExPASy server, as at present used for
MIDI-TOP MS and to generate the data in a form suitable for
computers.
[0178] Other techniques for improving the mass accuracy and
sensitivity of the MALDI-TOF MS can be used to analyze the
fragments of protein obtained on the collection membrane. These
include the use of delayed ion extraction, energy reflectors and
ion-trap modules. In addition, post source decay and MS-MS analysis
are useful to provide further structural analysis. With ESI, the
sample is in the liquid phase and the analysis can be by ion-trap,
TOF, single quadrupole or multi-quadrupole mass spectrometers. The
use of such devices (other than a single quadrupole) allows MS-MS
or MS.sup.n analysis to be performed. Tandem mass spectrometry
allows multiple reactions to be monitored at the same time.
[0179] Capillary infusion may be employed to introduce the marker
to a desired MS implementation, for instance, because it can
efficiently introduce small quantities of a sample into a mass
spectrometer without destroying the vacuum. Capillary columns are
routinely used to interface the ionization source of a MS with
other separation techniques including gas chromatography (GC) and
liquid chromatography (LC). GC and LC can serve to separate a
solution into its different components prior to mass analysis. Such
techniques are readily combined with MS, for instance. One
variation of the technique is that high performance liquid
chromatography (HPLC) can now be directly coupled to mass
spectrometer for integrated sample separation/and mass spectrometer
analysis.
[0180] Quadrupole mass analyzers may also be employed as needed to
practice the invention. Fourier-transform ion cyclotron resonance
(FTMS) can also be used for some invention embodiments. It offers
high resolution and the ability of tandem MS experiments. FTMS is
based on the principle of a charged particle orbiting in the
presence of a magnetic field. Coupled to ESI and MALDI, FTMS offers
high accuracy with errors as low as 0.001%.
[0181] In one embodiment, the marker qualification methods of the
invention may further comprise identifying significant peaks from
combined spectra. The methods may also further comprise searching
for outlier spectra. In another embodiment, the method of the
invention further comprises determining distant dependent K-nearest
neighbors.
[0182] In another embodiment of the method of the invention, an ion
mobility spectrometer can be used to detect and characterize serum
peptide markers. The principle of ion mobility spectrometry is
based on different mobility of ions. Specifically, ions of a sample
produced by ionization move at different rates, due to their
difference in, e.g., mass, charge, or shape, through a tube under
the influence of an electric field. The ions (typically in the form
of a current) are registered at the detector which can then be used
to identify a marker or other substances in a sample. One advantage
of ion mobility spectrometry is that it can operate at atmospheric
pressure.
[0183] For the mass values of the markers disclosed herein, the
mass accuracy of the spectral instrument is considered to be about
within +/-0.15 percent of the disclosed molecular weight value.
Additionally, to such recognized accuracy variations of the
instrument, the spectral mass determination can vary within
resolution limits of from about 400 to 1000 m/dm, where m is mass
and dm is the mass spectral peak width at 0.5 peak height. Mass
accuracy and resolution variances and thus meaning of the term
"about" with respect to the mass of each of the markers described
herein is inclusive of variants of the markers as may exist due to
sex, genotype and/or ethnicity of the subject and the particular
cancer or origin or stage thereof.
[0184] In an additional embodiment of the methods of the present
invention, multiple markers are measured. The use of multiple
markers increases the predictive value of the test and provides
greater utility in diagnosis, toxicology, patient stratification
and patient monitoring. The process called "Pattern recognition"
detects the patterns formed by multiple markers greatly improves
the sensitivity and specificity of clinical proteomics for
predictive medicine. Subtle variations in data from clinical
samples indicate that certain patterns of protein expression can
predict phenotypes such as the presence or absence of a certain
disease, a particular stage of cancer-progression, or a positive or
adverse response to drug treatments.
[0185] C. Data Analysis
[0186] Data generated by desorption and detection of markers can be
analyzed using any suitable means. In one embodiment, data is
analyzed and/or stored by electronic means, such as with the use of
a programmable digital computer. The computer program generally
contains a readable medium that stores codes. Certain code can be
devoted to memory that includes the location of each feature on a
probe, the identity of the adsorbent at that feature and the
elution conditions used to wash the adsorbent. The computer also
contains code that receives as input, data on the strength of the
signal at various molecular masses received from a particular
addressable location on the probe. This data can indicate the
number of markers detected, including the strength of the signal
generated by each marker.
[0187] Data analysis can include the steps of determining signal
strength (e.g., height of peaks) of a marker detected and removing
"outliers" (data deviating from a predetermined statistical
distribution). The observed peaks can be normalized, a process
whereby the height of each peak relative to some reference is
calculated. For example, a reference can be background noise
generated by instrument and chemicals (e.g., energy absorbing
molecule) which is set as zero in the scale. Then the signal
strength detected for each marker or other biomolecules can be
displayed in the form of relative intensities in the scale desired
(e.g., 100). Alternatively, a standard (e.g., a serum protein) may
be admitted with the sample so that a peak from the standard can be
used as a reference to calculate relative intensities of the
signals observed for each marker or other markers detected.
[0188] The computer can transform the resulting data into various
formats for displaying, such as "spectrum view or retentate map,"
"peak map," "gel view," "3-D overlays," "difference map view," and
Spotfire Scatter Plot. For each sample, markers that are detected
and the amount of markers present in the sample can be saved in a
computer readable medium. This data can then be compared to a
control (e.g., a profile or quantity of markers detected in
control, e.g., subjects in whom human cancer is undetectable).
[0189] When the sample is measured and data is generated, e.g., by
mass spectrometry, the data is then analyzed by a computer software
program. Generally, the software can comprise code that converts
signal from the mass spectrometer into computer readable form. The
software also can include code that applies an algorithm to the
analysis of the signal to determine whether the signal represents a
"peak" in the signal corresponding to a marker of this invention,
or other useful markers. The software also can include code that
executes an algorithm that compares signal from a test sample to a
typical signal characteristic of "normal" and human cancer and
determines the closeness of fit between the two signals. The
software also can include code indicating which the test sample is
closest to, thereby providing a probable diagnosis.
[0190] TOF-to-M/Z transformation involves the application of an
algorithm that transforms times-of-flight into mass-to-charge ratio
(M/Z). In this step, the signals are converted from the time domain
to the mass domain. That is, each time-of-flight is converted into
mass-to-charge ratio, or M/Z. Calibration can be done internally or
externally. In internal calibration, the sample analyzed contains
one or more analytes of known M/Z. Signal peaks at times-of-flight
representing these massed analytes are assigned the known M/Z.
Based on these assigned M/Z ratios, parameters are calculated for a
mathematical function that converts times-of-flight to M/Z. In
external calibration, a function that converts times-of-flight to
M/Z, such as one created by prior internal calibration, is applied
to a time-of-flight spectrum without the use of internal
calibrants.
[0191] Baseline subtraction improves data quantification by
eliminating artificial, reproducible instrument offsets that
perturb the spectrum. It involves calculating a spectrum baseline
using an algorithm that incorporates parameters such as peak width,
and then subtracting the baseline from the mass spectrum.
[0192] High frequency noise signals are eliminated by the
application of a smoothing function. A typical smoothing function
applies a moving average function to each time-dependent bin. In an
improved version, the moving average filter is a variable width
digital filter in which the bandwidth of the filter varies as a
function of, e.g., peak bandwidth, generally becoming broader with
increased time-of-flight. See, e.g., WO 00/70648, Nov. 23, 2000
(Gavin et al., "Variable Width Digital Filter for Time-of-flight
Mass Spectrometry").
[0193] As mentioned briefly above, analysis generally involves the
identification of peaks in the spectrum that represent signal from
an analyte. Peak data from one or more spectra can be subject to
further analysis by, for example, creating a spreadsheet in which
each row represents a particular mass spectrum, each column
represents a peak in the spectra defined by mass, and each cell
includes the intensity of the peak in that particular spectrum.
Various statistical or pattern recognition approaches can applied
to the data.
[0194] The spectra that are generated in embodiments of the
invention can be classified using a pattern recognition process
that uses a classification model. In some embodiments, data derived
from the spectra (e.g., mass spectra or time-of-flight spectra)
that are generated using samples such as "known samples" can then
be used to "train" a classification model. A "known sample" is a
sample that is pre-classified (e.g., cancer or not cancer). The
data that are derived from the spectra and are used to form the
classification model can be referred to as a "training data set".
Once trained, the classification model can recognize patterns in
data derived from spectra generated using unknown samples. The
classification model can then be used to classify the unknown
samples into classes. This can be useful, for example, in
predicting whether or not a particular biological sample is
associated with a certain biological condition (e.g., diseased vs.
non diseased).
[0195] The classification models can be formed on and used on any
suitable digital computer. The digital computer that is used may be
physically separate from the mass spectrometer that is used to
create the spectra of interest, or it may be coupled to the mass
spectrometer.
[0196] MALDI-TOF MS-Based Quantitative Profiling
[0197] Relative quantitation of serum peptides of interest can be
done by comparing the MS-ion intensities to those of added,
exogenous, isotopically labeled, reference peptides, having the
exact same sequence and otherwise same chemical properties as the
endogenous ones (i.e. distinguishable by molecular mass only). As
such, all peptide pairs will display the exact same MALD-ionization
characteristics. Comparing ion intensities will therefore provide a
means of normalizing the values for each peptide. For instance,
when the ion intensity of peptide A is two-fold higher than the
spiked reference in sample X but two-fold lower in sample Y, then
the difference would be about 4-fold between the same peptide in
the two samples. When done on a systematic, larger scale, this
approach can be referred to as relative "quantitative" profiling.
Of note, the reference peptides will be added to the raw serum
(i.e., before peptide extraction and MALDI sample prep), so that
putative losses during processing are accounted for.
[0198] 66 reference peptides (listed in FIG. 31) can be
synthesized, 44 of which have been determined to be surrogate
markers for either prostate or breast cancer, 18 additional ones
for bladder or thyroid cancer, and 4 non-marker control peptides.
These reference peptides should not degrade in serum, and are,
thus, synthesized using D-amino acids (i.e., D-stereo-isomers). One
amino acid (Leu, Val, or Phe) of each reference peptide is labeled
by incorporation of 6 (L, F) or 5 (V).sup.13C isotopes, and one
additional .sup.15N isotope (V only). .sup.13C-labeled, FMOC-amino
acids (for solid phase-peptide synthesis) are only commercially
available in the L-form, which should not compromise stability as
peptide bonds between a D- and L-amino acids are not protease
sensitive.
[0199] MALDI-TOF MS-Based Protease Assays
[0200] A large part of the human serum `peptidome`, as detected by
MALDI-TOF MS, is generated ex vivo (i.e., after blood collection)
by protease degradation of blood proteins. Endoproteases produce
`founder peptides` which are then pared down by exoproteases into
ladder-like clusters. Panels of proteolytic activity in the blood
contribute important cancer type-specific information, and that the
resulting metabolic patterns have utility as surrogate markers for
detection and classification of cancer. Degradation occurs during
clotting. The use of exogenous synthetic peptides, identical to
previously observed founder peptides, can be used to monitor
cancer-specific proteolytic degradation in plasma or serum that
contains proteases. Conditions in terms of time, temperature and
added amounts of substrates can hereby be readily controlled.
Coupled to a MALDI-based read-out, such analyses are blood
"protease assays" to monitor the tumor-dependent activities
inferred from prior studies. Simultaneous addition of
non-degradable, exogenous reference peptides also enables relative
quantitation of all rungs in the ladders.
[0201] Exogenous peptide degradation assays can be done, for
example, in plasma, where there are no endogenous peptides that
clutter the spectra, therefore simplifying interpretation. Thus, in
addition to serving as (i) an alternative to endogenous serum
peptide profiling, and as (ii) a highly reproducible, functional
proteomics approach, the external peptide degradation assay (iii)
permit analysis of plasma by the NY consortium, which is important
as plasma is preferred by many for proteomic studies.
[0202] 15 founder peptides (listed in FIG. 33) can be synthesized,
all `double-isotopically` labeled to be 12 Da heavier in molecular
mass than their endogenous counterparts and 6 Da heavier than the
non-degradable reference peptides. Selection is based on a sequence
comparison of all previously observed peptide ladders in serum,
most of which contain some known surrogate marker peptides.
Synthesis, QC, quantitation and storage of the peptides will be
done as described previously.
[0203] The degradation conditions and times are studied and
optimized for each of the 15 synthetic founder peptides in each of
the plasmas from the different groups of cancer patients and
controls. The permissible inter-mixability of the different
founders, and, particularly, of their resulting degradation ladders
is determined in order to avoid disturbing the peak patterns (by
ion suppression effects) and to avoid overlapping isotopic
envelopes (when the peaks are too close).
[0204] As aminopeptidases come in varieties that remove one two or
three amino acids, shorter endogenous peptides may have conceivably
been derived from another precursor by leapfrogging over the
stalled position. For non-degradable "founder" peptides, limited
N-terminal ladders can be synthesized (by sequential sampling of
resin during a pilot scale synthesis of unlabeled peptides), for
instance, as shown in FIG. 33 (founder #7; five alternative `test`
founder peptides), and degradability can be tested in pooled cancer
patient plasma in a time course (15 min to 4 hours) experiment.
Similar tests are performed for founder peptides 8, 9, 10 and 12A
in FIG. 33. Each time, synthesis is carried out of the
"full-length" founder, but resin sampled at 5, 4, 3, 2 and 1 amino
acid away from the N-terminus, or as appropriate. The longest
peptide is cleaved from the resin, purified, and tested. If no
degradation in plasma is observed, the shorter versions are also
cleaved, purified and tested. An isotope-labeled version of the
peptide with the best founder properties (i.e., generating the best
ladder in plasma) is then produced.
[0205] The assay may be divided into `founder pools` if two or more
time points are too far apart or in the case of peptide
inter-mixability problems. Once the ideal conditions and founder
pools have been selected, and the resulting degradation products
are identified, a relative quantitation aspect can be added to the
blood protease assay by using the same non-degradable reference
peptides as shown in FIG. 31.
[0206] D. Diagnosis
[0207] As indicated above, the invention provides methods for
aiding a human cancer diagnosis using one or more markers, as
specified herein. These markers can be used alone, in combination
with other markers in any set, or with entirely different markers
in aiding human cancer diagnosis. The markers are differentially
present in samples of a human cancer patient and a normal subject
in whom human cancer is undetectable. For example, some of the
markers are expressed at an elevated level and/or are present at a
higher frequency in human prostate cancer subjects than in normal
subjects, while some of the markers are expressed at a decreased
level and/or are present at a lower frequency in human prostate
cancer subjects than in normal subjects. Therefore, detection of
one or more of these markers in a person would provide useful
information regarding the probability that the person may have
prostate cancer.
[0208] The detection of the peptide marker is then correlated with
a probable diagnosis of cancer. In some embodiments, the detection
of the mere presence or absence of a marker, without quantifying
the amount thereof, is useful and can be correlated with a probable
diagnosis of cancer. The measurement of markers may also involve
quantifying the markers to correlate the detection of markers with
a probable diagnosis of cancer. Thus, if the amount of the markers
detected in a subject being tested is different compared to a
control amount (i.e., higher or lower than the control, depending
on the marker), then the subject being tested has a higher
probability of having cancer.
[0209] The correlation may take into account the amount of the
marker or markers in the sample compared to a control amount of the
marker or markers (up or down regulation of the marker or markers)
(e.g., in normal subjects or in non-cancer subjects such as where
cancer is undetectable). A control can be, e.g., the average or
median amount of marker present in comparable samples of normal
subjects in normal subjects or in non-cancer subjects such as where
cancer is undetectable. The control amount is measured under the
same or substantially similar experimental conditions as in
measuring the test amount. As a result, the control can be employed
as a reference standard, where the normal (non-cancer) phenotype is
known, and each result can be compared to that standard, rather
than re-running a control.
[0210] Accordingly, a marker profile may be obtained from a subject
sample and compared to a reference marker profile obtained from a
reference population, so that it is possible to classify the
subject as belonging to or not belonging to the reference
population. The correlation may take into account the presence or
absence of the markers in a test sample and the frequency of
detection of the same markers in a control. The correlation may
take into account both of such factors to facilitate determination
of cancer status.
[0211] In certain embodiments of the methods of qualifying cancer
status, the methods further comprise managing subject treatment
based on the status. The invention also provides for such methods
where the markers (or specific combination of markers) are measured
again after subject management. In these cases, the methods are
used to monitor the status of the cancer, e.g., response to cancer
treatment, remission of the disease or progression of the
disease.
[0212] The markers of the present invention have a number of other
uses. For example, they can be used to monitor responses to certain
treatments of human cancer. In yet another example, the markers can
be used in heredity studies. For instance, certain markers may be
genetically linked. This can be determined by, e.g., analyzing
samples from a population of human cancer subjects whose families
have a history of cancer. The results can then be compared with
data obtained from, e.g., cancer subjects whose families do not
have a history of cancer. The markers that are genetically linked
may be used as a tool to determine if a subject whose family has a
history of cancer is pre-disposed to having cancer.
[0213] Any marker, individually, is useful in aiding in the
determination of cancer status. First, the selected marker is
detected in a subject sample using the methods described herein
(e.g. mass spectrometry). Then, the result is compared with a
control that distinguishes cancer status from non-cancer status. As
is well understood in the art, the techniques can be adjusted to
increase sensitivity or specificity of the diagnostic assay
depending on the preference of the diagnostician.
[0214] While individual markers are useful diagnostic markers, in
some instances, a combination of markers provides greater
predictive value than single markers alone. The detection of a
plurality of markers (or absence thereof, as the case may be) in a
sample can increase the percentage of true positive and true
negative diagnoses and decrease the percentage of false positive or
false negative diagnoses. Thus, preferred methods of the present
invention comprise the measurement of more than one marker.
[0215] E. Kits
[0216] In one aspect, the invention provides kits for monitoring
and diagnosing cancer, wherein the kits can be used to detect the
markers described herein. For example, the kits can be used to
detect any one or more of the markers potentially differentially
present in samples of cancer subjects vs. normal subjects. The kits
of the invention have many applications. For example, the kits can
be used to differentiate if a subject has cancer or has a negative
diagnosis, thus aiding a cancer diagnosis. In another embodiment,
the invention provides kits for aiding the diagnosis of cancer or
the diagnosis of a specific type of cancer such as, for example,
cancer of the prostate, of the bladder, or of the breast. The kits
can also be used to identify compounds that modulate expression of
one or more of the herein-described markers in in vitro or in vivo
animal models for cancer.
[0217] In specific embodiments, kits of the invention contain an
exogenous reference peptide, which is optionally isotopically
labeled, for use in conducting the diagnostic assays of the
invention.
[0218] The kits of the invention may include instructions for the
assay, reagents, testing equipment (test tubes, reaction vessels,
needles, syringes, etc.), standards for calibrating the assay,
and/or equipment provided or used to conduct the assay. Reagents
may include acids, bases, oxidizing agents, marker species. The
instructions provided in a kit according to the invention may be
directed to suitable operational parameters in the form of a label
or a separate insert.
[0219] The kits may also include an adsorbent, wherein the
adsorbent retains one or more markers selected from one or more of
the markers described herein, and written instructions for use of
the kit for detection of cancer. Such a kit could, for example,
comprise: (a) a substrate comprising an adsorbent thereon, wherein
the adsorbent is suitable for binding a marker, and (b)
instructions to detect the marker or markers by contacting a sample
with the adsorbent and detecting the marker or markers retained by
the adsorbent. Accordingly, the kit could comprise (a) a DNA probe
that specifically binds to a marker; and (b) a detection reagent.
Such a kit could further comprise an eluant (as an alternative or
in combination with instructions) or instructions for making an
eluant, wherein the combination of the adsorbent and the eluant
allows detection of the markers using gas phase ion
spectrometry.
[0220] Optionally, the kit may further comprise a standard or
control information so that the test sample can be compared with
the control information standard to determine if the test amount of
a marker detected in a sample is a diagnostic amount consistent
with a diagnosis of cancer.
[0221] This invention is further illustrated by the following
examples, which should not be construed as limiting. A skilled
artisan should readily understand that other similar instruments
with equivalent function/specification, either commercially
available or user modified, are suitable for practicing the instant
invention. Rather, the invention should be construed to include any
and all applications provided herein and all equivalent variations
within the skill of the ordinary artisan.
EXAMPLES
Example 1
Unsupervised Hierarchical Clustering and PCA of Mass
Spectrometry-Based Serum Peptide Profiling Data
[0222] In order to determine if selected patterns of serum peptides
with known sequences can (i) separate cancer from non-cancer, (ii)
distinguish between different types of solid tumors, and (iii)
allow class prediction with an independent validation set, the
serum peptide profiles were analyzed from patients with advanced
prostate, breast, or bladder cancer, as well as control sera from
healthy volunteers, all collected using a standardized protocol
(Villanueva, J., et al., 2005. J. Proteome Res: 4:1060-1072).
[0223] A. Methods
[0224] Serum Samples
[0225] Blood samples from n=33 healthy volunteers (mixed gender;
ages 23 to 49) with no known malignancies and from patients
diagnosed with advanced prostate cancer (n=32), bladder cancer
(n=20), or breast cancer (n=21) were collected following a standard
clinical protocol (Villanueva, J., et al., 2005. J Proteome Res:
4:1060-1072) and approved by the MSKCC Institutional Review and
Privacy Board. Blood samples were obtained in 8.5-mL, BD
Vacutainer, glass `red-top` tubes (Becton Dickinson #366430,
Franklin Lakes, N.J.), allowed to clot at room temperature for 1
hour, and centrifuged at 1400-2000 RCF for 10 min, at RT.
[0226] Sera (upper phase) were transferred to four 4-mL cryovials
(Fisher #0566966), .about.1 mL serum in each, and stored frozen at
-80.degree. C. until further use (Villanueva, J., et al. 2005. J
Proteome Res: 4:1060-1072). A similar procedure was followed for
preparation of plasma in heparin-containing `green-top` tubes (BD
#366480), except that centrifugation was done immediately after
blood collection. Upon delivery at the mass spectrometry (MS)
laboratory, the cryovials (source vials) were barcoded. One
cryovial of each sample was thawed on ice and used to generate nine
smaller aliquots (50 .mu.L each) in barcoded micro-eppendorf tubes
and stored at -80.degree. C. in barcoded freezer boxes. In the
present study, every serum sample underwent two freeze/thaw cycles,
the second thawing step occurring immediately prior to peptide
extraction and MS analysis.
[0227] All 106 serum samples were processed automatically as a
single batch with a robot liquid handler followed within one hour
by automated MALDI-TOF mass spectrometric analysis. The four
clinical groups were randomized before automated solid-phase
peptide extraction and MALDI-TOF mass spectrometry.
[0228] Automated, Solid-Phase Peptide Extraction
[0229] Serum peptide profiling was accomplished using a technology
platform developed for simultaneous measurement of large numbers of
serum polypeptides (Villanueva, J., et al. 2004. Anal Chem
76:1560-1570). It uses magnetic bead-based, solid-phase extraction
of predominantly small peptides followed by a MALDI-TOF MS
read-out. The system is intrinsically more sensitive than any
surface capture on chips, as spherical particles have larger
combined surface areas than small-diameter spots. When combined
with high-resolution MS, hundreds of peptides are detected in a
single droplet of serum.
[0230] For the present analysis, peptides were captured and
concentrated using SiMAG-C8/K superparamagnetic, silica-based
particles (.ltoreq.1 micron diameter; 80% iron oxide; non-porous),
bearing C8 reversed-phase (RP) ligands (Chemicell, Berlin,
Germany). All analyses were performed in a 96-well format, using
the same batch of C8 magnetic particles, in 0.2-mL polypropylene
tubes (8.times.12-tube `Temp Plate II`; USA Scientific, Ocala,
Fla.).
[0231] The protocol is based on a detailed investigation of serum
handling, RP ligand and eluant selection (Villanueva, J., et al.
2004. Anal Chem 76:1560-1570), and is automated using a `Genesis
Freedom 100` (Tecan; Research Triangle Park, N.C.) liquid handling
workstation for throughput and reproducibility. The system was
programmed either directly via its standard software or, when
individual wells needed to be accessed independently, indirectly
through its work-lister capability. This system automates all of
the liquid-handling steps, including magnetic separation via a
robotic manipulating arm, mixing of eluates with MALDI matrix and
deposition onto the Bruker 384-spot MALDI target plates. A computer
randomization program was used to position case and control samples
for both solid-phase extraction and mass spectrometry.
[0232] Mass Spectrometry
[0233] Peptide profiles were analyzed with an Autoflex MALDI-TOF
mass spectrometer (Bruker; Bremen, Germany) equipped with a 337 nm
nitrogen laser, a gridless ion source, delayed-extraction (DE)
electronics, a high-resolution timed ion selector (TIS), and a 2
GHz digitizer. Separate spectra were obtained for two restricted
mass-to-charge (m/z) ranges, corresponding to polypeptides with
molecular mass of 0.7-4 kDa (".ltoreq.4 kD") and 4-15 kDa
(".gtoreq.4 kD") (assuming z=1), under specifically optimized
instrument settings. Each spectrum was the result of 400 laser
shots, per m/z segment per sample, delivered in four sets of 100
shots (at 50-Hz frequency) to each of four different locations on
the surface of the matrix spot.
[0234] The peak list (normalized intensities of 651 m/z-peaks,
i.e., peptide-ions, in all 106 samples) generated was subjected to
a Mann-Whitney U test, for each of the cancer groups individually
versus the control. In a first selection, 196 peaks with adjusted
p-values <0.00001 (arbitrarily chosen) for at least one cancer
type were retained. This number was reduced to 68 by applying an
arbitrary threshold (500 `units`) to the median intensities of each
individual peptide peak within a group. An m/z-peak was selected if
it passed the threshold in at least one of the cancer groups or the
control (FIG. 13).
[0235] A weekly performance test was carried out with commercial
human reference serum (# S-7023, lot 034K8937; Sigma, St Louis,
Mo.), and the effective laser energy delivered to the target was
adjusted when necessary. The entire irradiation program was
automated using the instrument's `AutoXecute` function. Spectra
were acquired in linear mode geometry under 20 kV (18.6 kV during
DE) of ion accelerating and -1.3 kV multiplier potentials, and with
gating of mass ions .ltoreq.400 m/z (.ltoreq.4 kD segment) or
.ltoreq.3,000 m/z (.gtoreq.4 kD segment). DE was maintained for 80
(.ltoreq.4 kD) or 50 nanoseconds (.gtoreq.4 kD) to give appropriate
time-lag focusing after each laser shot.
[0236] Peptide samples were consistently mixed with two volumes of
pre-made a-cyano-4-hydroxycinnamic acid (ACCA) matrix solution
(Agilent; Palo Alto, Calif.), deposited onto the stainless steel
target surface, in every other column of the 384-spot layout, and
allowed to dry at room temperature. Thirty fmoles (per peptide) and
500 fmoles (per protein) of commercially available calibration
standards (Bruker Daltonics #206195 (<4 kD) and #206355 (>4
kD)) were also mixed with ACCA matrix and separately deposited onto
the target plates, adjacent to each spotted serum sample (one
sample/one standard), in the alternating columns. All spectra were
acquired within less than 1-2 hours after completion of robotic
sample processing, as an adverse effect had previously been
observed upon increasing times between crystallization and mass
spectral acquisition.
[0237] The AutoFlex MALDI-TOF has a probe at the output of the
laser, before the attenuator. The accuracy of this monitoring
device was verified prior to the calibration of the settings of the
attenuator (displayed on the computer screen as an arbitrary scale
of 100-0%) by measuring transmitted energy at varying %. This
allowed the generation of a calibration curve to convert
before-to-after attenuation laser energy. The optimal laser setting
that had been empirically determined was then measured to yield
16-.mu.J energy per pulse, post-attenuation. Laser output energy
was measured and documented on a weekly basis, and adjustments were
made accordingly to compensate for fading laser energy over
time.
[0238] Samples from patients with different cancers and from
controls were randomly distributed during processing and
analysis.
[0239] Signal Processing
[0240] Once acquired, all data were stored with a naming convention
that allows each sample to be associated with its calibrant. The
spectra were first converted from binary format to ASCII files
containing two columns of data (x: m/z, y: intensity) by a custom
written macro in FlexAnalysis (Bruker). For the lower mass range
(700-4,000 Da), about 48,000 x,y-points were generated, while for
the upper mass range (4-15 kDa), there were about 77,000
points.
[0241] Further data processing was carried out in MATLAB with a
custom script called `Qcealign` using only the ASCII versions of
the raw spectra. `Qcealign` used the `Qpeaks` program (Spectrum
Square Associates, Ithaca, N.Y.) for smoothing, baseline
subtraction and peak labeling. The singletwidth parameter required
by `Qpeaks` was set to -400 for the lower mass range and -200 for
the upper mass range, thereby specifying the resolution,
(m/z)/.DELTA.(m/z), for processing. This peak information was used
automatically by `Qpeaks` in setting the parameters for smoothing,
baseline-subtraction, and binning. The noise statistics were
assumed `Normal`.
[0242] Following parameter selection, a setup file was created.
`Qcealign` then queries the setup file to obtain a list of all the
directories for processing. During a single processing run, all
data files in all listed directories are aligned with each other.
For each directory, singletwidth information is provided in the
setup file, along with parameters controlling calibration, peak
labeling sensitivity, alignment, etc. The files containing the
polypeptide standards are calibrated first. The centroid positions
of peaks in these calibration files are obtained from the peak
table created by `Qpeaks`, compared to the known polypeptide peak
positions, and a quadratic calibration equation for correcting the
measured masses in each calibration file is created. The
calibration equations are saved to disk for use in calibrating the
mass axes of the sample files.
[0243] `Qcealign` subsequently creates a reference file to which
all sample spectra will later be aligned. The first data file is
loaded and calibrated by applying the curve calculated from its
associated calibrant spectrum. This file's x-axis (m/z) becomes the
x-axis (and thus the calibration) used in the reference file.
`Qcealign` then loads all other sample files, calibrates them, and
adds their intensities to the reference file's intensity. After all
samples have been added, the reference spectrum becomes the average
of all the sample files. The reference is processed with `Qpeaks`
to find a baseline, which is subtracted, and is then normalized to
unit size by dividing each intensity value by the Total Ion Count
(TIC). Once normalized, a scaling factor is added by multiplying
each intensity value by a user-selected number (e.g., 10.sup.7).
This scaling factor is constant within a data set and is used to
convert the normalized spectrum to a "user friendly" scale, where
most peak heights are greater than one. Next, `Qcealign` processes
each sample file with `Qpeaks` to create a peak table, smoothed
curve and a baseline. This spectrum is then taken for
alignment.
[0244] Alignment
[0245] Processed spectra were aligned using the custom `Entropycal`
program described herein above. A custom alignment algorithm,
`Entropycal`, aligns sample data files to a reference file using a
minimum entropy algorithm by taking unsmoothed (`raw`),
baseline-corrected data. Taking raw spectra for alignment
facilitates the use of all statistical information in the data;
processed data contains less information. The alignment is
performed in two steps: `Entropycal` and binning. `Entropycal`
slides each data file by `n` data points to the right or left along
the x-axis of the reference file. At each relative position n, the
Shannon entropy of the sum of the two files is computed. The
optimal alignment occurs at the shift that produces the minimum
Shannon entropy. Second, the aligned peak lists are binned by using
the resolution of the peaks: all peaks in rows within .DELTA.(m/z)
of the strongest peak at a given value of m/z are binned together,
and a spreadsheet is created for further statistical analysis.
[0246] Three software modules, developed in MATLAB, were used for
visualization and signal processing of the spectra. (I) Signal
Processing & Preview (SPP), a graphical viewer for spectra in
ASCII format, allows to plot raw and processed spectra side-by-side
to review the outcome of signal processing. Furthermore, parameters
of `Qpeaks` (the signal processing software) can be adjusted. (II)
Mass Spectra Viewer (MSV), a visual interface for processed
spectral data, plots spectra as X-Y curves (mass vs. magnitude) for
examining the signatures of several groups of samples. MSV supports
regular browsing functions such as scroll, zoom, highlighting, etc.
(III) HeatMap (HM) displays spectra as a 2D heat map images, in
which the magnitude of the peaks are color-coded on a continuous
scale. In addition to browsing functions such as zoom and scroll,
the rank of X- and Y-position coordinates can be reorganized
without the constraints of statistical correlation that are
enforced by most HeatMap commercial software packages.
[0247] Ratios were calculated by dividing the median normalized
intensity of each m/z-peak in each cancer group by the median of
the same m/z-peak in the control group. To avoid having to divide
by zero, any median value of less than was converted to 1; this was
applied to all groups. For hierarchical clustering, the 651
m/z-values were subjected to average-linkage hierarchical
clustering analysis using the available algorithm in `GeneSpring`.
The peaks were organized by creating mock-phylogenetic trees
(dendrograms) termed `gene trees` and `experiment tree` in the
software. The trees were displayed with the samples along the
X-axis and the masses along the Y-axis. The clustering method for
both trees also measured similarity by Standard Correlation (also
known as `Pearson correlation around zero`) as the distance
matrix.
[0248] A spreadsheet (`peak list`), containing the normalized
intensities of all 651 peaks for each of the samples was taken for
unsupervised, average-linkage hierarchical clustering using
standard correlation. This resulted in a high degree of separation
between each of the cancer types and the controls in either binary
or multi-class comparisons (FIGS. 1B and 1C). Recognizing that
correlations between patient samples involving 651 features would
be difficult at different times and locations, statistical feature
selection was performed to identify the most discriminant
peaks.
[0249] The binned spreadsheet, containing data from spectra
obtained for all samples of cancer patients or healthy subjects
(106 samples total; 651 m/z values, with normalized intensities for
each sample; >70,000 data points), as well as the test set for
prostate (`Prostate #2`; 41 samples; .about.27,000 data points),
were imported into the `GeneSpring` program (Agilent; Palo Alto,
Calif.) and analyzed using various statistical algorithms, such as
one-way ANOVA, PCA, hierarchical clustering, K-NN and SVM.
[0250] Different "experiments" were created in `GeneSpring` to
represent the masses. No normalizations were applied to the
experiment, since the masses were normalized by the database that
binned them. In the parameter section of the experiments, a
parameter called `cancertype` was created to label samples as
prostate cancer, breast cancer, bladder cancer, or control. In the
Experiment's Interpretation section, the Analysis mode was set to
"Ratio (signal/control)", and all measurements were used. No
Cross-Gene Error model was used.
[0251] For ANOVA, once the experiments were created, the m/z-values
(`peaks`) were filtered by using non-parametric tests: Mann-Whitney
test (for binary comparisons) and Kluskal-Wallis test (for
multi-class comparisons) with Benjamini and Hochberg False
Discovery Rate at p<1e-5. These tests are meant to find peaks
that show statistically significant differences between the
clinical groups studied.
[0252] For class prediction, K-nearest-neighbor (K-NN) analysis and
Support Vector Machine (SVM) were carried out by using the Class
Prediction Tool in `GeneSpring`. The training groups constituted
either a binary comparison (prostate #1 and Control) or a
multi-class comparison (prostate #1, breast, bladder and control).
The test set was `prostate #2`. The Parameter to Predict was set to
Cancertype. The Gene selection was set to use different groups of
masses previously selected (e.g., 651, 68, 14, 13). In K-NN, the
number of neighbors was set to five with a p-value decision cutoff
of 1. The SVM was done with the same training sets and parameters
and set to predict the Prostate #2 test set. The kernel used was
polynomial dot product (Order 1) with a diagonal scaling of 0.
[0253] B. Results
[0254] 1. Distribution of serum peptides, detected by MALDI-TOF MS,
as a function of mass-to-charge (m/z) range and normalized
intensity.
[0255] Peptides were extracted from 106 different serum samples (50
.mu.L), drawn from one of three groups of cancer patients or
healthy controls, analyzed by MALDI-TOF MS and the m/z-peaks were
exported from the aligned spectra, as described earlier. In FIG.
11A, a total of 651 unique m/z-peaks, i.e., peptide-ions, derived
from the combined spectra, are grouped in successive bins of 250
amu, starting at m/z=700.
[0256] In FIG. 11B, all peak intensities of all samples (i.e.,
651.times.106 peaks) are grouped in successive bins of 100
arbitrary units, starting at zero. The intensities refer to
normalized units that were calculated for each peak by dividing its
raw intensity by the total of all the intensities in that spectrum
(TIC--Total Ion Count). The resultant values were then multiplied
by fixed scaling factor (1.times.10.sup.7) to convert the data to a
`user-friendly` scale (i.e. most values .gtoreq.1) Serum peptide
profiling resulted in a total of 651 distinct mass/charge (m/z)
values resolved in the 800-15,000 Dalton range (FIG. 16A).
[0257] 2. Serum peptides, determined by MALDI-OF MS, before and
after two successive feature selection steps for candidate
markers.
[0258] One-way ANOVA Mann-Whitney test, for each individual cancer
versus control, selected 196 peaks (red bars, FIG. 12) with a false
positive rate of p<0.00001 (arbitrarily chosen) for at least one
cancer type. This number was further reduced to 68 (yellow bars,
FIG. 12) by applying an arbitrary threshold of 500 `units` to the
median intensities of each individual peptide peak within a group.
The threshold was set high enough to select only robust peaks in
the spectra, with intensities that would permit MALDI MS/MS-based
tandem mass spectrometric sequencing and to exclude closely
positioned neighboring peaks or `shoulders`.
[0259] An m/z-peak was selected if this criterion was met in at
least one of the cancer groups or the control (FIG. 13). When
feature selection was repeated using a multi-class Kluskal-Wallis
test (adjusted p<1e-5) and the same median intensity threshold
as above, 214 and 67 peaks were selected (data not shown). The
majority of selected peaks corresponded to peptides with molecular
mass <2,000 Da; most peptides with a mass >4,000 Da were
removed (FIG. 2A; FIG. 13). Thus, significance levels (p-values)
were calculated for each m/z-peak using the Mann-Whitney rank sum
test (for binary comparisons) or the Kruskal-Wallis test (for
multi-class comparisons) (FIG. 16B).
Example 2
Feature Selection and Comparative Analysis of Serum Peptide
Profiling Data
[0260] Feature Selection
[0261] The peak list (normalized intensities of 651 m/z-peaks in
all 106 samples), generated as described in Example 1, above, was
subjected to one-way ANOVA Mann-Whitney test for each of the three
previously identified cancer groups individually vs. the control.
For each of the three cancer groups versus the control, 196 peaks
with a p-value <1e-5 were arbitrarily selected and retained
(FIG. 12). This number was subsequently reduced to 68 by applying
an arbitrary threshold (500 `units`) to the median intensities of
each individual peptide peak within a group. The threshold was set
high enough to only select robust peaks in the spectra, with
intensities that would permit MALDI TOF/TOF-based tandem mass
spectrometric sequencing and to exclude closely positioned
neighboring peaks or `shoulders`. An m/z peak was selected if it
passed the threshold in at least one of the cancer groups or the
control.
[0262] The pie-charts depicted in FIG. 2A illustrate the effect of
using a significance level (p<0.00001) cutoff by itself, or in
combination with a cutoff for the median of normalized intensities
(.gtoreq.500) within any one group, on the m/z distribution of the
candidate biomarker peptides. After the first filter, the 196
remaining peptides were redistributed in groups of 92, 76 and 28
for the increasing mass ranges. Sixty eight peptides passed the
second filter; 39, 22 and merely 7 in the low-, medium- and
high-mass ranges, respectively (right panel, FIG. 2A).
[0263] Examples are shown in FIGS. 2D and 3. The majority of the
selected peaks corresponded to peptides with molecular mass
<2,000 Da; most peptides with a mass >4,000 Da were
eliminated (FIGS. 12 and 2A). Color-coded spectra from all samples
were subsequently overlaid to visually inspect the 68 peaks for
correct assignment, degree of separation, and overall difference
between cancer and control. Of the peptides that passed the
above-delineated two selection steps, 47 m/z peaks had higher
intensities in one or more of the cancer groups, as compared to the
controls, and 23 had lower intensities, as compared with the
control. Of those, two were higher in breast cancer but lower in
bladder cancer.
[0264] The total numbers of peptides of a specific cancer group
that were observed to be up or down (have specific biomarker
potential) were as follows: 3 peptides were up and 11 down (14
total--1 unique, 3 shared) in serum samples from prostate cancer
patients, 12 up/2 down (14 total--11 unique) in breast cancer, and
36 up/22 down (58 total--43 unique) in bladder cancer (FIG.
2B).
[0265] Comparative analysis via heat map display and mass spectral
overlay: Comparison of the selected features (Tables 17A-C) of the
three cancer groups with controls in multi-class and binary formats
was accomplished with heat maps. Heat map displays were generated
using a MATLAB custom software tool.
[0266] Three software modules, developed in MATLAB, were used for
visualization and signal processing of the spectra. (I) Signal
Processing & Preview (SPP), a graphical viewer for spectra in
ASCII format, allows the plotting of raw and processed spectra
side-by-side to review the outcome of signal processing.
Furthermore, parameters of `Qpeaks` (the signal processing
software) can be adjusted. (II) Mass Spectra Viewer (MSV), a visual
interface for processed spectral data, plots spectra as X-Y curves
(mass vs. magnitude) for examining the signatures of several groups
of samples. MSV supports regular browsing functions such as scroll,
zoom, highlighting, etc. (III) HeatMap (HM) displays spectra as 2D
heat map images, in which the magnitude of the peaks are
color-coded on a continuous scale. In addition to browsing
functions such as zoom and scroll, the rank of X- and Y-position
coordinates can be reorganized without the constraints of
statistical correlation that are enforced by most HeatMap
commercial software packages.
[0267] The results, when represented in the form of heat maps in
FIG. 2C, indicated that data reduction (by .about.90%) did not
adversely affect the separation of the clinical groups.
[0268] Subsequently, mass spectra for the three binary comparisons
(cancer vs. control) were processed as described earlier and
displayed using Mass Spectra Viewer (MSV) (FIG. 2C).
Example 3
Serum Peptide Barcodes for Advanced Prostate, Bladder, and Breast
Cancer
[0269] A. Methods
[0270] Assigning Peptide Sequences
[0271] A set of peptides previously selected on the basis of
statistical differences in intensity between cancers and control
groups was analyzed by MALDI-TOF/TOF tandem mass spectrometry,
using an UltraFlex TOF/TOF instrument (Bruker; Bremen, Germany)
operated in `LIFT` mode. The mono-isotopic masses were first
assigned by one-dimensional reflectron-TOF MS, in the presence of
three peptide calibrants (6 fmoles each; calculated monoisotopic
masses of 2,108.155 Da, 1,307.762 Da and 969.575 Da in the
protonated form), as previously described (Winkler, G. S., et al;
2002, Methods 26:260-269).
[0272] Spectra were obtained by averaging multiple signals; laser
irradiance and number of acquisitions (typically 100-150) were
operator-adjusted to yield maximal peak deflections derived from
the digitizer in real time. Mono-isotopic masses were assigned for
all selected and other prominent peaks after visual inspection, and
the low- and high-end internal standards were used for
recalibration. The pass/fail criterion for recalibration is a
correct assignment of an m/z value for the `middle` calibrant with
a mass accuracy equal or better than 12 ppm.
[0273] Alternatively, a QSTAR XL Hybrid quadrupole (Q)
time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex;
Concord, Canada), equipped with an o-MALDI ion source, was used for
both duplicate and additional tandem-MS analyses. By selecting
precursor ions of interest in `Q1` (operated in the mass-filter
mode), mass measurements of fragment ions could be obtained in the
TOF detector following collision-induced dissociation (CID) in
`Q2`. Typically, a mass window of 3 Da was selected in order to
transmit the entire isotopic envelope of the precursor ion species.
Collision energy was operator adjusted to yield maximum number and
intensities of the fragment ions.
[0274] Fragment ion spectra resulting from TOF/TOF analyses
(300-1,000 acquisitions averaged per spectrum) were taken to search
a "non-redundant" human database (`NCBInr`; release data: Sep. 20,
2004; 106,486 entries; National Center for Biotechnology
Information, Bethesda, Md.) using the MASCOT MS/MS ion search
program, version 2.0.04 for Windows (Matrix Science Ltd., London,
UK) with the following search parameters: mono-isotopic precursor
mass tolerance of 35 ppm, fragment mass tolerance of 0.5 Da, and
without a specified protease cleavage site.
[0275] Mascot `mowse` scores greater than 35 were considered
significant. Any identification thus obtained was verified
independently by two different people, by comparing the
computer-generated fragment ion series of the predicted peptide
with the experimental MS/MS data. Some sequence assignments had
below-threshold scores but could, nonetheless, be unequivocally
assigned, as the precursor ion mass and selected fragment ion
masses (b'' or y'') matched a particular peptide, representing a
rung in one of the serum peptide sequence ladders.
[0276] B. Results
[0277] Peptide sequence assignment: 46 of the 68 previously
selected peptides (FIGS. 2B and 17) were positively identified by
MALDI-TOF/TOF MS/MS and MALDI-Q/TOF MS/MS analysis and database
searches (FIG. 5A, additionally showing others (including
m/z=1786.86, 2021.05, 2305.20, 2627.48)). Note that the m/z values
listed in FIG. 5A are mono-isotopic and therefore smaller than the
corresponding average isotopic values listed in FIGS. 16 and 17. Of
note, all but a few of the peptide sequences clustered into the
sets of overlapping fragments, lined up within each group at either
the C- or N-terminal end, and with ladder-like truncations at the
opposite ends. Some sequence assignments had below-threshold scores
but could, nonetheless, be unequivocally assigned, as the precursor
ion mass and selected fragment ion masses (b'' or y'') matched a
particular rung in one of the ladders, taking into account whether
the limited CID patterns were in agreement with established rules
(Kapp, E. A. et al., 2003. Anal Chem 75:6251-6264) of preferential
peptide bond cleavage (e.g., Xaa-Pro or Asp/Glu-Xaa) and the
putative sequence.
[0278] Furthermore, 23 additional peptides, outside the original
group of 68, could also be matched to certain sequence clusters by
hypothesis-driven, targeted MS/MS analysis. Fifteen of those had
significant discriminant analysis adjusted p-values (<0.0002)
for at least on cancer type but typically lower ion intensities
(FIG. 5B). Two others (`2553` and `2021`; yellow-coded in FIGS. 5A
and 5B) displayed very high but similar MS ion intensities across
all cancer groups and the control, with adjusted p-values >0.04,
and can therefore be regarded as quasi-internal controls. Six more
peptides (pink-coded in FIGS. 5A and 5B) that fit into the clusters
were randomly observed in samples of the cancer and control groups
and have neither discriminant nor internal control value. It should
be noted that we used an unbiased approach to identify `marker
peptides`, in which the peptides were selected first on the basis
of discriminant analysis and then sequenced. This approach,
commonly referred to as `ion mapping`, can be taken using any type
of mass spectrometric platform (Gao, J. et al., 2003. J. Proteome
Res 2:643-649; Fach, E. M. et al. 2004. Mol Cell Proteomics
3:1200-1210).
[0279] Three clusters derived from naturally occurring serum
peptides, fibrinopeptide A (FPA), complement C3f and bradykinin,
that are themselves generated from various plasma proteins through
endoproteolytic cleavage, either before (bradykinin, cleaved from
H-kininogen by a kallekrein) or during (FPA, N-terminally cleaved
from fibrinogen by thrombin to form fibrin; C3f, released by
Factors I and H after prior conversion of C3 to C3b) serum
preparation (Jandl, J. H. 1996. Blood: Textbook of hematology. New
York, N.Y.: Little, Brown and Co.; Sahu, A., and Lambris, J. D.
2001. Immunol Rev 180:35-48).
[0280] The full-length `founder` peptides end with Arg, preceded by
a hydrophobic amino acid (Val, Leu or Phe). Arg is partially
removed from C3f and bradykinin (to form desArg-bradykinin).
Similar `trypsin-like` cleavages (Arg/Lys-Xaa) underlie formation
of all other peptide clusters as well (see below). The C-terminal
basic amino acid is preceded by a hydrophobic amino acid (F, L, V,
I, W, A) in 21 and by S, Q or N in 15 out of the 39 observed
cleavage sites (FIG. 15). Arg/Lys is typically removed (fully or in
part) by a carboxypeptidase, except when preceded by Pro (3 out of
3 cases) or sometimes when preceded by Val (2 out 4). Further
exoprotease degradation then proceeds at the N-terminal or
C-terminal ends, either to completion or until it stalls; many or
all of the `intermediates` are typically represented (FIGS. 5A and
14). Of note, full-length C3f (m/z=2021.05) was found to be present
at equally high concentrations in all patient and control sera (see
B), and therefore represents a virtual internal standard.
[0281] Diagnostic MALDI-TOF spectral patterns consisting of
N-terminal FPA and C3f truncations have previously been found in
sera of myocardial infarction patients (Marshall, J. et al., 2003.
J. Proteome Res 2:361-372). In contrast, nearly all of these
peptides (19 total) were detected in control sera (FIG. 3B), and
their presence was shown to be either consistently lower (all FPA
fragments in all cancers; three C3f fragments in breast cancer)
and/or higher (several Cf3 fragments in bladder and prostate
cancer; one FPA fragment in breast cancer) in patient sera (FIG.
5A). Full-length C3f was present in all samples at equally high
concentrations. Full-length FPA was virtually absent in sera from
bladder cancer patients; no fibrinopeptide B or fragments thereof
were found in any of the samples.
[0282] Decreased levels of FPA (fragments) in prostate, bladder and
breast cancer patients, as shown here, also contrast with earlier
findings indicating elevated levels of phospho-FPA in sera of
ovarian cancer patients (measured by ESI-MS (Bergen, H. R., 3rd, et
al., 2003. Dis Markers 19:239-249) and of FPA in gastrointestinal
and breast cancers (measured immunochemically (Abbasciano, V. et
al., 1987. Med Oncol Tumor Pharmacother 4:75-79; Auger, M. J. et
al., 1987. Haemostasis 17:336-339).
[0283] Bradykinin and desArg-bradykinin levels were higher in sera
of breast cancer patients and lower in bladder cancer patients. Of
note, the pro-hydroxylated forms of each peptide also followed that
trend (data not shown). The bradykinin and FPA parent proteins,
fibrinogen alpha and HMW-kininogen, each contributed one additional
sequence cluster, located in a different section of the precursor
sequence, to the cancer serum peptide barcodes (FIGS. 5A and 6;
FIGS. 14 and 15). Interestingly, the bradykinin and `other`
kininogen-derived peptides have very different marker properties.
For example, whereas bradykinin and desArg-bradykinin were
generally of lower ion intensity in bladder cancer than in control
sera, the other two peptides (`1944` and `2209`) actually showed
higher relative intensities in bladder cancer (FIGS. 5A and
16).
[0284] One of the peptides (`2724`, FIG. 5A) in a cluster of
sequences is derived from the inter-alpha-trypsin inhibitor heavy
chain H4 (ITIH4) precursor (Salier, J. P. et al., 1996. Biochem J
315 (Pt 1):1-9) and covers amino acids 662-687 (FIG. 17) and is
bracketed by two kallikrein cleavage sites (Phe-Arg-Xaa). Residues
662-688 likely represent a `propeptide` of unknown function
(Nishimura, H. et al., 1995. FEBS Lett 357:207-211). Like
bradykinin, it ends with Pro-Phe-Arg. Several longer ITIH4
precursor fragments actually span the first kallikrein cleavage
site, including `3272` at 658-687, that has been reported as a
biomarker for early stage ovarian cancer (Zhang, Z. et al., 2004.
Cancer Res 64:5882-5890). Variations in N-terminal truncation by
just a few amino acids in the ITIH4 cluster were found to produce
relatively selective `markers` for each of the three different
cancers. Median ion intensities of peptides `3971` and `3273`, for
instance, were clearly highest in bladder cancer samples, peptides
`2358` and `2184` were highest in breast cancer, and `2271` was
highest in prostate cancer. Also of note, peptide `2115` matches
the sequence of an ITIH4 splice variant (PRO1851; FIG. 15) and
appears to have strong marker capacity for each cancer type,
particularly for bladder and breast (FIG. 16).
[0285] A seventh cluster of 8 sequences, 4 on either site of a
single Ile-Arg-Xaa cleavage site, is derived from the complement
C4a precursor (Belt, K. T. et al., 1984. Cell 36:907-914) (FIGS.
5A, 14, and 15). This C4a-cluster has the highest incidence of ion
markers for breast cancer; more than any in other cluster and also
more than C4a-derived bladder cancer markers (FIG. 16). Only a
single ion (`1763`) of this cluster is an ion marker for prostate
cancer, and is shared in that capacity with the other two cancer
types. On the other hand, all but one ion marker derived from
apolipoproteins (APO) A-I, A-IV and E are bladder cancer specific,
all with appreciably higher ion intensities; the exception (APO
A-IV, peptide `1971`) is actually highly selective and
statistically the most significant (p=5.5e-13) ion marker for
breast cancer (FIGS. 5A and 16).
[0286] Up-regulation of clusterin, i.e., `APO J`, has been
correlated, by immuno-histochemistry, with progression of both
prostate and bladder cancer (July, L. V. et al., 2002. Prostate
50:179-188; Scaltriti, M. et al., 2004. Int J Cancer 108:23-30;
Miyake, H. et al., 2002. Urology 59:150-154). The 10-amino acid
clusterin fragment detected at elevated concentrations in sera of
bladder and prostate cancer patients is located at the C-terminus
of the beta-chain. A single cut is, therefore, sufficient to
release this peptide, following separation of the clusterin beta
(N-t) and alpha (C-t) chains by cleavage of a Val-Arg-Xaa bond. A
6-amino acid sub-fragment has statistically relevant marker
potential for bladder cancer (FIGS. 5A and 16), which is in keeping
with the trend for most other peptides from APO A-I, A-IV, and E.
Two ions (`2602`; `2451`), each with significantly higher median
intensities in breast cancer samples than in controls, corresponded
to peptides derived from, respectively, Factor XIIIa and
thransthyretin (FIGS. 5A and 5B). In contrast to the aforementioned
clusters, each peptide was the only fragment from the respective
precursors that we observed. Peptide `2602` actually represents the
C-terminal 25 amino acids of the Factor XIIIa propeptide
(37-residues long) (FIGS. 14 and 15). Interestingly, Factor XIII
itself has been found significantly down-regulated in breast tumors
compared to normal mammary tissues (Jiang, W. G. et al., 2003.
Oncol Rep 10:2039-2044).
Example 4
MALDI-TOF Mass Spectral Overlays of Selected Peaks Derived from
Serum Peptide Profiling of Three Groups of Cancer Patients and
Healthy Controls
[0287] All spectra were obtained and aligned as described above,
and subsequently displayed using the Mass Spectra Viewer (MSV)
(FIGS. 3A and 3B). Overlays of mass spectra of selected peptides of
known sequence (FIG. 5) that showed statistically significant
differences between peak intensities in one or more of the three
binary comparisons are shown in FIG. 3A. Peptide `2021.05` (i.e.,
C3f) is shown as an example of a peptide that is present in about
equal concentrations in all serum samples analyzed in this study.
Overlays of mass spectra of some as yet unidentified peptides that
also showed statistically significant differences between peak
intensities in one or more of the three binary comparisons are
shown in FIG. 3B.
[0288] Peptides from a serum sample obtained from a breast cancer
patient were extracted and analyzed by MS, and the ion of choice
selected for MS/MS analysis. The fragment ion spectrum shown herein
was taken for a MASCOT MS/MS Ion Search of the human segment of NR
database, and retrieved a peptide sequence, GLEEELQFSLGSKINVKVGGNS
(SEQ ID NO:76) ([MH].sup.+=2305.19; .DELTA.=4 ppm) with a Mascot
score of 38.
[0289] Taken together, a total of 69 serum peptides are listed in
FIG. 5A (with matching information provided in FIG. 5B; all 79
sequenced peptides listed in FIG. 14). Of those, 61 have clear
MALDI-TOF MS-ion marker potential (adjusted p<0.0002; and, in
most cases, much lower) for at least one type of cancer and are
color-coded in blue (prostate cancer), green (bladder cancer) or
red (breast cancer). The resulting `barcodes` for the three cancer
types consist of 26 (prostate), 50 (bladder) and 25 (breast)
`bars`, i.e., peptides, several in common between any two or all
three. Compared to healthy control samples, median intensities of
ion markers could be up or down (represented by black dots in the
colored barcodes in FIG. 5A) in any particular cancer group; 16
higher and 10 lower (16+/10-) in prostate cancer, 31+/-19 in
bladder cancer, and 19+/6- in breast cancer. Only three peptides in
each of the up- or down-categories were shared by all cancer
groups.
[0290] One peptide from the C4a- and two from the ITIH4-cluster had
consistently higher ion intensities in all cancers than in healthy
controls; three FPA fragments were lower in all cancers. The rest
of the ion markers were either in common between 2 groups or, more
often, unique to a single patient cohort (FIG. 5A). Twenty six
(17+/9-) of those were unique for bladder cancer and 16 (13+/3-)
for breast cancer. To be noted are the nine APO[A-I, A-IV, E,
J]-peptides and three C3f-peptides exclusively of higher ion
intensities in bladder cancer, and the four C4a- two bradykinin-
and one transthyretin-peptides in breast cancer. All three serum
peptide ions that were uniquely of lower intensity in the breast
cancer cohort each derived from C3f. Interestingly, a number of
`shared` marker ions had, in fact, higher median intensities than
the controls in one type cancer and lower in another (FIGS. 5A and
5B). For instance, one ITIH4-peptide (`842`) and one C3f-peptide
(`1865`) had higher median ion intensities in sera from prostate
cancer patients than in, respectively, bladder and breast cancer.
Five peptide ions (including those corresponding to bradykinin and
desArg-bradykinin) that had higher median intensities in breast
cancer samples were lower in bladder cancer and had no appreciable
marker value for prostate cancer.
[0291] In an attempt to find trends in what clusters might have ion
marker value for a type of cancer, or to at least better visualize
any global differences that might exist, we plotted the ratios of
the median ion intensities were plotted, for each of the peptides
in the four major clusters, between each cancer group and the
healthy controls (i.e., r=case/control). The center line in the
panels of FIG. 6 represents no difference (r=1); bars pointing to
the left (r<1) or right (r>1) indicate, respectively, lower
or higher median. Even in case of the FPA ladder where nearly all
peptides in cancer sera produced ion signals of lower intensities
than in controls, the actual ratios vary for each `rung` and for
each cancer type. Of particular note is the seemingly total absence
(r=0) of full-length FPA in sera of bladder cancer patients. The
three other clusters exhibit an even more pronounced `internal`
variability, with median intensity ratios that were mostly over,
but also equal to or under 1.
[0292] Visual inspection of the 4 color-coded graphs (33.times.3
total data points) in FIG. 6 readily distinguishes the three cancer
types. There is a trend for peptides in bladder cancer sera to
exhibit relatively high ion intensities in the C3f cluster and
rather variable intensities in the C4a and ITIH4 clusters, and for
some peptides in the C3f-cluster to be of lower intensity and
others in the C4a-cluster to be of higher intensity in breast
cancer sera. Ion intensities of peptides in prostate cancer sera
don't seem to follow those trends, but are selectively more
pronounced in some of the smaller peptides of the ITIH4-cluster.
Interestingly, there is one rung in each of the C3f-, C4a- and
ITIH4-ladders (respectively the 6.sup.th, 5.sup.th and 5.sup.th
rung in the corresponding panels in FIG. 6) for which median ion
intensities in the control samples were virtually zero, yet much
higher in all three cancer types, resulting in very high ratios for
each.
[0293] Taken together, the data in FIG. 6, based in parts on
statistical analysis (FIG. 5B), visual inspection of spectra
overlays (FIG. 3), peptide sequencing (FIGS. 4 and 5A) and relative
ion intensity analysis, now strongly indicate that the human serum
peptidome holds information, in the form of barcodes consisting of
a few dozen peptides each, that can distinguish three different
cancers from controls as well as from each other.
Example 5
Independent Set of Prostate Cancer Serum Samples for Validation of
Established `Peptide-Signature` Biomarkers
[0294] It was next tested whether the identified markers would
correctly predict the class of an external validation set.
[0295] Sample Groups
[0296] An initial set of 32 serum samples from patients with
advanced prostate cancer (Prostate #1) were analyzed together with
33 samples from healthy controls and two additional groups of
cancer patient samples (FIG. 1A). One month later, an entirely
different group of 41 advanced prostate cancer patients (Prostate
#2), none previously studied, was analyzed using identical
methodology (FIG. 8A), and a new spreadsheet with all data from the
original 106 subjects and the new validation set, was generated.
The assignment of the prostate cancer samples into the training set
(Prostate 1--`PR1`) or the test set (Prostate 2--`PR2`) was random,
but preserving the same demographic/pathological parameters (e.g.,
age, PSA levels, Gleason score, survival time).
[0297] Peptide ions from `feature list #2` (68 peptides; see FIGS.
2A and 7) and from the `prostate cancer barcode` (26 sequenced
peptides; blue `barcode` in FIGS. 5A and 5B) were then selectively
used for comparison of the control, PR1 and PR2 groups by
hierarchical clustering and principal component analysis. While not
a perfect fit, samples from prostate cancer sets #1 and #2 were
mixed to some extent but for the most part separated from the
controls. Individual comparisons of each of these 26 peptide ions
between the three sample groups indicated that the intensities of
26 out of 26 were statistically different (adjusted p<0.0002;
i.e. the p-value to create the barcode--FIG. 5B) between PR1 and
control, 23 out of 26 between PR2 and control, and only 1 out of 26
between PR1 and PR2.
[0298] Class Prediction Analysis of the Prostate Cancer
Validation
[0299] Support vector machine (SVM)-based class predictions, in
either binary or multi-class formats, were carried out using all
651, or the 68 or 14 previously selected peptides. Analyses were
carried out using linear kernel (as described earlier). Similar
sensitivities were obtained in all three instances, namely 100%
(41/41) and 97.5% (40/41) accuracy for, respectively, binary and
multi-group class predictions.
Example 6
Aminoprotease Activities in Plasma
[0300] The serum peptidome is likely largely the product of
resident substrates, more specifically their proteolytic breakdown
products (Koomen, J. M. et al., 2005. J Proteome Res 4:972-981);
findings herein), and therefore, represents a read-out of the
repertoire of proteases that exist in plasma and/or become
activated during clotting. With the exception of bradykinin, much
higher peptide concentrations were consistently observed in serum
than in plasma (FIG. 9; and data not shown). The data presented
herein indicate that cancer cells contribute unique proteases,
perhaps exoproteases, which result in subtle but signature
alterations of the complex equation of hundreds of peptides that
can be resolved from human serum.
[0301] In an effort to begin to understand the presence and roles
of exoproteases, synthetic C3f was added to fresh plasma at a
concentration close to that observed in serum. As shown in FIG. 9,
degradation is very fast. C-terminal Arg was removed within
seconds, and the N-terminal truncations occurred in 10-15 min. The
resulting pattern was similar to the endogenous one observed in
serum and also illustrated the disparate ion intensities for
different rungs in the ladder. However, most of the C3f ladder,
except its smallest rung, disappeared upon prolonged incubation
(data not shown). Exoproteolytic degradation of synthetic FPA in
plasma followed a similar time course, but FPB was completely
degraded in just a few minutes (data not shown. The results suggest
that the operative exoprotease concentrations and activities are
roughly equivalent in plasma and serum, and therefore not the
consequence of coagulation.
[0302] As per Example 6, above, it is indicated that a sizable part
of the human serum `peptidome`, as detected by MALDI-TOF MS, is
generated by degradation of endogenous substrates by endogenous
proteases. Peptide profiling is, therefore, a form of
activity-based proteomics, by using a `metabolomic` read-out that
is subject to variations in enzyme panels, cofactors and
inhibitors. Here, proteolytic activities of the ex-vivo coagulation
and complement-degradation pathways, in combination with
exoproteases, have been shown to contribute to generation of not
only cancer-specific, but also `cancer type`-specific serum
peptides. The specificity derives largely from aminopeptidase
panels in serum, which is consistent with previous observations
(van Hensbergen, Y., et al., 2002, Clin Cancer Res 8:3747-3754;
Matrisian, L. M., et al., 2003, Cancer Res 63:6105-6109; Moffatt,
S., et al., 2005, Hum Gene Ther 16:57-67; Kehlen, A., et al., 2003,
Cancer Res 63:8500-8506; Rocken, C., et al., 2004, Int J Oncol
24:487-495; Carl-McGrath, S, et al., 2004, Int J Oncol
25:1223-1232; Kojima, K., et al., 1987, Biochem Med Metab Biol
37:35-41; Essler, M., et al., 2002, Proc Natl Acad Sci USA
99:2252-2257; Carrera, M. P., et al., 2005, Anticancer Res
25:193-196; Pulido-Cejudo, G., et al., 2004, Biotechnol Lett
26:1335-1339; Suganuma, T., et al., 2004, Lab Invest 84:639-648;
Selvakumar, P., et al., 2004, Clin Cancer Res 10:2771-2775; Ni, R.
Z., et al., 2003, World J Gastroenterol 9:710-713; Sheppard, G. S.,
et al., 2004, Bioorg Med Chem Lett 14:865-868; Griffith, E. C., et
al., 1998, Proc Natl Acad Sci USA 95:15183-15188; Pasqualini, R.,
et al., 2000, Cancer Res 60:722-727; Petrovic, N., et al., 2003, J
Biol Chem 278:49358-49368; O'Malley, P. G., et al., 2005, Biochem
J; Fair, W. R., et al., 1997, Prostate 32:140-148).
[0303] In the discovery phase of the present studies, hundreds of
features were sorted through to identify several that are most
predictive of outcome. Reduction in the number of key peptides to
only a few that are easily recognized between samples has been
shown not to adversely affect class predictions. Focused mass
spectrometric quantitation of key peptides should facilitate
introduction of this technology into general clinical practice.
Example 7
MALDI-TOF MS-Based Quantitative Profiling
[0304] Relative quantitation of the rungs of a C3f ladder in a pool
of 50 serum samples from thyroid carcinoma patients and a pool of
50 healthy controls was carried out. Ten reference peptides (FIG.
31) were added to the raw sera (2 picomoles/50 .mu.L), peptides
extracted on magnetic beads, MALDI spectra taken and ion intensity
ratios calculated for each pair, for each pool. The relative ion
intensities (ratio: endogenous/REF) were consistently higher for
the peptides in the `cancer sera` compared to the controls
(.about.20% to 100% higher) (FIG. 32, panel C). These results are
in agreement with the normalized ion intensity comparisons of 40
individual cancer and 40 individual control samples; presented as
spectral overlays and a heat plot in FIG. 32, panels A and B.
Example 8
MALDI-TOF MS-Based Protease Assays
[0305] The degradation conditions and times were studied for C3f
and FPA in serum and plasma as described above. Synthetic C3f and
FPA readily degraded in control serum and plasma; C3f rapidly
(within 15-30 min), FPA rather slowly (up to 4 hours). 2 picomoles
[.sup.13C-Leu]-labeled C3f was incubated for 30 min at RT with
50-.mu.L aliquots of serum from 20 different breast cancer patients
and 20 control samples. Four rungs (m/z=942, 1212, 1563, 1865) of
the endogenous C3f degradation ladder were previously found to have
a lower median ion intensity in MALDI spectra taken of breast
cancer sera than control sera (FIG. 34, top panel). Upon overlay of
the 40 color-coded spectra (FIG. 34, bottom panel), the equivalent
four rungs in the ladder resulting from degradation of exogenous
[.sup.13C-Leu]C3f had also generally lower ion intensities in the
spectra of cancer patient sera compared to the controls, thus
closely matching the endogenous patterns.
[0306] A synthetic version of the longest ITIH4-derived founder
peptide (FIG. 33; #7, with N-t Pro) did not degrade in serum or
plasma (data not shown), indicating that it probably is not a
founder but rather a stalled degradation product of a bigger
peptide.
[0307] Labeled C3f was added to two pools of serum, one from 50
samples obtained from thyroid carcinoma patients, and one from age-
and gender-matched healthy controls. Aliquots were retrieved at
various time points, ranging from 5 min to 5 hours, and analyzed by
magnetic bead processing and a MALDI read-out; in triplicate. The
10 peptide-triplets (one for each rung in the C3f ladder) were then
selected for each time point and each of the triplicates, the
ratios between exogenously derived peptide and reference peptide
calculated and plotted (FIG. 35).
[0308] The exogenous peptide was singly labeled (.sup.13C-Leu), and
the reference peptide doubly labeled with .sup.13C/.sup.15N-Leu,
hence the 14 Da mass difference from the endogenous peptide. The
time course results indicate that during the first 5 or so minutes,
peptide degradation (removal of the C-t Arg) kinetics are faster in
the cancer sera than in the controls. Furthermore, after 1-2 hours
of incubation, clear differences in relative ion intensity were
observed for the two smallest peptides in the ladder between the
two samples; both higher in the cancer sample, indicating that the
founder peptide was either more rapidly degraded in the cancer
serum or that, alternatively, it was completely degraded to single
amino acids in the control serum.
Sequence CWU 1
1
148115PRTHomo sapiens 1Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly
Gly Gly Val Arg 1 5 10 15 214PRTHomo sapiens 2Ser Gly Glu Gly Asp
Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 313PRTHomo sapiens 3Gly
Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 412PRTHomo
sapiens 4Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10
511PRTHomo sapiens 5Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5
10 610PRTHomo sapiens 6Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5
10 716PRTHomo sapiens 7Glu Glu Glu Leu Gln Phe Ser Gly Leu Ser Phe
Asn Val Lys Val Ser 1 5 10 15 816PRTHomo sapiens 8Ser Ser Lys Ile
Thr His Arg Ile His Trp Glu Ser Ala Ser Leu Leu 1 5 10 15
915PRTHomo sapiens 9Ser Lys Ile Thr His Arg Ile His Trp Glu Ser Ala
Ser Leu Leu 1 5 10 15 1014PRTHomo sapiens 10Lys Ile Thr His Arg Ile
His Trp Glu Ser Ala Ser Leu Leu 1 5 10 1112PRTHomo sapiens 11Thr
His Arg Ile His Trp Glu Ser Ala Ser Leu Leu 1 5 10 128PRTHomo
sapiens 12His Trp Glu Ser Ala Ser Leu Leu 1 5 1326PRTHomo sapiens
13Pro Gly Val Leu Ser Ser Arg Gln Leu Gly Leu Pro Gly Pro Pro Asp 1
5 10 15 Val Pro Asp His Ala Ala Tyr His Pro Phe 20 25 1425PRTHomo
sapiens 14Gly Val Leu Ser Ser Arg Gln Leu Gly Leu Pro Gly Pro Pro
Asp Val 1 5 10 15 Pro Asp His Ala Ala Tyr His Pro Phe 20 25
1521PRTHomo sapiens 15Ser Arg Gln Leu Gly Leu Pro Gly Pro Pro Asp
Val Pro Asp His Ala 1 5 10 15 Ala Tyr His Pro Phe 20 1617PRTHomo
sapiens 16Gly Leu Pro Gly Pro Pro Asp Val Pro Asp His Ala Ala Tyr
His Pro 1 5 10 15 Phe 1710PRTHomo sapiens 17His Phe Phe Phe Pro Lys
Ser Arg Ile Val 1 5 10 186PRTHomo sapiens 18His Phe Phe Phe Pro Lys
1 5 199PRTHomo sapiens 19Arg Pro Pro Gly Phe Ser Pro Phe Arg 1 5
208PRTHomo sapiens 20Arg Pro Pro Gly Phe Ser Pro Phe 1 5
2116PRTHomo sapiens 21Arg Asn Gly Phe Lys Ser His Ala Leu Gln Leu
Asn Asn Arg Gln Ile 1 5 10 15 2215PRTHomo sapiens 22Asn Gly Phe Lys
Ser His Ala Leu Gln Leu Asn Asn Arg Gln Ile 1 5 10 15 2322PRTHomo
sapiens 23Gly Leu Glu Glu Glu Leu Gln Phe Ser Leu Gly Ser Lys Ile
Asn Val 1 5 10 15 Lys Val Gly Gly Asn Ser 20 249PRTHomo sapiens
24Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 258PRTHomo sapiens 25Leu
Ala Glu Gly Gly Gly Val Arg 1 5 2613PRTHomo sapiens 26Ile Thr His
Arg Ile His Trp Glu Ser Ala Ser Leu Leu 1 5 10 2710PRTHomo sapiens
27Arg Ile His Trp Glu Ser Ala Ser Leu Leu 1 5 10 289PRTHomo sapiens
28Ile His Trp Glu Ser Ala Ser Leu Leu 1 5 2915PRTHomo sapiens 29Ser
Ser Lys Ile Thr His Arg Ile His Trp Glu Ser Ala Ser Leu 1 5 10 15
3014PRTHomo sapiens 30Asn Gly Phe Lys Ser His Ala Leu Gln Leu Asn
Asn Arg Gln 1 5 10 3113PRTHomo sapiens 31Asn Gly Phe Lys Ser His
Ala Leu Gln Leu Asn Asn Arg 1 5 10 3226PRTHomo sapiens 32Gly Leu
Glu Glu Glu Leu Gln Phe Ser Leu Gly Ser Lys Ile Asn Val 1 5 10 15
Lys Val Gly Gly Asn Ser Lys Gly Thr Leu 20 25 3316PRTHomo sapiens
33Gly Leu Glu Glu Glu Leu Gln Phe Ser Leu Gly Ser Lys Ile Asn Val 1
5 10 15 348PRTHomo sapiens 34His Ala Ala Tyr His Pro Phe Arg 1 5
3520PRTHomo sapiens 35Gln Leu Gly Leu Pro Gly Pro Pro Asp Val Pro
Asp His Ala Ala Tyr 1 5 10 15 His Pro Phe Arg 20 3638PRTHomo
sapiens 36Gln Ala Gly Ala Ala Gly Ser Arg Met Asn Phe Arg Pro Gly
Val Leu 1 5 10 15 Ser Ser Arg Gln Leu Gly Leu Pro Gly Pro Pro Asp
Val Pro Asp His 20 25 30 Ala Ala Tyr His Pro Phe 35 3730PRTHomo
sapiens 37Met Asn Phe Arg Pro Gly Val Leu Ser Ser Arg Gln Leu Gly
Leu Pro 1 5 10 15 Gly Pro Pro Asp Val Pro Asp His Ala Ala Tyr His
Pro Phe 20 25 30 3822PRTHomo sapiens 38Ser Ser Arg Gln Leu Gly Leu
Pro Gly Pro Pro Asp Val Pro Asp His 1 5 10 15 Ala Ala Tyr His Pro
Phe 20 397PRTHomo sapiens 39His Ala Ala Tyr His Pro Phe 1 5
4028PRTHomo sapiens 40Asn Val His Ser Gly Ser Thr Phe Phe Lys Tyr
Tyr Leu Gln Gly Ala 1 5 10 15 Lys Ile Pro Lys Pro Glu Ala Ser Phe
Ser Pro Arg 20 25 4121PRTHomo sapiens 41Asn Val His Ser Ala Gly Ala
Ala Gly Ser Arg Met Asn Phe Arg Pro 1 5 10 15 Gly Val Leu Ser Ser
20 4228PRTHomo sapiens 42Gln Gly Leu Leu Pro Val Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser 1 5 10 15 Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr Gln 20 25 4317PRTHomo sapiens 43Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr 1 5 10 15 Gln 4419PRTHomo
sapiens 44Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro
Ala Leu 1 5 10 15 Glu Asp Leu 4523PRTHomo sapiens 45Ile Ser Ala Ser
Ala Glu Glu Leu Arg Gln Arg Leu Ala Pro Leu Ala 1 5 10 15 Glu Asp
Val Arg Gly Asn Leu 20 4625PRTHomo sapiens 46Gly Asn Thr Glu Gly
Leu Gln Lys Ser Leu Ala Glu Leu Gly Gly His 1 5 10 15 Leu Asp Gln
Gln Val Glu Glu Phe Arg 20 25 4717PRTHomo sapiens 47Ser Leu Ala Glu
Leu Gly Gly His Leu Asp Gln Gln Val Glu Glu Phe 1 5 10 15 Arg
4816PRTHomo sapiens 48Ser Leu Ala Glu Leu Gly Gly His Leu Asp Gln
Gln Val Glu Glu Phe 1 5 10 15 4924PRTHomo sapiens 49Ala Ala Thr Val
Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala 1 5 10 15 Gln Ala
Trp Gly Glu Arg Leu Arg 20 5023PRTHomo sapiens 50Ala Ala Thr Val
Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala 1 5 10 15 Gln Ala
Trp Gly Glu Arg Leu 20 5119PRTHomo sapiens 51Lys His Asn Leu Gly
His Gly His Lys His Glu Arg Asp Gln Gly His 1 5 10 15 Gly His Gln
5217PRTHomo sapiens 52Asn Leu Gly His Gly His Lys His Glu Arg Asp
Gln Gly His Gly His 1 5 10 15 Gln 5325PRTHomo sapiens 53Ala Val Pro
Pro Asn Asn Ser Asn Ala Ala Glu Asp Asp Leu Pro Thr 1 5 10 15 Val
Glu Leu Gln Gly Val Val Pro Arg 20 25 5423PRTHomo sapiens 54Ala Leu
Gly Ile Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr 1 5 10 15
Ala Asn Asp Ser Gly Pro Arg 20 5529PRTHomo sapiens 55Ser Ser Ser
Tyr Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr Asn Arg 1 5 10 15 Gly
Asp Ser Thr Phe Glu Ser Lys Ser Tyr Lys Met Ala 20 25 5628PRTHomo
sapiens 56Ser Ser Ser Tyr Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr
Asn Arg 1 5 10 15 Gly Asp Ser Thr Phe Glu Ser Lys Ser Tyr Lys Met
20 25 5726PRTHomo sapiens 57Ser Ser Ser Tyr Ser Lys Gln Phe Thr Ser
Ser Thr Ser Tyr Asn Arg 1 5 10 15 Gly Asp Ser Thr Phe Glu Ser Lys
Ser Tyr 20 25 5825PRTHomo sapiens 58Ser Ser Ser Tyr Ser Lys Gln Phe
Thr Ser Ser Thr Ser Tyr Asn Arg 1 5 10 15 Gly Asp Ser Thr Phe Glu
Ser Lys Ser 20 25 5925PRTHomo sapiens 59Gly Leu Glu Glu Glu Leu Gln
Phe Ser Leu Gly Ser Lys Ile Asn Val 1 5 10 15 Lys Gly Gly Asn Ser
Lys Gly Thr Ile 20 25 6021PRTHomo sapiens 60Ser Ser Tyr Ser Lys Gln
Phe Thr Ser Ser Thr Ser Tyr Asn Arg Gly 1 5 10 15 Asp Ser Thr Phe
Glu 20 6127PRTHomo sapiens 61Gly Ser Glu Ser Gly Ile Phe Thr Asn
Thr Lys Glu Ser Ser Ser His 1 5 10 15 His Pro Gly Ile Ala Glu Phe
Pro Ser Arg Gly 20 25 6225PRTHomo sapiens 62Asp Glu Ala Gly Ser Glu
Ala Asp His Glu Gly Thr His Ser Thr Lys 1 5 10 15 Arg Gly His Ala
Lys Ser Arg Pro Val 20 25 63436PRTHomo sapiens 63Tyr Pro Phe Ala
Leu Phe Tyr Arg His Tyr Leu Phe Tyr Lys Glu Thr 1 5 10 15 Tyr Leu
Ile His Leu Phe His Thr Phe Thr Gly Leu Ser Ile Ala Tyr 20 25 30
Phe Asn Phe Gly Asn Gln Leu Tyr His Ser Leu Leu Cys Ile Val Leu 35
40 45 Gln Phe Leu Ile Leu Arg Leu Met Gly Arg Thr Ile Thr Ala Val
Leu 50 55 60 Thr Thr Phe Cys Phe Gln Met Ala Tyr Leu Leu Ala Gly
Tyr Tyr Tyr 65 70 75 80 Thr Ala Thr Gly Asn Tyr Asp Ile Lys Trp Thr
Met Pro His Cys Val 85 90 95 Leu Thr Leu Lys Leu Ile Gly Leu Ala
Val Asp Tyr Phe Asp Gly Gly 100 105 110 Lys Asp Gln Asn Ser Leu Ser
Ser Glu Gln Gln Lys Tyr Ala Ile Arg 115 120 125 Gly Val Pro Ser Leu
Leu Glu Val Ala Gly Phe Ser Tyr Phe Tyr Gly 130 135 140 Ala Phe Leu
Val Gly Pro Gln Phe Ser Met Asn His Tyr Met Lys Leu 145 150 155 160
Val Gln Gly Glu Leu Ile Asp Ile Pro Gly Lys Ile Pro Asn Ser Ile 165
170 175 Ile Pro Ala Leu Lys Arg Leu Ser Leu Gly Leu Phe Tyr Leu Val
Gly 180 185 190 Tyr Thr Leu Leu Ser Pro His Ile Thr Glu Asp Tyr Leu
Leu Thr Glu 195 200 205 Asp Tyr Asp Asn His Pro Phe Trp Phe Arg Cys
Met Tyr Met Leu Ile 210 215 220 Trp Gly Lys Phe Val Leu Tyr Lys Tyr
Val Thr Cys Trp Leu Val Thr 225 230 235 240 Glu Gly Val Cys Ile Leu
Thr Gly Leu Gly Phe Asn Gly Phe Glu Glu 245 250 255 Lys Gly Lys Ala
Lys Trp Asp Ala Cys Ala Asn Met Lys Val Trp Leu 260 265 270 Phe Glu
Thr Asn Pro Arg Phe Thr Gly Thr Ile Ala Ser Phe Asn Ile 275 280 285
Asn Thr Asn Ala Trp Val Ala Arg Tyr Ile Phe Lys Arg Leu Lys Phe 290
295 300 Leu Gly Asn Lys Glu Leu Ser Gln Gly Leu Ser Leu Leu Phe Leu
Ala 305 310 315 320 Leu Trp His Gly Leu His Ser Gly Tyr Leu Val Cys
Phe Gln Met Lys 325 330 335 Phe Leu Ile Val Ile Val Glu Arg Gln Ala
Ala Arg Leu Ile Gln Glu 340 345 350 Ser Pro Thr Leu Ser Lys Leu Ala
Ala Ile Thr Val Leu Gln Pro Phe 355 360 365 Tyr Tyr Leu Val Gln Gln
Thr Ile His Trp Leu Phe Met Gly Tyr Ser 370 375 380 Met Thr Ala Phe
Cys Leu Phe Thr Trp Asp Lys Trp Leu Lys Val Tyr 385 390 395 400 Lys
Ser Ile Tyr Phe Leu Gly His Ile Phe Phe Leu Ser Leu Leu Phe 405 410
415 Ile Leu Pro Tyr Ile His Lys Ala Met Val Pro Arg Lys Glu Lys Leu
420 425 430 Lys Lys Met Glu 435 64930PRTHomo sapiens 64Met Lys Pro
Pro Arg Pro Val Arg Thr Cys Ser Lys Val Leu Val Leu 1 5 10 15 Leu
Ser Leu Leu Ala Ile His Gln Thr Thr Thr Ala Glu Lys Asn Gly 20 25
30 Ile Asp Ile Tyr Ser Leu Thr Val Asp Ser Arg Val Ser Ser Arg Phe
35 40 45 Ala His Thr Val Val Thr Ser Arg Val Val Asn Arg Ala Asn
Thr Val 50 55 60 Gln Glu Ala Thr Phe Gln Met Glu Leu Pro Lys Lys
Ala Phe Ile Thr 65 70 75 80 Asn Phe Ser Met Asn Ile Asp Gly Met Thr
Tyr Pro Gly Ile Ile Lys 85 90 95 Glu Lys Ala Glu Ala Gln Ala Gln
Tyr Ser Ala Ala Val Ala Lys Gly 100 105 110 Lys Ser Ala Gly Leu Val
Lys Ala Thr Gly Arg Asn Met Glu Gln Phe 115 120 125 Gln Val Ser Val
Ser Val Ala Pro Asn Ala Lys Ile Thr Phe Glu Leu 130 135 140 Val Tyr
Glu Glu Leu Leu Lys Arg Arg Leu Gly Val Tyr Glu Leu Leu 145 150 155
160 Leu Lys Val Arg Pro Gln Gln Leu Val Lys His Leu Gln Met Asp Ile
165 170 175 His Ile Phe Glu Pro Gln Gly Ile Ser Phe Leu Glu Thr Glu
Ser Thr 180 185 190 Phe Met Thr Asn Gln Leu Val Asp Ala Leu Thr Thr
Trp Gln Asn Lys 195 200 205 Thr Lys Ala His Ile Arg Phe Lys Pro Thr
Leu Ser Gln Gln Gln Lys 210 215 220 Ser Pro Glu Gln Gln Glu Thr Val
Leu Asp Gly Asn Leu Ile Ile Arg 225 230 235 240 Tyr Asp Val Asp Arg
Ala Ile Ser Gly Gly Ser Ile Gln Ile Glu Asn 245 250 255 Gly Tyr Phe
Val His Tyr Phe Ala Pro Glu Gly Leu Thr Thr Met Pro 260 265 270 Lys
Asn Val Val Phe Val Ile Asp Lys Ser Gly Ser Met Ser Gly Arg 275 280
285 Lys Ile Gln Gln Thr Arg Glu Ala Leu Ile Lys Ile Leu Asp Asp Leu
290 295 300 Ser Pro Arg Asp Gln Phe Asn Leu Ile Val Phe Ser Thr Glu
Ala Thr 305 310 315 320 Gln Trp Arg Pro Ser Leu Val Pro Ala Ser Ala
Glu Asn Val Asn Lys 325 330 335 Ala Arg Ser Phe Ala Ala Gly Ile Gln
Ala Leu Gly Gly Thr Asn Ile 340 345 350 Asn Asp Ala Met Leu Met Ala
Val Gln Leu Leu Asp Ser Ser Asn Gln 355 360 365 Glu Glu Arg Leu Pro
Glu Gly Ser Val Ser Leu Ile Ile Leu Leu Thr 370 375 380 Asp Gly Asp
Pro Thr Val Gly Glu Thr Asn Pro Arg Ser Ile Gln Asn 385 390 395 400
Asn Val Arg Glu Ala Val Ser Gly Arg Tyr Ser Leu Phe Cys Leu Gly 405
410 415 Phe Gly Phe Asp Val Ser Tyr Ala Phe Leu Glu Lys Leu Ala Leu
Asp 420 425 430 Asn Gly Gly Leu Ala Arg Arg Ile His Glu Asp Ser Asp
Ser Ala Leu 435 440 445 Gln Leu Gln Asp Phe Tyr Gln Glu Val Ala Asn
Pro Leu Leu Thr Ala 450 455 460 Val Thr Phe Glu Tyr Pro Ser Asn Ala
Val Glu Glu Val Thr Gln Asn 465 470 475 480 Asn Phe Arg Leu Leu Phe
Lys Gly Ser Glu Met Val Val Ala Gly Lys 485 490 495 Leu Gln Asp Arg
Gly Pro Asp Val Leu Thr Ala Thr Val Ser Gly Lys 500 505 510 Leu Pro
Thr Gln Asn Ile Thr Phe Gln Thr Glu Ser Ser Val Ala Glu 515 520 525
Gln Glu Ala Glu Phe Gln Ser Pro Lys Tyr Ile Phe His Asn Phe Met 530
535 540 Glu Arg Leu Trp Ala Tyr Leu Thr Ile Gln Gln Leu Leu Glu Gln
Thr 545 550 555 560 Val Ser Ala Ser Asp Ala Asp Gln Gln Ala Leu Arg
Asn Gln Ala Leu 565
570 575 Asn Leu Ser Leu Ala Tyr Ser Phe Val Thr Pro Leu Thr Ser Met
Val 580 585 590 Val Thr Lys Pro Asp Asp Gln Glu Gln Ser Gln Val Ala
Glu Lys Pro 595 600 605 Met Glu Gly Glu Ser Arg Asn Arg Asn Val His
Ser Gly Ser Thr Phe 610 615 620 Phe Lys Tyr Tyr Leu Gln Gly Ala Lys
Ile Pro Lys Pro Glu Ala Ser 625 630 635 640 Phe Ser Pro Arg Arg Gly
Trp Asn Arg Gln Ala Gly Ala Ala Gly Ser 645 650 655 Arg Met Asn Phe
Arg Pro Gly Val Leu Ser Ser Arg Gln Leu Gly Leu 660 665 670 Pro Gly
Pro Pro Asp Val Pro Asp His Ala Ala Tyr His Pro Phe Arg 675 680 685
Arg Leu Ala Ile Leu Pro Ala Ser Ala Pro Pro Ala Thr Ser Asn Pro 690
695 700 Asp Pro Ala Val Ser Arg Val Met Asn Met Lys Ile Glu Glu Thr
Thr 705 710 715 720 Met Thr Thr Gln Thr Pro Ala Pro Ile Gln Ala Pro
Ser Ala Ile Leu 725 730 735 Pro Leu Pro Gly Gln Ser Val Glu Arg Leu
Cys Val Asp Pro Arg His 740 745 750 Arg Gln Gly Pro Val Asn Leu Leu
Ser Asp Pro Glu Gln Gly Val Glu 755 760 765 Val Thr Gly Gln Tyr Glu
Arg Glu Lys Ala Gly Phe Ser Trp Ile Glu 770 775 780 Val Thr Phe Lys
Asn Pro Leu Val Trp Val His Ala Ser Pro Glu His 785 790 795 800 Val
Val Val Thr Arg Asn Arg Arg Ser Ser Ala Tyr Lys Trp Lys Glu 805 810
815 Thr Leu Phe Ser Val Met Pro Gly Leu Lys Met Thr Met Asp Lys Thr
820 825 830 Gly Leu Leu Leu Leu Ser Asp Pro Asp Lys Val Thr Ile Gly
Leu Leu 835 840 845 Phe Trp Asp Gly Arg Gly Glu Gly Leu Arg Leu Leu
Leu Arg Asp Thr 850 855 860 Asp Arg Phe Ser Ser His Val Gly Gly Thr
Leu Gly Gln Phe Tyr Gln 865 870 875 880 Glu Val Leu Trp Gly Ser Pro
Ala Ala Ser Asp Asp Gly Arg Arg Thr 885 890 895 Leu Arg Val Gln Gly
Asn Asp His Ser Ala Thr Arg Glu Arg Arg Leu 900 905 910 Asp Tyr Gln
Glu Gly Pro Pro Gly Val Glu Ile Ser Cys Trp Ser Val 915 920 925 Glu
Leu 930 65447PRTHomo sapiens 65Met Met Lys Thr Leu Leu Leu Phe Val
Gly Leu Leu Leu Thr Trp Glu 1 5 10 15 Ser Gly Gln Val Leu Gly Asp
Gln Thr Val Ser Asp Asn Glu Leu Gln 20 25 30 Glu Met Ser Asn Gln
Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn 35 40 45 Ala Val Asn
Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn 50 55 60 Glu
Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys 65 70
75 80 Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu
Lys 85 90 95 Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu
Trp Glu Glu 100 105 110 Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys
Phe Tyr Ala Arg Val 115 120 125 Cys Arg Ser Gly Ser Gly Leu Val Gly
Arg Gln Leu Glu Glu Phe Leu 130 135 140 Asn Gln Ser Ser Pro Phe Tyr
Phe Trp Met Asn Gly Asp Arg Ile Asp 145 150 155 160 Ser Leu Leu Glu
Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met 165 170 175 Gln Asp
His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln 180 185 190
Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro 195
200 205 Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser
Arg 210 215 220 Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro
Leu Asn Phe 225 230 235 240 His Ala Met Phe Gln Pro Phe Leu Glu Met
Ile His Glu Ala Gln Gln 245 250 255 Ala Met Asp Ile His Phe His Ser
Pro Ala Phe Gln His Pro Pro Thr 260 265 270 Glu Phe Ile Arg Glu Gly
Asp Asp Asp Arg Thr Val Cys Arg Glu Ile 275 280 285 Arg His Asn Ser
Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys 290 295 300 Cys Arg
Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln 305 310 315
320 Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg
325 330 335 Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp
Lys Met 340 345 350 Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu
Gln Phe Asn Trp 355 360 365 Val Ser Arg Leu Ala Asn Leu Thr Gln Gly
Glu Asp Gln Tyr Tyr Leu 370 375 380 Arg Val Thr Thr Val Ala Ser His
Thr Ser Asp Ser Asp Val Pro Ser 385 390 395 400 Gly Val Thr Glu Val
Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr 405 410 415 Val Thr Val
Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu 420 425 430 Thr
Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg 435 440 445
66534PRTHomo sapiensMOD_RES(485)..(485)Any amino acid 66Gly Gln Tyr
Ala Ser Pro Thr Ala Lys Arg Cys Cys Gln Asp Gly Val 1 5 10 15 Thr
Arg Leu Pro Met Met Arg Ser Cys Glu Gln Arg Ala Ala Arg Val 20 25
30 Gln Gln Pro Asp Cys Arg Glu Pro Phe Leu Ser Cys Cys Gln Phe Ala
35 40 45 Glu Ser Leu Arg Lys Lys Ser Arg Asp Lys Gly Gln Ala Gly
Leu Gln 50 55 60 Arg Ala Leu Glu Ile Leu Gln Glu Glu Asp Leu Ile
Asp Glu Asp Asp 65 70 75 80 Ile Pro Val Arg Ser Phe Phe Pro Glu Asn
Trp Leu Trp Arg Val Glu 85 90 95 Thr Val Asp Arg Phe Gln Ile Leu
Thr Leu Trp Leu Pro Asp Ser Leu 100 105 110 Thr Thr Trp Glu Ile His
Gly Leu Ser Leu Ser Lys Thr Lys Gly Leu 115 120 125 Cys Val Ala Thr
Pro Val Gln Leu Arg Val Phe Arg Glu Phe His Leu 130 135 140 His Leu
Arg Leu Pro Met Ser Val Arg Arg Phe Glu Gln Leu Glu Leu 145 150 155
160 Arg Pro Val Leu Tyr Asn Tyr Leu Asp Lys Asn Leu Thr Val Ser Val
165 170 175 His Val Ser Pro Val Glu Gly Leu Cys Leu Ala Gly Gly Gly
Gly Leu 180 185 190 Ala Gln Gln Val Leu Val Pro Ala Gly Ser Ala Arg
Pro Val Ala Phe 195 200 205 Ser Val Val Pro Thr Ala Ala Thr Ala Val
Ser Leu Lys Val Val Ala 210 215 220 Arg Gly Ser Phe Glu Phe Pro Val
Gly Asp Ala Val Ser Lys Val Leu 225 230 235 240 Gln Ile Glu Lys Glu
Gly Ala Ile His Arg Glu Glu Leu Val Tyr Glu 245 250 255 Leu Asn Pro
Leu Asp His Arg Gly Arg Thr Leu Glu Ile Pro Gly Asn 260 265 270 Ser
Asp Pro Asn Met Ile Pro Asp Gly Asp Phe Asn Ser Tyr Val Arg 275 280
285 Val Thr Ala Ser Asp Pro Leu Asp Thr Leu Gly Ser Glu Gly Ala Leu
290 295 300 Ser Pro Gly Gly Val Ala Ser Leu Leu Arg Leu Pro Arg Gly
Cys Gly 305 310 315 320 Glu Gln Thr Met Ile Tyr Leu Ala Pro Thr Leu
Ala Ala Ser Arg Tyr 325 330 335 Leu Asp Lys Thr Glu Gln Trp Ser Thr
Leu Pro Pro Glu Thr Lys Asp 340 345 350 His Ala Val Asp Leu Ile Gln
Lys Gly Tyr Met Arg Ile Gln Gln Phe 355 360 365 Arg Lys Ala Asp Gly
Ser Tyr Ala Ala Trp Leu Ser Arg Gly Ser Ser 370 375 380 Thr Trp Leu
Thr Ala Phe Val Leu Lys Val Leu Ser Leu Ala Gln Glu 385 390 395 400
Gln Val Gly Gly Ser Pro Glu Lys Leu Gln Glu Thr Ser Asn Trp Leu 405
410 415 Leu Ser Gln Gln Gln Ala Asp Gly Ser Phe Gln Asp Pro Cys Pro
Val 420 425 430 Leu Asp Arg Ser Met Gln Gly Gly Leu Val Gly Asn Asp
Glu Thr Val 435 440 445 Ala Leu Thr Ala Phe Val Thr Ile Ala Leu His
His Gly Leu Ala Val 450 455 460 Phe Gln Asp Glu Gly Ala Glu Pro Leu
Lys Gln Arg Val Glu Ala Ser 465 470 475 480 Ile Ser Lys Ala Xaa Ser
Phe Leu Gly Glu Lys Ala Ser Ala Gly Leu 485 490 495 Leu Gly Ala His
Ala Ala Ala Ile Thr Ala Tyr Ala Leu Thr Leu Thr 500 505 510 Lys Ala
Pro Val Asp Leu Leu Gly Val Ala His Asn Asn Leu Met Ala 515 520 525
Met Ala Gln Glu Thr Gly 530 67644PRTHomo sapiens 67Met Phe Ser Met
Arg Ile Val Cys Leu Val Leu Ser Val Val Gly Thr 1 5 10 15 Ala Trp
Thr Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly 20 25 30
Gly Val Arg Gly Pro Arg Val Val Glu Arg His Gln Ser Ala Cys Lys 35
40 45 Asp Ser Asp Trp Pro Phe Cys Ser Asp Glu Asp Trp Asn Tyr Lys
Cys 50 55 60 Pro Ser Gly Cys Arg Met Lys Gly Leu Ile Asp Glu Val
Asn Gln Asp 65 70 75 80 Phe Thr Asn Arg Ile Asn Lys Leu Lys Asn Ser
Leu Phe Glu Tyr Gln 85 90 95 Lys Asn Asn Lys Asp Ser His Ser Leu
Thr Thr Asn Ile Met Glu Ile 100 105 110 Leu Arg Gly Asp Phe Ser Ser
Ala Asn Asn Arg Asp Asn Thr Tyr Asn 115 120 125 Arg Val Ser Glu Asp
Leu Arg Ser Arg Ile Glu Val Leu Lys Arg Lys 130 135 140 Val Ile Glu
Lys Val Gln His Ile Gln Leu Leu Gln Lys Asn Val Arg 145 150 155 160
Ala Gln Leu Val Asp Met Lys Arg Leu Glu Val Asp Ile Asp Ile Lys 165
170 175 Ile Arg Ser Cys Arg Gly Ser Cys Ser Arg Ala Leu Ala Arg Glu
Val 180 185 190 Asp Leu Lys Asp Tyr Glu Asp Gln Gln Lys Gln Leu Glu
Gln Val Ile 195 200 205 Ala Lys Asp Leu Leu Pro Ser Arg Asp Arg Gln
His Leu Pro Leu Ile 210 215 220 Lys Met Lys Pro Val Pro Asp Leu Val
Pro Gly Asn Phe Lys Ser Gln 225 230 235 240 Leu Gln Lys Val Pro Pro
Glu Trp Lys Ala Leu Thr Asp Met Pro Gln 245 250 255 Met Arg Met Glu
Leu Glu Arg Pro Gly Gly Asn Glu Ile Thr Arg Gly 260 265 270 Gly Ser
Thr Ser Tyr Gly Thr Gly Ser Glu Thr Glu Ser Pro Arg Asn 275 280 285
Pro Ser Ser Ala Gly Ser Trp Asn Ser Gly Ser Ser Gly Pro Gly Ser 290
295 300 Thr Gly Asn Arg Asn Pro Gly Ser Ser Gly Thr Gly Gly Thr Ala
Thr 305 310 315 320 Trp Lys Pro Gly Ser Ser Gly Pro Gly Ser Thr Gly
Ser Trp Asn Ser 325 330 335 Gly Ser Ser Gly Thr Gly Ser Thr Gly Asn
Gln Asn Pro Gly Ser Pro 340 345 350 Arg Pro Gly Ser Thr Gly Thr Trp
Asn Pro Gly Ser Ser Glu Arg Gly 355 360 365 Ser Ala Gly His Trp Thr
Ser Glu Ser Ser Val Ser Gly Ser Thr Gly 370 375 380 Gln Trp His Ser
Glu Ser Gly Ser Phe Arg Pro Asp Ser Pro Gly Ser 385 390 395 400 Gly
Asn Ala Arg Pro Asn Asn Pro Asp Trp Gly Thr Phe Glu Glu Val 405 410
415 Ser Gly Asn Val Ser Pro Gly Thr Arg Arg Glu Tyr His Thr Glu Lys
420 425 430 Leu Val Thr Ser Lys Gly Asp Lys Glu Leu Arg Thr Gly Lys
Glu Lys 435 440 445 Val Thr Ser Gly Ser Thr Thr Thr Thr Arg Arg Ser
Cys Ser Lys Thr 450 455 460 Val Thr Lys Thr Val Ile Gly Pro Asp Gly
His Lys Glu Val Thr Lys 465 470 475 480 Glu Val Val Thr Ser Glu Asp
Gly Ser Asp Cys Pro Glu Ala Met Asp 485 490 495 Leu Gly Thr Leu Ser
Gly Ile Gly Thr Leu Asp Gly Phe Arg His Arg 500 505 510 His Pro Asp
Glu Ala Ala Phe Phe Asp Thr Ala Ser Thr Gly Lys Thr 515 520 525 Phe
Pro Gly Phe Phe Ser Pro Met Leu Gly Glu Phe Val Ser Glu Thr 530 535
540 Glu Ser Arg Gly Ser Glu Ser Gly Ile Phe Thr Asn Thr Lys Glu Ser
545 550 555 560 Ser Ser His His Pro Gly Ile Ala Glu Phe Pro Ser Arg
Gly Lys Ser 565 570 575 Ser Ser Tyr Ser Lys Gln Phe Thr Ser Ser Thr
Ser Tyr Asn Arg Gly 580 585 590 Asp Ser Thr Phe Glu Ser Lys Ser Tyr
Lys Met Ala Asp Glu Ala Gly 595 600 605 Ser Glu Ala Asp His Glu Gly
Thr His Ser Thr Lys Arg Gly His Ala 610 615 620 Lys Ser Arg Pro Val
Arg Gly Ile His Thr Ser Pro Leu Gly Lys Pro 625 630 635 640 Ser Leu
Ser Pro 68644PRTHomo sapiens 68Met Lys Leu Ile Thr Ile Leu Phe Leu
Cys Ser Arg Leu Leu Leu Ser 1 5 10 15 Leu Thr Gln Glu Ser Gln Ser
Glu Glu Ile Asp Cys Asn Asp Lys Asp 20 25 30 Leu Phe Lys Ala Val
Asp Ala Ala Leu Lys Lys Tyr Asn Ser Gln Asn 35 40 45 Gln Ser Asn
Asn Gln Phe Val Leu Tyr Arg Ile Thr Glu Ala Thr Lys 50 55 60 Thr
Val Gly Ser Asp Thr Phe Tyr Ser Phe Lys Tyr Glu Ile Lys Glu 65 70
75 80 Gly Asp Cys Pro Val Gln Ser Gly Lys Thr Trp Gln Asp Cys Glu
Tyr 85 90 95 Lys Asp Ala Ala Lys Ala Ala Thr Gly Glu Cys Thr Ala
Thr Val Gly 100 105 110 Lys Arg Ser Ser Thr Lys Phe Ser Val Ala Thr
Gln Thr Cys Gln Ile 115 120 125 Thr Pro Ala Glu Gly Pro Val Val Thr
Ala Gln Tyr Asp Cys Leu Gly 130 135 140 Cys Val His Pro Ile Ser Thr
Gln Ser Pro Asp Leu Glu Pro Ile Leu 145 150 155 160 Arg His Gly Ile
Gln Tyr Phe Asn Asn Asn Thr Gln His Ser Ser Leu 165 170 175 Phe Met
Leu Asn Glu Val Lys Arg Ala Gln Arg Gln Val Val Ala Gly 180 185 190
Leu Asn Phe Arg Ile Thr Tyr Ser Ile Val Gln Thr Asn Cys Ser Lys 195
200 205 Glu Asn Phe Leu Phe Leu Thr Pro Asp Cys Lys Ser Leu Trp Asn
Gly 210 215 220 Asp Thr Gly Glu Cys Thr Asp Asn Ala Tyr Ile Asp Ile
Gln Leu Arg 225 230 235 240 Ile Ala Ser Phe Ser Gln Asn Cys Asp Ile
Tyr Pro Gly Lys Asp Phe 245 250 255 Val Gln Pro Pro Thr Lys Ile Cys
Val Gly Cys Pro Arg Asp Ile Pro 260 265 270 Thr Asn Ser Pro Glu Leu
Glu Glu Thr Leu Thr His Thr Ile Thr Lys 275 280 285 Leu Asn Ala Glu
Asn Asn Ala Thr Phe Tyr Phe Lys Ile Asp Asn Val 290 295 300 Lys Lys
Ala Arg Val Gln Val Val Ala Gly Lys
Lys Tyr Phe Ile Asp 305 310 315 320 Phe Val Ala Arg Glu Thr Thr Cys
Ser Lys Glu Ser Asn Glu Glu Leu 325 330 335 Thr Glu Ser Cys Glu Thr
Lys Lys Leu Gly Gln Ser Leu Asp Cys Asn 340 345 350 Ala Glu Val Tyr
Val Val Pro Trp Glu Lys Lys Ile Tyr Pro Thr Val 355 360 365 Asn Cys
Gln Pro Leu Gly Met Ile Ser Leu Met Lys Arg Pro Pro Gly 370 375 380
Phe Ser Pro Phe Arg Ser Ser Arg Ile Gly Glu Ile Lys Glu Glu Thr 385
390 395 400 Thr Val Ser Pro Pro His Thr Ser Met Ala Pro Ala Gln Asp
Glu Glu 405 410 415 Arg Asp Ser Gly Lys Glu Gln Gly His Thr Arg Arg
His Asp Trp Gly 420 425 430 His Glu Lys Gln Arg Lys His Asn Leu Gly
His Gly His Lys His Glu 435 440 445 Arg Asp Gln Gly His Gly His Gln
Arg Gly His Gly Leu Gly His Gly 450 455 460 His Glu Gln Gln His Gly
Leu Gly His Gly His Lys Phe Lys Leu Asp 465 470 475 480 Asp Asp Leu
Glu His Gln Gly Gly His Val Leu Asp His Gly His Lys 485 490 495 His
Lys His Gly His Gly His Gly Lys His Lys Asn Lys Gly Lys Lys 500 505
510 Asn Gly Lys His Asn Gly Trp Lys Thr Glu His Leu Ala Ser Ser Ser
515 520 525 Glu Asp Ser Thr Thr Pro Ser Ala Gln Thr Gln Glu Lys Thr
Glu Gly 530 535 540 Pro Thr Pro Ile Pro Ser Leu Ala Lys Pro Gly Val
Thr Val Thr Phe 545 550 555 560 Ser Asp Phe Gln Asp Ser Asp Leu Ile
Ala Thr Met Met Pro Pro Ile 565 570 575 Ser Pro Ala Pro Ile Gln Ser
Asp Asp Asp Trp Ile Pro Asp Ile Gln 580 585 590 Thr Asp Pro Asn Gly
Leu Ser Phe Asn Pro Ile Ser Asp Phe Pro Asp 595 600 605 Thr Thr Ser
Pro Lys Cys Pro Gly Arg Pro Trp Lys Ser Val Ser Glu 610 615 620 Ile
Asn Pro Thr Thr Gln Met Lys Glu Ser Tyr Tyr Phe Asp Leu Thr 625 630
635 640 Asp Gly Leu Ser 69644PRTHomo sapiens 69Met Phe Ser Met Arg
Ile Val Cys Leu Val Leu Ser Val Val Gly Thr 1 5 10 15 Ala Trp Thr
Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly 20 25 30 Gly
Val Arg Gly Pro Arg Val Val Glu Arg His Gln Ser Ala Cys Lys 35 40
45 Asp Ser Asp Trp Pro Phe Cys Ser Asp Glu Asp Trp Asn Tyr Lys Cys
50 55 60 Pro Ser Gly Cys Arg Met Lys Gly Leu Ile Asp Glu Val Asn
Gln Asp 65 70 75 80 Phe Thr Asn Arg Ile Asn Lys Leu Lys Asn Ser Leu
Phe Glu Tyr Gln 85 90 95 Lys Asn Asn Lys Asp Ser His Ser Leu Thr
Thr Asn Ile Met Glu Ile 100 105 110 Leu Arg Gly Asp Phe Ser Ser Ala
Asn Asn Arg Asp Asn Thr Tyr Asn 115 120 125 Arg Val Ser Glu Asp Leu
Arg Ser Arg Ile Glu Val Leu Lys Arg Lys 130 135 140 Val Ile Glu Lys
Val Gln His Ile Gln Leu Leu Gln Lys Asn Val Arg 145 150 155 160 Ala
Gln Leu Val Asp Met Lys Arg Leu Glu Val Asp Ile Asp Ile Lys 165 170
175 Ile Arg Ser Cys Arg Gly Ser Cys Ser Arg Ala Leu Ala Arg Glu Val
180 185 190 Asp Leu Lys Asp Tyr Glu Asp Gln Gln Lys Gln Leu Glu Gln
Val Ile 195 200 205 Ala Lys Asp Leu Leu Pro Ser Arg Asp Arg Gln His
Leu Pro Leu Ile 210 215 220 Lys Met Lys Pro Val Pro Asp Leu Val Pro
Gly Asn Phe Lys Ser Gln 225 230 235 240 Leu Gln Lys Val Pro Pro Glu
Trp Lys Ala Leu Thr Asp Met Pro Gln 245 250 255 Met Arg Met Glu Leu
Glu Arg Pro Gly Gly Asn Glu Ile Thr Arg Gly 260 265 270 Gly Ser Thr
Ser Tyr Gly Thr Gly Ser Glu Thr Glu Ser Pro Arg Asn 275 280 285 Pro
Ser Ser Ala Gly Ser Trp Asn Ser Gly Ser Ser Gly Pro Gly Ser 290 295
300 Thr Gly Asn Arg Asn Pro Gly Ser Ser Gly Thr Gly Gly Thr Ala Thr
305 310 315 320 Trp Lys Pro Gly Ser Ser Gly Pro Gly Ser Thr Gly Ser
Trp Asn Ser 325 330 335 Gly Ser Ser Gly Thr Gly Ser Thr Gly Asn Gln
Asn Pro Gly Ser Pro 340 345 350 Arg Pro Gly Ser Thr Gly Thr Trp Asn
Pro Gly Ser Ser Glu Arg Gly 355 360 365 Ser Ala Gly His Trp Thr Ser
Glu Ser Ser Val Ser Gly Ser Thr Gly 370 375 380 Gln Trp His Ser Glu
Ser Gly Ser Phe Arg Pro Asp Ser Pro Gly Ser 385 390 395 400 Gly Asn
Ala Arg Pro Asn Asn Pro Asp Trp Gly Thr Phe Glu Glu Val 405 410 415
Ser Gly Asn Val Ser Pro Gly Thr Arg Arg Glu Tyr His Thr Glu Lys 420
425 430 Leu Val Thr Ser Lys Gly Asp Lys Glu Leu Arg Thr Gly Lys Glu
Lys 435 440 445 Val Thr Ser Gly Ser Thr Thr Thr Thr Arg Arg Ser Cys
Ser Lys Thr 450 455 460 Val Thr Lys Thr Val Ile Gly Pro Asp Gly His
Lys Glu Val Thr Lys 465 470 475 480 Glu Val Val Thr Ser Glu Asp Gly
Ser Asp Cys Pro Glu Ala Met Asp 485 490 495 Leu Gly Thr Leu Ser Gly
Ile Gly Thr Leu Asp Gly Phe Arg His Arg 500 505 510 His Pro Asp Glu
Ala Ala Phe Phe Asp Thr Ala Ser Thr Gly Lys Thr 515 520 525 Phe Pro
Gly Phe Phe Ser Pro Met Leu Gly Glu Phe Val Ser Glu Thr 530 535 540
Glu Ser Arg Gly Ser Glu Ser Gly Ile Phe Thr Asn Thr Lys Glu Ser 545
550 555 560 Ser Ser His His Pro Gly Ile Ala Glu Phe Pro Ser Arg Gly
Lys Ser 565 570 575 Ser Ser Tyr Ser Lys Gln Phe Thr Ser Ser Thr Ser
Tyr Asn Arg Gly 580 585 590 Asp Ser Thr Phe Glu Ser Lys Ser Tyr Lys
Met Ala Asp Glu Ala Gly 595 600 605 Ser Glu Ala Asp His Glu Gly Thr
His Ser Thr Lys Arg Gly His Ala 610 615 620 Lys Ser Arg Pro Val Arg
Gly Ile His Thr Ser Pro Leu Gly Lys Pro 625 630 635 640 Ser Leu Ser
Pro 70267PRTHomo sapiens 70Met Lys Ala Ala Val Leu Thr Leu Ala Val
Leu Phe Leu Thr Gly Ser 1 5 10 15 Gln Ala Arg His Phe Trp Gln Gln
Asp Glu Pro Pro Gln Ser Pro Trp 20 25 30 Asp Arg Val Lys Asp Leu
Ala Thr Val Tyr Val Asp Val Leu Lys Asp 35 40 45 Ser Gly Arg Asp
Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys 50 55 60 Gln Leu
Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr 65 70 75 80
Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp 85
90 95 Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser
Lys 100 105 110 Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu
Asp Asp Phe 115 120 125 Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr
Arg Gln Lys Val Glu 130 135 140 Pro Leu Arg Ala Glu Leu Gln Glu Gly
Ala Arg Gln Lys Leu His Glu 145 150 155 160 Leu Gln Glu Lys Leu Ser
Pro Leu Gly Glu Glu Met Arg Asp Arg Ala 165 170 175 Arg Ala His Val
Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp 180 185 190 Glu Leu
Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn 195 200 205
Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu 210
215 220 Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg
Gln 225 230 235 240 Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala 245 250 255 Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln 260 265 71396PRTHomo sapiens 71Met Phe Leu Lys Ala Val Val Leu
Thr Leu Ala Leu Val Ala Val Ala 1 5 10 15 Gly Ala Arg Ala Glu Val
Ser Ala Asp Gln Val Ala Thr Val Met Trp 20 25 30 Asp Tyr Phe Ser
Gln Leu Ser Asn Asn Ala Lys Glu Ala Val Glu His 35 40 45 Leu Gln
Lys Ser Glu Leu Thr Gln Gln Leu Asn Ala Leu Phe Gln Asp 50 55 60
Lys Leu Gly Glu Val Asn Thr Tyr Ala Gly Asp Leu Gln Lys Lys Leu 65
70 75 80 Val Pro Phe Ala Thr Glu Leu His Glu Arg Leu Ala Lys Asp
Ser Glu 85 90 95 Lys Leu Lys Glu Glu Ile Gly Lys Glu Leu Glu Glu
Leu Arg Ala Arg 100 105 110 Leu Leu Pro His Ala Asn Glu Val Ser Gln
Lys Ile Gly Asp Asn Leu 115 120 125 Arg Glu Leu Gln Gln Arg Leu Glu
Pro Tyr Ala Asp Gln Leu Arg Thr 130 135 140 Gln Val Asn Thr Gln Ala
Glu Gln Leu Arg Arg Gln Leu Thr Pro Tyr 145 150 155 160 Ala Gln Arg
Met Glu Arg Val Leu Arg Glu Asn Ala Asp Ser Leu Gln 165 170 175 Ala
Ser Leu Arg Pro His Ala Asp Glu Leu Lys Ala Lys Ile Asp Gln 180 185
190 Asn Val Glu Glu Leu Lys Gly Arg Leu Thr Pro Tyr Ala Asp Glu Phe
195 200 205 Lys Val Lys Ile Asp Gln Thr Val Glu Glu Leu Arg Arg Ser
Leu Ala 210 215 220 Pro Tyr Ala Gln Asp Thr Gln Glu Lys Leu Asn His
Gln Leu Glu Gly 225 230 235 240 Leu Thr Phe Gln Met Lys Lys Asn Ala
Glu Glu Leu Lys Ala Arg Ile 245 250 255 Ser Ala Ser Ala Glu Glu Leu
Arg Gln Arg Leu Ala Pro Leu Ala Glu 260 265 270 Asp Val Arg Gly Asn
Leu Arg Gly Asn Thr Glu Gly Leu Gln Lys Ser 275 280 285 Leu Ala Glu
Leu Gly Gly His Leu Asp Gln Gln Val Glu Glu Phe Arg 290 295 300 Arg
Arg Val Glu Pro Tyr Gly Glu Asn Phe Asn Lys Ala Leu Val Gln 305 310
315 320 Gln Met Glu Gln Leu Arg Thr Lys Leu Gly Pro His Ala Gly Asp
Val 325 330 335 Glu Gly His Leu Ser Phe Leu Glu Lys Asp Leu Arg Asp
Lys Val Asn 340 345 350 Ser Phe Phe Ser Thr Phe Lys Glu Lys Glu Ser
Gln Asp Lys Thr Leu 355 360 365 Ser Leu Pro Glu Leu Glu Gln Gln Gln
Glu Gln His Gln Glu Gln Gln 370 375 380 Gln Glu Gln Val Gln Met Leu
Ala Pro Leu Glu Ser 385 390 395 72317PRTHomo sapiens 72Met Lys Val
Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys 1 5 10 15 Gln
Ala Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu 20 25
30 Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu
35 40 45 Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser
Glu Gln 50 55 60 Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln
Glu Leu Arg Ala 65 70 75 80 Leu Met Asp Glu Thr Met Lys Glu Leu Lys
Ala Tyr Lys Ser Glu Leu 85 90 95 Glu Glu Gln Leu Thr Pro Val Ala
Glu Glu Thr Arg Ala Arg Leu Ser 100 105 110 Lys Glu Leu Gln Ala Ala
Gln Ala Arg Leu Gly Ala Asp Met Glu Asp 115 120 125 Val Cys Gly Arg
Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu 130 135 140 Gly Gln
Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg 145 150 155
160 Lys Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg
165 170 175 Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg
Gly Leu 180 185 190 Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu
Gln Gly Arg Val 195 200 205 Arg Ala Ala Thr Val Gly Ser Leu Ala Gly
Gln Pro Leu Gln Glu Arg 210 215 220 Ala Gln Ala Trp Gly Glu Arg Leu
Arg Ala Arg Met Glu Glu Met Gly 225 230 235 240 Ser Arg Thr Arg Asp
Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu 245 250 255 Val Arg Ala
Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala 260 265 270 Glu
Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu 275 280
285 Asp Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala
290 295 300 Val Gly Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His 305
310 315 73427PRTHomo sapiens 73Met Lys Leu Ile Thr Ile Leu Phe Leu
Cys Ser Arg Leu Leu Leu Ser 1 5 10 15 Leu Thr Gln Glu Ser Gln Ser
Glu Glu Ile Asp Cys Asn Asp Lys Asp 20 25 30 Leu Phe Lys Ala Val
Asp Ala Ala Leu Lys Lys Tyr Asn Ser Gln Asn 35 40 45 Gln Ser Asn
Asn Gln Phe Val Leu Tyr Arg Ile Thr Glu Ala Thr Lys 50 55 60 Thr
Val Gly Ser Asp Thr Phe Tyr Ser Phe Lys Tyr Glu Ile Lys Glu 65 70
75 80 Gly Asp Cys Pro Val Gln Ser Gly Lys Thr Trp Gln Asp Cys Glu
Tyr 85 90 95 Lys Asp Ala Ala Lys Ala Ala Thr Gly Glu Cys Thr Ala
Thr Val Gly 100 105 110 Lys Arg Ser Ser Thr Lys Phe Ser Val Ala Thr
Gln Thr Cys Gln Ile 115 120 125 Thr Pro Ala Glu Gly Pro Val Val Thr
Ala Gln Tyr Asp Cys Leu Gly 130 135 140 Cys Val His Pro Ile Ser Thr
Gln Ser Pro Asp Leu Glu Pro Ile Leu 145 150 155 160 Arg His Gly Ile
Gln Tyr Phe Asn Asn Asn Thr Gln His Ser Ser Leu 165 170 175 Phe Met
Leu Asn Glu Val Lys Arg Ala Gln Arg Gln Val Val Ala Gly 180 185 190
Leu Asn Phe Arg Ile Thr Tyr Ser Ile Val Gln Thr Asn Cys Ser Lys 195
200 205 Glu Asn Phe Leu Phe Leu Thr Pro Asp Cys Lys Ser Leu Trp Asn
Gly 210 215 220 Asp Thr Gly Glu Cys Thr Asp Asn Ala Tyr Ile Asp Ile
Gln Leu Arg 225 230 235 240 Ile Ala Ser Phe Ser Gln Asn Cys Asp Ile
Tyr Pro Gly Lys Asp Phe 245 250 255 Val Gln Pro Pro Thr Lys Ile Cys
Val Gly Cys Pro Arg Asp Ile Pro 260 265 270 Thr Asn Ser Pro Glu Leu
Glu Glu Thr Leu Thr His Thr Ile Thr Lys 275 280 285 Leu Asn Ala Glu
Asn Asn Ala Thr Phe Tyr Phe Lys Ile Asp Asn Val 290 295 300 Lys Lys
Ala Arg Val Gln Val Val Ala Gly Lys Lys Tyr Phe Ile Asp 305 310 315
320 Phe Val Ala Arg Glu Thr Thr Cys Ser Lys Glu Ser Asn Glu Glu Leu
325 330
335 Thr Glu Ser Cys Glu Thr Lys Lys Leu Gly Gln Ser Leu Asp Cys Asn
340 345 350 Ala Glu Val Tyr Val Val Pro Trp Glu Lys Lys Ile Tyr Pro
Thr Val 355 360 365 Asn Cys Gln Pro Leu Gly Met Ile Ser Leu Met Lys
Arg Pro Pro Gly 370 375 380 Phe Ser Pro Phe Arg Ser Ser Arg Ile Gly
Glu Ile Lys Glu Glu Thr 385 390 395 400 Thr Ser His Leu Arg Ser Cys
Glu Tyr Lys Gly Arg Pro Pro Lys Ala 405 410 415 Gly Ala Glu Pro Ala
Ser Glu Arg Glu Val Ser 420 425 74732PRTHomo sapiens 74Met Ser Glu
Thr Ser Arg Thr Ala Phe Gly Gly Arg Arg Ala Val Pro 1 5 10 15 Pro
Asn Asn Ser Asn Ala Ala Glu Asp Asp Leu Pro Thr Val Glu Leu 20 25
30 Gln Gly Val Val Pro Arg Gly Val Asn Leu Gln Glu Phe Leu Asn Val
35 40 45 Thr Ser Val His Leu Phe Lys Glu Arg Trp Asp Thr Asn Lys
Val Asp 50 55 60 His His Thr Asp Lys Tyr Glu Asn Asn Lys Leu Ile
Val Arg Arg Gly 65 70 75 80 Gln Ser Phe Tyr Val Gln Ile Asp Leu Ser
Arg Pro Tyr Asp Pro Arg 85 90 95 Arg Asp Leu Phe Arg Val Glu Tyr
Val Ile Gly Arg Tyr Pro Gln Glu 100 105 110 Asn Lys Gly Thr Tyr Ile
Pro Val Pro Ile Val Ser Glu Leu Gln Ser 115 120 125 Gly Lys Trp Gly
Ala Lys Ile Val Met Arg Glu Asp Arg Ser Val Arg 130 135 140 Leu Ser
Ile Gln Ser Ser Pro Lys Cys Ile Val Gly Lys Phe Arg Met 145 150 155
160 Tyr Val Ala Val Trp Thr Pro Tyr Gly Val Leu Arg Thr Ser Arg Asn
165 170 175 Pro Glu Thr Asp Thr Tyr Ile Leu Phe Asn Pro Trp Cys Glu
Asp Asp 180 185 190 Ala Val Tyr Leu Asp Asn Glu Lys Glu Arg Glu Glu
Tyr Val Leu Asn 195 200 205 Asp Ile Gly Val Ile Phe Tyr Gly Glu Val
Asn Asp Ile Lys Thr Arg 210 215 220 Ser Trp Ser Tyr Gly Gln Phe Glu
Asp Gly Ile Leu Asp Thr Cys Leu 225 230 235 240 Tyr Val Met Asp Arg
Ala Gln Met Asp Leu Ser Gly Arg Gly Asn Pro 245 250 255 Ile Lys Val
Ser Arg Val Gly Ser Ala Met Val Asn Ala Lys Asp Asp 260 265 270 Glu
Gly Val Leu Val Gly Ser Trp Asp Asn Ile Tyr Ala Tyr Gly Val 275 280
285 Pro Pro Ser Ala Trp Thr Gly Ser Val Asp Ile Leu Leu Glu Tyr Arg
290 295 300 Ser Ser Glu Asn Pro Val Arg Tyr Gly Gln Cys Trp Val Phe
Ala Gly 305 310 315 320 Val Phe Asn Thr Phe Leu Arg Cys Leu Gly Ile
Pro Ala Arg Ile Val 325 330 335 Thr Asn Tyr Phe Ser Ala His Asp Asn
Asp Ala Asn Leu Gln Met Asp 340 345 350 Ile Phe Leu Glu Glu Asp Gly
Asn Val Asn Ser Lys Leu Thr Lys Asp 355 360 365 Ser Val Trp Asn Tyr
His Cys Trp Asn Glu Ala Trp Met Thr Arg Pro 370 375 380 Asp Leu Pro
Val Gly Phe Gly Gly Trp Gln Ala Val Asp Ser Thr Pro 385 390 395 400
Gln Glu Asn Ser Asp Gly Met Tyr Arg Cys Gly Pro Ala Ser Val Gln 405
410 415 Ala Ile Lys His Gly His Val Cys Phe Gln Phe Asp Ala Pro Phe
Val 420 425 430 Phe Ala Glu Val Asn Ser Asp Leu Ile Tyr Ile Thr Ala
Lys Lys Asp 435 440 445 Gly Thr His Val Val Glu Asn Val Asp Ala Thr
His Ile Gly Lys Leu 450 455 460 Ile Val Thr Lys Gln Ile Gly Gly Asp
Gly Met Met Asp Ile Thr Asp 465 470 475 480 Thr Tyr Lys Phe Gln Glu
Gly Gln Glu Glu Glu Arg Leu Ala Leu Glu 485 490 495 Thr Ala Leu Met
Tyr Gly Ala Lys Lys Pro Leu Asn Thr Glu Gly Val 500 505 510 Met Lys
Ser Arg Ser Asn Val Asp Met Asp Phe Glu Val Glu Asn Ala 515 520 525
Val Leu Gly Lys Asp Phe Lys Leu Ser Ile Thr Phe Arg Asn Asn Ser 530
535 540 His Asn Arg Tyr Thr Ile Thr Ala Tyr Leu Ser Ala Asn Ile Thr
Phe 545 550 555 560 Tyr Thr Gly Val Pro Lys Ala Glu Phe Lys Lys Glu
Thr Phe Asp Val 565 570 575 Thr Leu Glu Pro Leu Ser Phe Lys Lys Glu
Ala Val Leu Ile Gln Ala 580 585 590 Gly Glu Tyr Met Gly Gln Leu Leu
Glu Gln Ala Ser Leu His Phe Phe 595 600 605 Val Thr Ala Arg Ile Asn
Glu Thr Arg Asp Val Leu Ala Lys Gln Lys 610 615 620 Ser Thr Val Leu
Thr Ile Pro Glu Ile Ile Ile Lys Val Arg Gly Thr 625 630 635 640 Gln
Val Val Gly Ser Asp Met Thr Val Thr Val Gln Phe Thr Asn Pro 645 650
655 Leu Lys Glu Thr Leu Arg Asn Val Trp Val His Leu Asp Gly Pro Gly
660 665 670 Val Thr Arg Pro Met Lys Lys Met Phe Arg Glu Ile Arg Pro
Asn Ser 675 680 685 Thr Val Gln Trp Glu Glu Val Cys Arg Pro Trp Val
Ser Gly His Arg 690 695 700 Lys Leu Ile Ala Ser Met Ser Ser Asp Ser
Leu Arg His Val Tyr Gly 705 710 715 720 Glu Leu Asp Val Gln Ile Gln
Arg Arg Pro Ser Met 725 730 75147PRTHomo sapiens 75Met Ala Ser His
Arg Leu Leu Leu Leu Cys Leu Ala Gly Leu Val Phe 1 5 10 15 Val Ser
Glu Ala Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu 20 25 30
Met Val Lys Val Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val 35
40 45 Ala Val His Val Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro
Phe 50 55 60 Ala Ser Gly Lys Thr Ser Glu Ser Gly Glu Leu His Gly
Leu Thr Thr 65 70 75 80 Glu Glu Glu Phe Val Glu Gly Ile Tyr Lys Val
Glu Ile Asp Thr Lys 85 90 95 Ser Tyr Trp Lys Ala Leu Gly Ile Ser
Pro Phe His Glu His Ala Glu 100 105 110 Val Val Phe Thr Ala Asn Asp
Ser Gly Pro Arg Arg Tyr Thr Ile Ala 115 120 125 Ala Leu Leu Ser Pro
Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn 130 135 140 Pro Lys Glu
145 7617PRTHomo sapiens 76Arg Asn Gly Phe Lys Ser His Ala Leu Gln
Leu Asn Asn Arg Gln Ile 1 5 10 15 Arg 7716PRTHomo sapiens 77Asn Gly
Phe Lys Ser His Ala Leu Gln Leu Asn Asn Arg Gln Ile Arg 1 5 10 15
7831PRTHomo sapiens 78Arg Gly Leu Glu Glu Glu Leu Gln Phe Ser Leu
Gly Ser Lys Ile Asn 1 5 10 15 Val Lys Val Gly Gly Asn Ser Lys Gly
Thr Leu Lys Val Leu Arg 20 25 30 7927PRTHomo sapiens 79Arg Gly Leu
Glu Glu Glu Leu Gln Phe Ser Leu Gly Ser Lys Ile Asn 1 5 10 15 Val
Lys Val Gly Gly Asn Ser Lys Gly Thr Leu 20 25 8023PRTHomo sapiens
80Arg Gly Leu Glu Glu Glu Leu Gln Phe Ser Leu Gly Ser Lys Ile Asn 1
5 10 15 Val Lys Val Gly Gly Asn Ser 20 8117PRTHomo sapiens 81Arg
Gly Leu Glu Glu Glu Leu Gln Phe Ser Leu Gly Ser Lys Ile Asn 1 5 10
15 Val 8239PRTHomo sapiens 82Arg Gln Ala Gly Ala Ala Gly Ser Arg
Met Asn Phe Arg Pro Gly Val 1 5 10 15 Leu Ser Ser Arg Gln Leu Gly
Leu Pro Gly Pro Pro Asp Val Pro Asp 20 25 30 His Ala Ala Tyr His
Pro Phe 35 8319PRTHomo sapiens 83Gln Leu Gly Leu Pro Gly Pro Pro
Asp Val Pro Asp His Ala Ala Tyr 1 5 10 15 His Pro Phe 8429PRTHomo
sapiens 84Arg Asn Val His Ser Gly Ser Thr Phe Phe Lys Tyr Tyr Leu
Gln Gly 1 5 10 15 Ala Lys Ile Pro Lys Pro Glu Ala Ser Phe Ser Pro
Arg 20 25 8523PRTHomo sapiens 85Arg Asn Val His Ser Ala Gly Ala Ala
Gly Ser Arg Met Asn Phe Arg 1 5 10 15 Pro Gly Val Leu Ser Ser Arg
20 8631PRTHomo sapiensMOD_RES(26)..(26)Oxidated Met 86Arg Ala Glu
Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln 1 5 10 15 Glu
Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg 20 25 30
8715PRTHomo sapiens 87Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His
Glu Leu Gln Glu 1 5 10 15 8829PRTHomo sapiens 88Arg Gln Gly Leu Leu
Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu 1 5 10 15 Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln 20 25 8921PRTHomo sapiens
89Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala 1
5 10 15 Leu Glu Asp Leu Arg 20 9024PRTHomo sapiens 90Ile Ser Ala
Ser Ala Glu Glu Leu Arg Gln Arg Leu Ala Pro Leu Ala 1 5 10 15 Glu
Asp Val Arg Gly Asn Leu Lys 20 9126PRTHomo sapiens 91Lys Gly Asn
Thr Glu Gly Leu Gln Lys Ser Leu Ala Glu Leu Gly Gly 1 5 10 15 His
Leu Asp Gln Gln Val Glu Glu Phe Arg 20 25 9225PRTHomo sapiens 92Arg
Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg 1 5 10
15 Ala Gln Ala Trp Gly Glu Arg Leu Arg 20 25 9324PRTHomo sapiens
93Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg 1
5 10 15 Ala Gln Ala Trp Gly Glu Arg Leu 20 9411PRTHomo sapiens
94His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg 1 5 10 9521PRTHomo
sapiens 95Arg Lys His Asn Leu Gly His Gly His Lys His Glu Arg Asp
Gln Gly 1 5 10 15 His Gly His Gln Arg 20 9618PRTHomo sapiens 96Asn
Leu Gly His Gly His Lys His Glu Arg Asp Gln Gly His Gly His 1 5 10
15 Gln Arg 9722PRTHomo sapiens 97Arg Gly His Gly Leu Gly His Gly
His Glu Gln Gln His Gly Leu Gly 1 5 10 15 His Gly His Lys Phe Lys
20 9826PRTHomo sapiens 98Arg Ala Val Pro Pro Asn Asn Ser Asn Ala
Ala Glu Asp Asp Leu Pro 1 5 10 15 Thr Val Glu Leu Gln Gly Val Val
Pro Arg 20 25 9924PRTHomo sapiens 99Lys Ala Leu Gly Ile Ser Pro Phe
His Glu His Ala Glu Val Val Phe 1 5 10 15 Thr Ala Asn Asp Ser Gly
Pro Arg 20 10021PRTHomo sapiens 100Gly Leu Glu Glu Glu Leu Gln Phe
Ser Leu Gly Ser Lys Ile Asn Val 1 5 10 15 Lys Gly Gly Asn Ser 20
10130PRTHomo sapiens 101Lys Ser Ser Ser Tyr Ser Lys Gln Phe Thr Ser
Ser Thr Ser Tyr Asn 1 5 10 15 Arg Gly Asp Ser Thr Phe Glu Ser Lys
Ser Tyr Lys Met Ala 20 25 30 10257PRTHomo sapiens 102Gly Ser Glu
Ser Gly Ile Phe Thr Asn Thr Lys Glu Ser Ser Ser His 1 5 10 15 His
Pro Gly Ile Ala Glu Phe Pro Ser Arg Gly Lys Ser Ser Ser Tyr 20 25
30 Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr Asn Arg Gly Asp Ser Thr
35 40 45 Phe Glu Ser Lys Ser Tyr Lys Met Ala 50 55 10319PRTHomo
sapiens 103Arg Gly Leu Glu Glu Glu Leu Gln Phe Ser Leu Gly Ser Lys
Ile Asn 1 5 10 15 Val Lys Val 10424PRTHomo sapiens 104Arg Thr Leu
Glu Ile Pro Gly Asn Ser Asp Pro Asn Met Ile Pro Asp 1 5 10 15 Gly
Asp Phe Asn Ser Tyr Val Arg 20 10525PRTHomo sapiens 105Lys Gly Asn
Thr Glu Gly Leu Gln Lys Ser Leu Ala Glu Leu Gly Gly 1 5 10 15 His
Leu Asp Gln Gln Val Glu Glu Phe 20 25 10626PRTHomo sapiens 106Asp
Val Ser Ser Ala Leu Asp Lys Leu Lys Glu Phe Gly Asn Thr Leu 1 5 10
15 Glu Asp Lys Ala Arg Glu Leu Ile Ser Arg 20 25 10721PRTHomo
sapiens 107Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala
Gln Ala 1 5 10 15 Trp Gly Glu Arg Leu 20 108644PRTHomo sapiens
108Met Phe Ser Met Arg Ile Val Cys Leu Val Leu Ser Val Val Gly Thr
1 5 10 15 Ala Trp Thr Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu
Gly Gly 20 25 30 Gly Val Arg Gly Pro Arg Val Val Glu Arg His Gln
Ser Ala Cys Lys 35 40 45 Asp Ser Asp Trp Pro Phe Cys Ser Asp Glu
Asp Trp Asn Tyr Lys Cys 50 55 60 Pro Ser Gly Cys Arg Met Lys Gly
Leu Ile Asp Glu Val Asn Gln Asp 65 70 75 80 Phe Thr Asn Arg Ile Asn
Lys Leu Lys Asn Ser Leu Phe Glu Tyr Gln 85 90 95 Lys Asn Asn Lys
Asp Ser His Ser Leu Thr Thr Asn Ile Met Glu Ile 100 105 110 Leu Arg
Gly Asp Phe Ser Ser Ala Asn Asn Arg Asp Asn Thr Tyr Asn 115 120 125
Arg Val Ser Glu Asp Leu Arg Ser Arg Ile Glu Val Leu Lys Arg Lys 130
135 140 Val Ile Glu Lys Val Gln His Ile Gln Leu Leu Gln Lys Asn Val
Arg 145 150 155 160 Ala Gln Leu Val Asp Met Lys Arg Leu Glu Val Asp
Ile Asp Ile Lys 165 170 175 Ile Arg Ser Cys Arg Gly Ser Cys Ser Arg
Ala Leu Ala Arg Glu Val 180 185 190 Asp Leu Lys Asp Tyr Glu Asp Gln
Gln Lys Gln Leu Glu Gln Val Ile 195 200 205 Ala Lys Asp Leu Leu Pro
Ser Arg Asp Arg Gln His Leu Pro Leu Ile 210 215 220 Lys Met Lys Pro
Val Pro Asp Leu Val Pro Gly Asn Phe Lys Ser Gln 225 230 235 240 Leu
Gln Lys Val Pro Pro Glu Trp Lys Ala Leu Thr Asp Met Pro Gln 245 250
255 Met Arg Met Glu Leu Glu Arg Pro Gly Gly Asn Glu Ile Thr Arg Gly
260 265 270 Gly Ser Thr Ser Tyr Gly Thr Gly Ser Glu Thr Glu Ser Pro
Arg Asn 275 280 285 Pro Ser Ser Ala Gly Ser Trp Asn Ser Gly Ser Ser
Gly Pro Gly Ser 290 295 300 Thr Gly Asn Arg Asn Pro Gly Ser Ser Gly
Thr Gly Gly Thr Ala Thr 305 310 315 320 Trp Lys Pro Gly Ser Ser Gly
Pro Gly Ser Thr Gly Ser Trp Asn Ser 325 330 335 Gly Ser Ser Gly Thr
Gly Ser Thr Gly Asn Gln Asn Pro Gly Ser Pro 340 345 350 Arg Pro Gly
Ser Thr Gly Thr Trp Asn Pro Gly Ser Ser Glu Arg Gly 355 360 365 Ser
Ala Gly His Trp Thr Ser Glu Ser Ser Val Ser Gly Ser Thr Gly 370 375
380 Gln Trp His Ser Glu Ser Gly Ser Phe Arg Pro Asp Ser Pro Gly Ser
385 390 395 400 Gly Asn Ala Arg Pro Asn Asn Pro Asp Trp Gly Thr Phe
Glu Glu Val 405 410 415 Ser Gly Asn Val Ser Pro Gly Thr Arg Arg Glu
Tyr His Thr Glu Lys 420 425 430 Leu Val Thr Ser Lys Gly Asp Lys Glu
Leu Arg Thr Gly Lys Glu Lys 435 440 445 Val Thr Ser Gly Ser Thr Thr
Thr Thr Arg Arg Ser Cys Ser
Lys Thr 450 455 460 Val Thr Lys Thr Val Ile Gly Pro Asp Gly His Lys
Glu Val Thr Lys 465 470 475 480 Glu Val Val Thr Ser Glu Asp Gly Ser
Asp Cys Pro Glu Ala Met Asp 485 490 495 Leu Gly Thr Leu Ser Gly Ile
Gly Thr Leu Asp Gly Phe Arg His Arg 500 505 510 His Pro Asp Glu Ala
Ala Phe Phe Asp Thr Ala Ser Thr Gly Lys Thr 515 520 525 Phe Pro Gly
Phe Phe Ser Pro Met Leu Gly Glu Phe Val Ser Glu Thr 530 535 540 Glu
Ser Arg Gly Ser Glu Ser Gly Ile Phe Thr Asn Thr Lys Glu Ser 545 550
555 560 Ser Ser His His Pro Gly Ile Ala Glu Phe Pro Ser Arg Gly Lys
Ser 565 570 575 Ser Ser Tyr Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr
Asn Arg Gly 580 585 590 Asp Ser Thr Phe Glu Ser Lys Ser Tyr Lys Met
Ala Asp Glu Ala Gly 595 600 605 Ser Glu Ala Asp His Glu Gly Thr His
Ser Thr Lys Arg Gly His Ala 610 615 620 Lys Ser Arg Pro Val Arg Gly
Ile His Thr Ser Pro Leu Gly Lys Pro 625 630 635 640 Ser Leu Ser Pro
1091663PRTHomo sapiens 109Met Gly Pro Thr Ser Gly Pro Ser Leu Leu
Leu Leu Leu Leu Thr His 1 5 10 15 Leu Pro Leu Ala Leu Gly Ser Pro
Met Tyr Ser Ile Ile Thr Pro Asn 20 25 30 Ile Leu Arg Leu Glu Ser
Glu Glu Thr Met Val Leu Glu Ala His Asp 35 40 45 Ala Gln Gly Asp
Val Pro Val Thr Val Thr Val His Asp Phe Pro Gly 50 55 60 Lys Lys
Leu Val Leu Ser Ser Glu Lys Thr Val Leu Thr Pro Ala Thr 65 70 75 80
Asn His Met Gly Asn Val Thr Phe Thr Ile Pro Ala Asn Arg Glu Phe 85
90 95 Lys Ser Glu Lys Gly Arg Asn Lys Phe Val Thr Val Gln Ala Thr
Phe 100 105 110 Gly Thr Gln Val Val Glu Lys Val Val Leu Val Ser Leu
Gln Ser Gly 115 120 125 Tyr Leu Phe Ile Gln Thr Asp Lys Thr Ile Tyr
Thr Pro Gly Ser Thr 130 135 140 Val Leu Tyr Arg Ile Phe Thr Val Asn
His Lys Leu Leu Pro Val Gly 145 150 155 160 Arg Thr Val Met Val Asn
Ile Glu Asn Pro Glu Gly Ile Pro Val Lys 165 170 175 Gln Asp Ser Leu
Ser Ser Gln Asn Gln Leu Gly Val Leu Pro Leu Ser 180 185 190 Trp Asp
Ile Pro Glu Leu Val Asn Met Gly Gln Trp Lys Ile Arg Ala 195 200 205
Tyr Tyr Glu Asn Ser Pro Gln Gln Val Phe Ser Thr Glu Phe Glu Val 210
215 220 Lys Glu Tyr Val Leu Pro Ser Phe Glu Val Ile Val Glu Pro Thr
Glu 225 230 235 240 Lys Phe Tyr Tyr Ile Tyr Asn Glu Lys Gly Leu Glu
Val Thr Ile Thr 245 250 255 Ala Arg Phe Leu Tyr Gly Lys Lys Val Glu
Gly Thr Ala Phe Val Ile 260 265 270 Phe Gly Ile Gln Asp Gly Glu Gln
Arg Ile Ser Leu Pro Glu Ser Leu 275 280 285 Lys Arg Ile Pro Ile Glu
Asp Gly Ser Gly Glu Val Val Leu Ser Arg 290 295 300 Lys Val Leu Leu
Asp Gly Val Gln Asn Leu Arg Ala Glu Asp Leu Val 305 310 315 320 Gly
Lys Ser Leu Tyr Val Ser Ala Thr Val Ile Leu His Ser Gly Ser 325 330
335 Asp Met Val Gln Ala Glu Arg Ser Gly Ile Pro Ile Val Thr Ser Pro
340 345 350 Tyr Gln Ile His Phe Thr Lys Thr Pro Lys Tyr Phe Lys Pro
Gly Met 355 360 365 Pro Phe Asp Leu Met Val Phe Val Thr Asn Pro Asp
Gly Ser Pro Ala 370 375 380 Tyr Arg Val Pro Val Ala Val Gln Gly Glu
Asp Thr Val Gln Ser Leu 385 390 395 400 Thr Gln Gly Asp Gly Val Ala
Lys Leu Ser Ile Asn Thr His Pro Ser 405 410 415 Gln Lys Pro Leu Ser
Ile Thr Val Arg Thr Lys Lys Gln Glu Leu Ser 420 425 430 Glu Ala Glu
Gln Ala Thr Arg Thr Met Gln Ala Leu Pro Tyr Ser Thr 435 440 445 Val
Gly Asn Ser Asn Asn Tyr Leu His Leu Ser Val Leu Arg Thr Glu 450 455
460 Leu Arg Pro Gly Glu Thr Leu Asn Val Asn Phe Leu Leu Arg Met Asp
465 470 475 480 Arg Ala His Glu Ala Lys Ile Arg Tyr Tyr Thr Tyr Leu
Ile Met Asn 485 490 495 Lys Gly Arg Leu Leu Lys Ala Gly Arg Gln Val
Arg Glu Pro Gly Gln 500 505 510 Asp Leu Val Val Leu Pro Leu Ser Ile
Thr Thr Asp Phe Ile Pro Ser 515 520 525 Phe Arg Leu Val Ala Tyr Tyr
Thr Leu Ile Gly Ala Ser Gly Gln Arg 530 535 540 Glu Val Val Ala Asp
Ser Val Trp Val Asp Val Lys Asp Ser Cys Val 545 550 555 560 Gly Ser
Leu Val Val Lys Ser Gly Gln Ser Glu Asp Arg Gln Pro Val 565 570 575
Pro Gly Gln Gln Met Thr Leu Lys Ile Glu Gly Asp His Gly Ala Arg 580
585 590 Val Val Leu Val Ala Val Asp Lys Gly Val Phe Val Leu Asn Lys
Lys 595 600 605 Asn Lys Leu Thr Gln Ser Lys Ile Trp Asp Val Val Glu
Lys Ala Asp 610 615 620 Ile Gly Cys Thr Pro Gly Ser Gly Lys Asp Tyr
Ala Gly Val Phe Ser 625 630 635 640 Asp Ala Gly Leu Thr Phe Thr Ser
Ser Ser Gly Gln Gln Thr Ala Gln 645 650 655 Arg Ala Glu Leu Gln Cys
Pro Gln Pro Ala Ala Arg Arg Arg Arg Ser 660 665 670 Val Gln Leu Thr
Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro Lys 675 680 685 Glu Leu
Arg Lys Cys Cys Glu Asp Gly Met Arg Glu Asn Pro Met Arg 690 695 700
Phe Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser Leu Gly Glu Ala Cys 705
710 715 720 Lys Lys Val Phe Leu Asp Cys Cys Asn Tyr Ile Thr Glu Leu
Arg Arg 725 730 735 Gln His Ala Arg Ala Ser His Leu Gly Leu Ala Arg
Ser Asn Leu Asp 740 745 750 Glu Asp Ile Ile Ala Glu Glu Asn Ile Val
Ser Arg Ser Glu Phe Pro 755 760 765 Glu Ser Trp Leu Trp Asn Val Glu
Asp Leu Lys Glu Pro Pro Lys Asn 770 775 780 Gly Ile Ser Thr Lys Leu
Met Asn Ile Phe Leu Lys Asp Ser Ile Thr 785 790 795 800 Thr Trp Glu
Ile Leu Ala Val Ser Met Ser Asp Lys Lys Gly Ile Cys 805 810 815 Val
Ala Asp Pro Phe Glu Val Thr Val Met Gln Asp Phe Phe Ile Asp 820 825
830 Leu Arg Leu Pro Tyr Ser Val Val Arg Asn Glu Gln Val Glu Ile Arg
835 840 845 Ala Val Leu Tyr Asn Tyr Arg Gln Asn Gln Glu Leu Lys Val
Arg Val 850 855 860 Glu Leu Leu His Asn Pro Ala Phe Cys Ser Leu Ala
Thr Thr Lys Arg 865 870 875 880 Arg His Gln Gln Thr Val Thr Ile Pro
Pro Lys Ser Ser Leu Ser Val 885 890 895 Pro Tyr Val Ile Val Pro Leu
Lys Thr Gly Leu Gln Glu Val Glu Val 900 905 910 Lys Ala Ala Val Tyr
His His Phe Ile Ser Asp Gly Val Arg Lys Ser 915 920 925 Leu Lys Val
Val Pro Glu Gly Ile Arg Met Asn Lys Thr Val Ala Val 930 935 940 Arg
Thr Leu Asp Pro Glu Arg Leu Gly Arg Glu Gly Val Gln Lys Glu 945 950
955 960 Asp Ile Pro Pro Ala Asp Leu Ser Asp Gln Val Pro Asp Thr Glu
Ser 965 970 975 Glu Thr Arg Ile Leu Leu Gln Gly Thr Pro Val Ala Gln
Met Thr Glu 980 985 990 Asp Ala Val Asp Ala Glu Arg Leu Lys His Leu
Ile Val Thr Pro Ser 995 1000 1005 Gly Cys Gly Glu Gln Asn Met Ile
Gly Met Thr Pro Thr Val Ile 1010 1015 1020 Ala Val His Tyr Leu Asp
Glu Thr Glu Gln Trp Glu Lys Phe Gly 1025 1030 1035 Leu Glu Lys Arg
Gln Gly Ala Leu Glu Leu Ile Lys Lys Gly Tyr 1040 1045 1050 Thr Gln
Gln Leu Ala Phe Arg Gln Pro Ser Ser Ala Phe Ala Ala 1055 1060 1065
Phe Val Lys Arg Ala Pro Ser Thr Trp Leu Thr Ala Tyr Val Val 1070
1075 1080 Lys Val Phe Ser Leu Ala Val Asn Leu Ile Ala Ile Asp Ser
Gln 1085 1090 1095 Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu
Lys Gln Lys 1100 1105 1110 Pro Asp Gly Val Phe Gln Glu Asp Ala Pro
Val Ile His Gln Glu 1115 1120 1125 Met Ile Gly Gly Leu Arg Asn Asn
Asn Glu Lys Asp Met Ala Leu 1130 1135 1140 Thr Ala Phe Val Leu Ile
Ser Leu Gln Glu Ala Lys Asp Ile Cys 1145 1150 1155 Glu Glu Gln Val
Asn Ser Leu Pro Gly Ser Ile Thr Lys Ala Gly 1160 1165 1170 Asp Phe
Leu Glu Ala Asn Tyr Met Asn Leu Gln Arg Ser Tyr Thr 1175 1180 1185
Val Ala Ile Ala Gly Tyr Ala Leu Ala Gln Met Gly Arg Leu Lys 1190
1195 1200 Gly Pro Leu Leu Asn Lys Phe Leu Thr Thr Ala Lys Asp Lys
Asn 1205 1210 1215 Arg Trp Glu Asp Pro Gly Lys Gln Leu Tyr Asn Val
Glu Ala Thr 1220 1225 1230 Ser Tyr Ala Leu Leu Ala Leu Leu Gln Leu
Lys Asp Phe Asp Phe 1235 1240 1245 Val Pro Pro Val Val Arg Trp Leu
Asn Glu Gln Arg Tyr Tyr Gly 1250 1255 1260 Gly Gly Tyr Gly Ser Thr
Gln Ala Thr Phe Met Val Phe Gln Ala 1265 1270 1275 Leu Ala Gln Tyr
Gln Lys Asp Ala Pro Asp His Gln Glu Leu Asn 1280 1285 1290 Leu Asp
Val Ser Leu Gln Leu Pro Ser Arg Ser Ser Lys Ile Thr 1295 1300 1305
His Arg Ile His Trp Glu Ser Ala Ser Leu Leu Arg Ser Glu Glu 1310
1315 1320 Thr Lys Glu Asn Glu Gly Phe Thr Val Thr Ala Glu Gly Lys
Gly 1325 1330 1335 Gln Gly Thr Leu Ser Val Val Thr Met Tyr His Ala
Lys Ala Lys 1340 1345 1350 Asp Gln Leu Thr Cys Asn Lys Phe Asp Leu
Lys Val Thr Ile Lys 1355 1360 1365 Pro Ala Pro Glu Thr Glu Lys Arg
Pro Gln Asp Ala Lys Asn Thr 1370 1375 1380 Met Ile Leu Glu Ile Cys
Thr Arg Tyr Arg Gly Asp Gln Asp Ala 1385 1390 1395 Thr Met Ser Ile
Leu Asp Ile Ser Met Met Thr Gly Phe Ala Pro 1400 1405 1410 Asp Thr
Asp Asp Leu Lys Gln Leu Ala Asn Gly Val Asp Arg Tyr 1415 1420 1425
Ile Ser Lys Tyr Glu Leu Asp Lys Ala Phe Ser Asp Arg Asn Thr 1430
1435 1440 Leu Ile Ile Tyr Leu Asp Lys Val Ser His Ser Glu Asp Asp
Cys 1445 1450 1455 Leu Ala Phe Lys Val His Gln Tyr Phe Asn Val Glu
Leu Ile Gln 1460 1465 1470 Pro Gly Ala Val Lys Val Tyr Ala Tyr Tyr
Asn Leu Glu Glu Ser 1475 1480 1485 Cys Thr Arg Phe Tyr His Pro Glu
Lys Glu Asp Gly Lys Leu Asn 1490 1495 1500 Lys Leu Cys Arg Asp Glu
Leu Cys Arg Cys Ala Glu Glu Asn Cys 1505 1510 1515 Phe Ile Gln Lys
Ser Asp Asp Lys Val Thr Leu Glu Glu Arg Leu 1520 1525 1530 Asp Lys
Ala Cys Glu Pro Gly Val Asp Tyr Val Tyr Lys Thr Arg 1535 1540 1545
Leu Val Lys Val Gln Leu Ser Asn Asp Phe Asp Glu Tyr Ile Met 1550
1555 1560 Ala Ile Glu Gln Thr Ile Lys Ser Gly Ser Asp Glu Val Gln
Val 1565 1570 1575 Gly Gln Gln Arg Thr Phe Ile Ser Pro Ile Lys Cys
Arg Glu Ala 1580 1585 1590 Leu Lys Leu Glu Glu Lys Lys His Tyr Leu
Met Trp Gly Leu Ser 1595 1600 1605 Ser Asp Phe Trp Gly Glu Lys Pro
Asn Leu Ser Tyr Ile Ile Gly 1610 1615 1620 Lys Asp Thr Trp Val Glu
His Trp Pro Glu Glu Asp Glu Cys Gln 1625 1630 1635 Asp Glu Glu Asn
Gln Lys Gln Cys Gln Asp Leu Gly Ala Phe Thr 1640 1645 1650 Glu Ser
Met Val Val Phe Gly Cys Pro Asn 1655 1660 1101744PRTHomo sapiens
110Met Arg Leu Leu Trp Gly Leu Ile Trp Ala Ser Ser Phe Phe Thr Leu
1 5 10 15 Ser Leu Gln Lys Pro Arg Leu Leu Leu Phe Ser Pro Ser Val
Val His 20 25 30 Leu Gly Val Pro Leu Ser Val Gly Val Gln Leu Gln
Asp Val Pro Arg 35 40 45 Gly Gln Val Val Lys Gly Ser Val Phe Leu
Arg Asn Pro Ser Arg Asn 50 55 60 Asn Val Pro Cys Ser Pro Lys Val
Asp Phe Thr Leu Ser Ser Glu Arg 65 70 75 80 Asp Phe Ala Leu Leu Ser
Leu Gln Val Pro Leu Lys Asp Ala Lys Ser 85 90 95 Cys Gly Leu His
Gln Leu Leu Arg Gly Pro Glu Val Gln Leu Val Ala 100 105 110 His Ser
Pro Trp Leu Lys Asp Ser Leu Ser Arg Thr Thr Asn Ile Gln 115 120 125
Gly Ile Asn Leu Leu Phe Ser Ser Arg Arg Gly His Leu Phe Leu Gln 130
135 140 Thr Asp Gln Pro Ile Tyr Asn Pro Gly Gln Arg Val Arg Tyr Arg
Val 145 150 155 160 Phe Ala Leu Asp Gln Lys Met Arg Pro Ser Thr Asp
Thr Ile Thr Val 165 170 175 Met Val Glu Asn Ser His Gly Leu Arg Val
Arg Lys Lys Glu Val Tyr 180 185 190 Met Pro Ser Ser Ile Phe Gln Asp
Asp Phe Val Ile Pro Asp Ile Ser 195 200 205 Glu Pro Gly Thr Trp Lys
Ile Ser Ala Arg Phe Ser Asp Gly Leu Glu 210 215 220 Ser Asn Ser Ser
Thr Gln Phe Glu Val Lys Lys Tyr Val Leu Pro Asn 225 230 235 240 Phe
Glu Val Lys Ile Thr Pro Gly Lys Pro Tyr Ile Leu Thr Val Pro 245 250
255 Gly His Leu Asp Glu Met Gln Leu Asp Ile Gln Ala Arg Tyr Ile Tyr
260 265 270 Gly Lys Pro Val Gln Gly Val Ala Tyr Val Arg Phe Gly Leu
Leu Asp 275 280 285 Glu Asp Gly Lys Lys Thr Phe Phe Arg Gly Leu Glu
Ser Gln Thr Lys 290 295 300 Leu Val Asn Gly Gln Ser His Ile Ser Leu
Ser Lys Ala Glu Phe Gln 305 310 315 320 Asp Ala Leu Glu Lys Leu Asn
Met Gly Ile Thr Asp Leu Gln Gly Leu 325 330 335 Arg Leu Tyr Val Ala
Ala Ala Ile Ile Glu Ser Pro Gly Gly Glu Met 340 345 350 Glu Glu Ala
Glu Leu Thr Ser Trp Tyr Phe Val Ser Ser Pro Phe Ser 355 360 365 Leu
Asp Leu Ser Lys Thr Lys Arg His Leu Val Pro Gly Ala Pro Phe 370 375
380 Leu Leu Gln Ala Leu Val Arg Glu Met Ser Gly Ser Pro Ala Ser Gly
385 390 395 400 Ile Pro Val Lys Val Ser Ala Thr Val Ser Ser Pro Gly
Ser Val Pro 405 410 415 Glu Val
Gln Asp Ile Gln Gln Asn Thr Asp Gly Ser Gly Gln Val Ser 420 425 430
Ile Pro Ile Ile Ile Pro Gln Thr Ile Ser Glu Leu Gln Leu Ser Val 435
440 445 Ser Ala Gly Ser Pro His Pro Ala Ile Ala Arg Leu Thr Val Ala
Ala 450 455 460 Pro Pro Ser Gly Gly Pro Gly Phe Leu Ser Ile Glu Arg
Pro Asp Ser 465 470 475 480 Arg Pro Pro Arg Val Gly Asp Thr Leu Asn
Leu Asn Leu Arg Ala Val 485 490 495 Gly Ser Gly Ala Thr Phe Ser His
Tyr Tyr Tyr Met Ile Leu Ser Arg 500 505 510 Gly Gln Ile Val Phe Met
Asn Arg Glu Pro Lys Arg Thr Leu Thr Ser 515 520 525 Val Ser Val Phe
Val Asp His His Leu Ala Pro Ser Phe Tyr Phe Val 530 535 540 Ala Phe
Tyr Tyr His Gly Asp His Pro Val Ala Asn Ser Leu Arg Val 545 550 555
560 Asp Val Gln Ala Gly Ala Cys Glu Gly Lys Leu Glu Leu Ser Val Asp
565 570 575 Gly Ala Lys Gln Tyr Arg Asn Gly Glu Ser Val Lys Leu His
Leu Glu 580 585 590 Thr Asp Ser Leu Ala Leu Val Ala Leu Gly Ala Leu
Asp Thr Ala Leu 595 600 605 Tyr Ala Ala Gly Ser Lys Ser His Lys Pro
Leu Asn Met Gly Lys Val 610 615 620 Phe Glu Ala Met Asn Ser Tyr Asp
Leu Gly Cys Gly Pro Gly Gly Gly 625 630 635 640 Asp Ser Ala Leu Gln
Val Phe Gln Ala Ala Gly Leu Ala Phe Ser Asp 645 650 655 Gly Asp Gln
Trp Thr Leu Ser Arg Lys Arg Leu Ser Cys Pro Lys Glu 660 665 670 Lys
Thr Thr Arg Lys Lys Arg Asn Val Asn Phe Gln Lys Ala Ile Asn 675 680
685 Glu Lys Leu Gly Gln Tyr Ala Ser Pro Thr Ala Lys Arg Cys Cys Gln
690 695 700 Asp Gly Val Thr Arg Leu Pro Met Met Arg Ser Cys Glu Gln
Arg Ala 705 710 715 720 Ala Arg Val Gln Gln Pro Asp Cys Arg Glu Pro
Phe Leu Ser Cys Cys 725 730 735 Gln Phe Ala Glu Ser Leu Arg Lys Lys
Ser Arg Asp Lys Gly Gln Ala 740 745 750 Gly Leu Gln Arg Ala Leu Glu
Ile Leu Gln Glu Glu Asp Leu Ile Asp 755 760 765 Glu Asp Asp Ile Pro
Val Arg Ser Phe Phe Pro Glu Asn Trp Leu Trp 770 775 780 Arg Val Glu
Thr Val Asp Arg Phe Gln Ile Leu Thr Leu Trp Leu Pro 785 790 795 800
Asp Ser Leu Thr Thr Trp Glu Ile His Gly Leu Ser Leu Ser Lys Thr 805
810 815 Lys Gly Leu Cys Val Ala Thr Pro Val Gln Leu Arg Val Phe Arg
Glu 820 825 830 Phe His Leu His Leu Arg Leu Pro Met Ser Val Arg Arg
Phe Glu Gln 835 840 845 Leu Glu Leu Arg Pro Val Leu Tyr Asn Tyr Leu
Asp Lys Asn Leu Thr 850 855 860 Val Ser Val His Val Ser Pro Val Glu
Gly Leu Cys Leu Ala Gly Gly 865 870 875 880 Gly Gly Leu Ala Gln Gln
Val Leu Val Pro Ala Gly Ser Ala Arg Pro 885 890 895 Val Ala Phe Ser
Val Val Pro Thr Ala Ala Ala Ala Val Ser Leu Lys 900 905 910 Val Val
Ala Arg Gly Ser Phe Glu Phe Pro Val Gly Asp Ala Val Ser 915 920 925
Lys Val Leu Gln Ile Glu Lys Glu Gly Ala Ile His Arg Glu Glu Leu 930
935 940 Val Tyr Glu Leu Asn Pro Leu Asp His Arg Gly Arg Thr Leu Glu
Ile 945 950 955 960 Pro Gly Asn Ser Asp Pro Asn Met Ile Pro Asp Gly
Asp Phe Asn Ser 965 970 975 Tyr Val Arg Val Thr Ala Ser Asp Pro Leu
Asp Thr Leu Gly Ser Glu 980 985 990 Gly Ala Leu Ser Pro Gly Gly Val
Ala Ser Leu Leu Arg Leu Pro Arg 995 1000 1005 Gly Cys Gly Glu Gln
Thr Met Ile Tyr Leu Ala Pro Thr Leu Ala 1010 1015 1020 Ala Ser Arg
Tyr Leu Asp Lys Thr Glu Gln Trp Ser Thr Leu Pro 1025 1030 1035 Pro
Glu Thr Lys Asp His Ala Val Asp Leu Ile Gln Lys Gly Tyr 1040 1045
1050 Met Arg Ile Gln Gln Phe Arg Lys Ala Asp Gly Ser Tyr Ala Ala
1055 1060 1065 Trp Leu Ser Arg Asp Ser Ser Thr Trp Leu Thr Ala Phe
Val Leu 1070 1075 1080 Lys Val Leu Ser Leu Ala Gln Glu Gln Val Gly
Gly Ser Pro Glu 1085 1090 1095 Lys Leu Gln Glu Thr Ser Asn Trp Leu
Leu Ser Gln Gln Gln Ala 1100 1105 1110 Asp Gly Ser Phe Gln Asp Pro
Cys Pro Val Leu Asp Arg Ser Met 1115 1120 1125 Gln Gly Gly Leu Val
Gly Asn Asp Glu Thr Val Ala Leu Thr Ala 1130 1135 1140 Phe Val Thr
Ile Ala Leu His His Gly Leu Ala Val Phe Gln Asp 1145 1150 1155 Glu
Gly Ala Glu Pro Leu Lys Gln Arg Val Glu Ala Ser Ile Ser 1160 1165
1170 Lys Ala Asn Ser Phe Leu Gly Glu Lys Ala Ser Ala Gly Leu Leu
1175 1180 1185 Gly Ala His Ala Ala Ala Ile Thr Ala Tyr Ala Leu Ser
Leu Thr 1190 1195 1200 Lys Ala Pro Val Asp Leu Leu Gly Val Ala His
Asn Asn Leu Met 1205 1210 1215 Ala Met Ala Gln Glu Thr Gly Asp Asn
Leu Tyr Trp Gly Ser Val 1220 1225 1230 Thr Gly Ser Gln Ser Asn Ala
Val Ser Pro Thr Pro Ala Pro Arg 1235 1240 1245 Asn Pro Ser Asp Pro
Met Pro Gln Ala Pro Ala Leu Trp Ile Glu 1250 1255 1260 Thr Thr Ala
Tyr Ala Leu Leu His Leu Leu Leu His Glu Gly Lys 1265 1270 1275 Ala
Glu Met Ala Asp Gln Ala Ser Ala Trp Leu Thr Arg Gln Gly 1280 1285
1290 Ser Phe Gln Gly Gly Phe Arg Ser Thr Gln Asp Thr Val Ile Ala
1295 1300 1305 Leu Asp Ala Leu Ser Ala Tyr Trp Ile Ala Ser His Thr
Thr Glu 1310 1315 1320 Glu Arg Gly Leu Asn Val Thr Leu Ser Ser Thr
Gly Arg Asn Gly 1325 1330 1335 Phe Lys Ser His Ala Leu Gln Leu Asn
Asn Arg Gln Ile Arg Gly 1340 1345 1350 Leu Glu Glu Glu Leu Gln Phe
Ser Leu Gly Ser Lys Ile Asn Val 1355 1360 1365 Lys Val Gly Gly Asn
Ser Lys Gly Thr Leu Lys Val Leu Arg Thr 1370 1375 1380 Tyr Asn Val
Leu Asp Met Lys Asn Thr Thr Cys Gln Asp Leu Gln 1385 1390 1395 Ile
Glu Val Thr Val Lys Gly His Val Glu Tyr Thr Met Glu Ala 1400 1405
1410 Asn Glu Asp Tyr Glu Asp Tyr Glu Tyr Asp Glu Leu Pro Ala Lys
1415 1420 1425 Asp Asp Pro Asp Ala Pro Leu Gln Pro Val Thr Pro Leu
Gln Leu 1430 1435 1440 Phe Glu Gly Arg Arg Asn Arg Arg Arg Arg Glu
Ala Pro Lys Val 1445 1450 1455 Val Glu Glu Gln Glu Ser Arg Val His
Tyr Thr Val Cys Ile Trp 1460 1465 1470 Arg Asn Gly Lys Val Gly Leu
Ser Gly Met Ala Ile Ala Asp Val 1475 1480 1485 Thr Leu Leu Ser Gly
Phe His Ala Leu Arg Ala Asp Leu Glu Lys 1490 1495 1500 Leu Thr Ser
Leu Ser Asp Arg Tyr Val Ser His Phe Glu Thr Glu 1505 1510 1515 Gly
Pro His Val Leu Leu Tyr Phe Asp Ser Val Pro Thr Ser Arg 1520 1525
1530 Glu Cys Val Gly Phe Glu Ala Val Gln Glu Val Pro Val Gly Leu
1535 1540 1545 Val Gln Pro Ala Ser Ala Thr Leu Tyr Asp Tyr Tyr Asn
Pro Glu 1550 1555 1560 Arg Arg Cys Ser Val Phe Tyr Gly Ala Pro Ser
Lys Ser Arg Leu 1565 1570 1575 Leu Ala Thr Leu Cys Ser Ala Glu Val
Cys Gln Cys Ala Glu Gly 1580 1585 1590 Lys Cys Pro Arg Gln Arg Arg
Ala Leu Glu Arg Gly Leu Gln Asp 1595 1600 1605 Glu Asp Gly Tyr Arg
Met Lys Phe Ala Cys Tyr Tyr Pro Arg Val 1610 1615 1620 Glu Tyr Gly
Phe Gln Val Lys Val Leu Arg Glu Asp Ser Arg Ala 1625 1630 1635 Ala
Phe Arg Leu Phe Glu Thr Lys Ile Thr Gln Val Leu His Phe 1640 1645
1650 Thr Lys Asp Val Lys Ala Ala Ala Asn Gln Met Arg Asn Phe Leu
1655 1660 1665 Val Arg Ala Ser Cys Arg Leu Arg Leu Glu Pro Gly Lys
Glu Tyr 1670 1675 1680 Leu Ile Met Gly Leu Asp Gly Ala Thr Tyr Asp
Leu Glu Gly His 1685 1690 1695 Pro Gln Tyr Leu Leu Asp Ser Asn Ser
Trp Ile Glu Glu Met Pro 1700 1705 1710 Ser Glu Arg Leu Cys Arg Ser
Thr Arg Gln Arg Ala Ala Cys Ala 1715 1720 1725 Gln Leu Asn Asp Phe
Leu Gln Glu Tyr Gly Thr Gln Gly Cys Gln 1730 1735 1740 Val
111930PRTHomo sapiens 111Met Lys Pro Pro Arg Pro Val Arg Thr Cys
Ser Lys Val Leu Val Leu 1 5 10 15 Leu Ser Leu Leu Ala Ile His Gln
Thr Thr Thr Ala Glu Lys Asn Gly 20 25 30 Ile Asp Ile Tyr Ser Leu
Thr Val Asp Ser Arg Val Ser Ser Arg Phe 35 40 45 Ala His Thr Val
Val Thr Ser Arg Val Val Asn Arg Ala Asn Thr Val 50 55 60 Gln Glu
Ala Thr Phe Gln Met Glu Leu Pro Lys Lys Ala Phe Ile Thr 65 70 75 80
Asn Phe Ser Met Asn Ile Asp Gly Met Thr Tyr Pro Gly Ile Ile Lys 85
90 95 Glu Lys Ala Glu Ala Gln Ala Gln Tyr Ser Ala Ala Val Ala Lys
Gly 100 105 110 Lys Ser Ala Gly Leu Val Lys Ala Thr Gly Arg Asn Met
Glu Gln Phe 115 120 125 Gln Val Ser Val Ser Val Ala Pro Asn Ala Lys
Ile Thr Phe Glu Leu 130 135 140 Val Tyr Glu Glu Leu Leu Lys Arg Arg
Leu Gly Val Tyr Glu Leu Leu 145 150 155 160 Leu Lys Val Arg Pro Gln
Gln Leu Val Lys His Leu Gln Met Asp Ile 165 170 175 His Ile Phe Glu
Pro Gln Gly Ile Ser Phe Leu Glu Thr Glu Ser Thr 180 185 190 Phe Met
Thr Asn Gln Leu Val Asp Ala Leu Thr Thr Trp Gln Asn Lys 195 200 205
Thr Lys Ala His Ile Arg Phe Lys Pro Thr Leu Ser Gln Gln Gln Lys 210
215 220 Ser Pro Glu Gln Gln Glu Thr Val Leu Asp Gly Asn Leu Ile Ile
Arg 225 230 235 240 Tyr Asp Val Asp Arg Ala Ile Ser Gly Gly Ser Ile
Gln Ile Glu Asn 245 250 255 Gly Tyr Phe Val His Tyr Phe Ala Pro Glu
Gly Leu Thr Thr Met Pro 260 265 270 Lys Asn Val Val Phe Val Ile Asp
Lys Ser Gly Ser Met Ser Gly Arg 275 280 285 Lys Ile Gln Gln Thr Arg
Glu Ala Leu Ile Lys Ile Leu Asp Asp Leu 290 295 300 Ser Pro Arg Asp
Gln Phe Asn Leu Ile Val Phe Ser Thr Glu Ala Thr 305 310 315 320 Gln
Trp Arg Pro Ser Leu Val Pro Ala Ser Ala Glu Asn Val Asn Lys 325 330
335 Ala Arg Ser Phe Ala Ala Gly Ile Gln Ala Leu Gly Gly Thr Asn Ile
340 345 350 Asn Asp Ala Met Leu Met Ala Val Gln Leu Leu Asp Ser Ser
Asn Gln 355 360 365 Glu Glu Arg Leu Pro Glu Gly Ser Val Ser Leu Ile
Ile Leu Leu Thr 370 375 380 Asp Gly Asp Pro Thr Val Gly Glu Thr Asn
Pro Arg Ser Ile Gln Asn 385 390 395 400 Asn Val Arg Glu Ala Val Ser
Gly Arg Tyr Ser Leu Phe Cys Leu Gly 405 410 415 Phe Gly Phe Asp Val
Ser Tyr Ala Phe Leu Glu Lys Leu Ala Leu Asp 420 425 430 Asn Gly Gly
Leu Ala Arg Arg Ile His Glu Asp Ser Asp Ser Ala Leu 435 440 445 Gln
Leu Gln Asp Phe Tyr Gln Glu Val Ala Asn Pro Leu Leu Thr Ala 450 455
460 Val Thr Phe Glu Tyr Pro Ser Asn Ala Val Glu Glu Val Thr Gln Asn
465 470 475 480 Asn Phe Arg Leu Leu Phe Lys Gly Ser Glu Met Val Val
Ala Gly Lys 485 490 495 Leu Gln Asp Arg Gly Pro Asp Val Leu Thr Ala
Thr Val Ser Gly Lys 500 505 510 Leu Pro Thr Gln Asn Ile Thr Phe Gln
Thr Glu Ser Ser Val Ala Glu 515 520 525 Gln Glu Ala Glu Phe Gln Ser
Pro Lys Tyr Ile Phe His Asn Phe Met 530 535 540 Glu Arg Leu Trp Ala
Tyr Leu Thr Ile Gln Gln Leu Leu Glu Gln Thr 545 550 555 560 Val Ser
Ala Ser Asp Ala Asp Gln Gln Ala Leu Arg Asn Gln Ala Leu 565 570 575
Asn Leu Ser Leu Ala Tyr Ser Phe Val Thr Pro Leu Thr Ser Met Val 580
585 590 Val Thr Lys Pro Asp Asp Gln Glu Gln Ser Gln Val Ala Glu Lys
Pro 595 600 605 Met Glu Gly Glu Ser Arg Asn Arg Asn Val His Ser Gly
Ser Thr Phe 610 615 620 Phe Lys Tyr Tyr Leu Gln Gly Ala Lys Ile Pro
Lys Pro Glu Ala Ser 625 630 635 640 Phe Ser Pro Arg Arg Gly Trp Asn
Arg Gln Ala Gly Ala Ala Gly Ser 645 650 655 Arg Met Asn Phe Arg Pro
Gly Val Leu Ser Ser Arg Gln Leu Gly Leu 660 665 670 Pro Gly Pro Pro
Asp Val Pro Asp His Ala Ala Tyr His Pro Phe Arg 675 680 685 Arg Leu
Ala Ile Leu Pro Ala Ser Ala Pro Pro Ala Thr Ser Asn Pro 690 695 700
Asp Pro Ala Val Ser Arg Val Met Asn Met Lys Ile Glu Glu Thr Thr 705
710 715 720 Met Thr Thr Gln Thr Pro Ala Pro Ile Gln Ala Pro Ser Ala
Ile Leu 725 730 735 Pro Leu Pro Gly Gln Ser Val Glu Arg Leu Cys Val
Asp Pro Arg His 740 745 750 Arg Gln Gly Pro Val Asn Leu Leu Ser Asp
Pro Glu Gln Gly Val Glu 755 760 765 Val Thr Gly Gln Tyr Glu Arg Glu
Lys Ala Gly Phe Ser Trp Ile Glu 770 775 780 Val Thr Phe Lys Asn Pro
Leu Val Trp Val His Ala Ser Pro Glu His 785 790 795 800 Val Val Val
Thr Arg Asn Arg Arg Ser Ser Ala Tyr Lys Trp Lys Glu 805 810 815 Thr
Leu Phe Ser Val Met Pro Gly Leu Lys Met Thr Met Asp Lys Thr 820 825
830 Gly Leu Leu Leu Leu Ser Asp Pro Asp Lys Val Thr Ile Gly Leu Leu
835 840 845 Phe Trp Asp Gly Arg Gly Glu Gly Leu Arg Leu Leu Leu Arg
Asp Thr 850 855 860 Asp Arg Phe Ser Ser His Val Gly Gly Thr Leu Gly
Gln Phe Tyr Gln 865 870 875 880 Glu Val Leu Trp Gly Ser Pro Ala Ala
Ser Asp Asp Gly Arg Arg Thr 885 890 895 Leu Arg Val Gln Gly Asn Asp
His Ser Ala Thr Arg Glu Arg Arg Leu 900 905 910 Asp Tyr Gln Glu Gly
Pro Pro Gly Val Glu Ile Ser Cys Trp Ser Val 915 920 925 Glu Leu 930
112644PRTHomo sapiens 112Met Pro Lys Asn Val Val Phe Val Ile Asp
Lys Ser
Gly Ser Met Ser 1 5 10 15 Gly Arg Lys Ile Gln Gln Thr Arg Glu Ala
Leu Ile Lys Ile Leu Asp 20 25 30 Asp Leu Ser Pro Arg Asp Gln Phe
Asn Leu Ile Val Phe Ser Thr Glu 35 40 45 Ala Thr Gln Trp Arg Pro
Ser Leu Val Pro Ala Ser Ala Glu Asn Val 50 55 60 Asn Lys Ala Arg
Ser Phe Ala Ala Gly Ile Gln Ala Leu Gly Gly Thr 65 70 75 80 Asn Ile
Asn Asp Ala Met Leu Met Ala Val Gln Leu Leu Asp Ser Ser 85 90 95
Asn Gln Glu Glu Arg Leu Pro Glu Gly Ser Val Ser Leu Ile Ile Leu 100
105 110 Leu Thr Asp Gly Asp Pro Thr Val Gly Glu Thr Asn Pro Arg Ser
Ile 115 120 125 Gln Asn Asn Val Arg Glu Ala Val Ser Gly Arg Tyr Ser
Leu Phe Cys 130 135 140 Leu Gly Phe Gly Phe Asp Val Ser Tyr Ala Phe
Leu Glu Lys Leu Ala 145 150 155 160 Leu Asp Asn Gly Gly Leu Ala Arg
Arg Ile His Glu Asp Ser Asp Ser 165 170 175 Ala Leu Gln Leu Gln Asp
Phe Tyr Gln Glu Val Ala Asn Pro Leu Leu 180 185 190 Thr Ala Val Thr
Phe Glu Tyr Pro Ser Asn Ala Val Glu Glu Val Thr 195 200 205 Gln Asn
Asn Phe Arg Leu Leu Phe Lys Gly Ser Glu Met Val Val Ala 210 215 220
Gly Lys Leu Gln Asp Arg Gly Pro Asp Val Leu Thr Ala Thr Val Ser 225
230 235 240 Gly Lys Leu Pro Thr Gln Asn Ile Thr Phe Gln Thr Glu Ser
Ser Val 245 250 255 Ala Glu Gln Glu Ala Glu Phe Gln Ser Pro Lys Tyr
Ile Phe His Asn 260 265 270 Phe Met Glu Arg Leu Trp Ala Tyr Leu Thr
Ile Gln Gln Leu Leu Glu 275 280 285 Gln Thr Val Ser Ala Ser Asp Ala
Asp Gln Gln Ala Leu Arg Asn Gln 290 295 300 Ala Leu Asn Leu Ser Leu
Ala Tyr Ser Phe Val Thr Pro Leu Thr Ser 305 310 315 320 Met Val Val
Thr Lys Pro Asp Asp Gln Glu Gln Ser Gln Val Ala Glu 325 330 335 Lys
Pro Met Glu Gly Glu Ser Arg Asn Arg Asn Val His Ser Ala Gly 340 345
350 Ala Ala Gly Ser Arg Met Asn Phe Arg Pro Gly Val Leu Ser Ser Arg
355 360 365 Gln Leu Gly Leu Pro Gly Pro Pro Asp Val Pro Asp His Ala
Ala Tyr 370 375 380 His Pro Phe Arg Arg Leu Ala Ile Leu Pro Ala Ser
Ala Pro Pro Ala 385 390 395 400 Thr Ser Asn Pro Asp Pro Ala Val Ser
Arg Val Met Asn Met Lys Ile 405 410 415 Glu Glu Thr Thr Met Thr Thr
Gln Thr Pro Ala Cys Pro Ser Cys Ser 420 425 430 Arg Ser Arg Ala Pro
Ala Val Pro Ala Pro Ile Gln Ala Pro Ser Ala 435 440 445 Ile Leu Pro
Leu Pro Gly Gln Ser Val Glu Arg Leu Cys Val Asp Pro 450 455 460 Arg
His Arg Gln Gly Pro Val Asn Leu Leu Ser Asp Pro Glu Gln Gly 465 470
475 480 Val Glu Val Thr Gly Gln Tyr Glu Arg Glu Lys Ala Gly Phe Ser
Trp 485 490 495 Ile Glu Val Thr Phe Lys Asn Pro Leu Val Trp Val His
Ala Ser Pro 500 505 510 Glu His Val Val Val Thr Arg Asn Arg Arg Ser
Ser Ala Tyr Lys Trp 515 520 525 Lys Glu Thr Leu Phe Ser Val Met Pro
Gly Leu Lys Met Thr Met Asp 530 535 540 Lys Thr Gly Leu Leu Leu Leu
Ser Asp Pro Asp Lys Val Thr Ile Gly 545 550 555 560 Leu Leu Phe Trp
Asp Gly Arg Gly Glu Gly Leu Arg Leu Leu Leu Arg 565 570 575 Asp Thr
Asp Arg Phe Ser Ser His Val Gly Gly Thr Leu Gly Gln Phe 580 585 590
Tyr Gln Glu Val Leu Trp Gly Ser Pro Ala Ala Ser Asp Asp Gly Arg 595
600 605 Arg Thr Leu Arg Val Gln Gly Asn Asp His Ser Ala Thr Arg Glu
Arg 610 615 620 Arg Leu Asp Tyr Gln Glu Gly Pro Pro Gly Val Glu Ile
Ser Cys Trp 625 630 635 640 Ser Val Glu Leu 113267PRTHomo sapiens
113Met Lys Ala Ala Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser
1 5 10 15 Gln Ala Arg His Phe Trp Gln Gln Asp Glu Pro Pro Gln Ser
Pro Trp 20 25 30 Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp
Val Leu Lys Asp 35 40 45 Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu
Gly Ser Ala Leu Gly Lys 50 55 60 Gln Leu Asn Leu Lys Leu Leu Asp
Asn Trp Asp Ser Val Thr Ser Thr 65 70 75 80 Phe Ser Lys Leu Arg Glu
Gln Leu Gly Pro Val Thr Gln Glu Phe Trp 85 90 95 Asp Asn Leu Glu
Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys 100 105 110 Asp Leu
Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe 115 120 125
Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu 130
135 140 Pro Leu Arg Ala Glu Leu Gly Glu Gly Ala Arg Gln Lys Leu His
Glu 145 150 155 160 Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met
Arg Asp Arg Ala 165 170 175 Arg Ala His Val Asp Ala Leu Arg Thr His
Leu Ala Pro Tyr Ser Asp 180 185 190 Glu Leu Arg Gln Arg Leu Ala Ala
Arg Leu Glu Ala Leu Lys Glu Asn 195 200 205 Gly Gly Ala Arg Leu Ala
Glu Tyr His Ala Lys Ala Thr Glu His Leu 210 215 220 Ser Thr Leu Ser
Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln 225 230 235 240 Gly
Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala 245 250
255 Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln 260 265
114396PRTHomo sapiens 114Met Phe Leu Lys Ala Val Val Leu Thr Leu
Ala Leu Val Ala Val Ala 1 5 10 15 Gly Ala Arg Ala Glu Val Ser Ala
Asp Gln Val Ala Thr Val Met Trp 20 25 30 Asp Tyr Phe Ser Gln Leu
Ser Asn Asn Ala Lys Glu Ala Val Glu His 35 40 45 Leu Gln Lys Ser
Glu Leu Thr Gln Gln Leu Asn Ala Leu Phe Gln Asp 50 55 60 Lys Leu
Gly Glu Val Asn Thr Tyr Ala Gly Asp Leu Gln Lys Lys Leu 65 70 75 80
Val Pro Phe Ala Thr Glu Leu His Glu Arg Leu Ala Lys Asp Ser Glu 85
90 95 Lys Leu Lys Glu Glu Ile Gly Lys Glu Leu Glu Glu Leu Arg Ala
Arg 100 105 110 Leu Leu Pro His Ala Asn Glu Val Ser Gln Lys Ile Gly
Asp Asn Leu 115 120 125 Arg Glu Leu Gln Gln Arg Leu Glu Pro Tyr Ala
Asp Gln Leu Arg Thr 130 135 140 Gln Val Asn Thr Gln Ala Glu Gln Leu
Arg Arg Gln Leu Thr Pro Tyr 145 150 155 160 Ala Gln Arg Met Glu Arg
Val Leu Arg Glu Asn Ala Asp Ser Leu Gln 165 170 175 Ala Ser Leu Arg
Pro His Ala Asp Glu Leu Lys Ala Lys Ile Asp Gln 180 185 190 Asn Val
Glu Glu Leu Lys Gly Arg Leu Thr Pro Tyr Ala Asp Glu Phe 195 200 205
Lys Val Lys Ile Asp Gln Thr Val Glu Glu Leu Arg Arg Ser Leu Ala 210
215 220 Pro Tyr Ala Gln Asp Thr Gln Glu Lys Leu Asn His Gln Leu Glu
Gly 225 230 235 240 Leu Thr Phe Gln Met Lys Lys Asn Ala Glu Glu Leu
Lys Ala Arg Ile 245 250 255 Ser Ala Ser Ala Glu Glu Leu Arg Gln Arg
Leu Ala Pro Leu Ala Glu 260 265 270 Asp Val Arg Gly Asn Leu Lys Gly
Asn Thr Glu Gly Leu Gln Lys Ser 275 280 285 Leu Ala Glu Leu Gly Gly
His Leu Asp Gln Gln Val Glu Glu Phe Arg 290 295 300 Arg Arg Val Glu
Pro Tyr Gly Glu Asn Phe Asn Lys Ala Leu Val Gln 305 310 315 320 Gln
Met Glu Gln Leu Arg Gln Lys Leu Gly Pro His Ala Gly Asp Val 325 330
335 Glu Gly His Leu Ser Phe Leu Glu Lys Asp Leu Arg Asp Lys Val Asn
340 345 350 Ser Phe Phe Ser Thr Phe Lys Glu Lys Glu Ser Gln Asp Lys
Thr Leu 355 360 365 Ser Leu Pro Glu Leu Glu Gln Gln Gln Glu Gln Gln
Gln Glu Gln Gln 370 375 380 Gln Glu Gln Val Gln Met Leu Ala Pro Glu
Leu Ser 385 390 395 115317PRTHomo sapiens 115Met Lys Val Leu Trp
Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys 1 5 10 15 Gln Ala Lys
Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu 20 25 30 Arg
Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu 35 40
45 Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln
50 55 60 Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu
Arg Ala 65 70 75 80 Leu Met Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr
Lys Ser Glu Leu 85 90 95 Glu Glu Gln Leu Thr Pro Val Ala Glu Glu
Thr Arg Ala Arg Leu Ser 100 105 110 Lys Glu Leu Gln Ala Ala Gln Ala
Arg Leu Gly Ala Asp Met Glu Asp 115 120 125 Val Cys Gly Arg Leu Val
Gln Tyr Arg Gly Glu Val Gln Ala Met Leu 130 135 140 Gly Gln Ser Thr
Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg 145 150 155 160 Lys
Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg 165 170
175 Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu
180 185 190 Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly
Arg Val 195 200 205 Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro
Leu Gln Glu Arg 210 215 220 Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala
Arg Met Glu Glu Met Gly 225 230 235 240 Ser Arg Thr Arg Asp Arg Leu
Asp Glu Val Lys Glu Gln Val Ala Glu 245 250 255 Val Arg Ala Lys Leu
Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala 260 265 270 Glu Ala Phe
Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu 275 280 285 Asp
Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala 290 295
300 Val Gly Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His 305 310 315
11616PRTHomo sapiens 116Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu
Gly Gly Gly Val Arg 1 5 10 15 11716PRTHomo sapiens 117Ala Asp Ser
Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15
11816PRTHomo sapiens 118Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu
Gly Gly Gly Val Arg 1 5 10 15 11930PRTHomo sapiens 119Lys Ser Ser
Ser Tyr Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr Asn 1 5 10 15 Arg
Gly Asp Ser Thr Phe Glu Ser Lys Ser Tyr Lys Met Ala 20 25 30
12029PRTHomo sapiens 120Lys Ser Ser Ser Tyr Ser Lys Gln Phe Thr Ser
Ser Thr Ser Tyr Asn 1 5 10 15 Arg Gly Asp Ser Thr Phe Glu Ser Lys
Ser Tyr Lys Met 20 25 12127PRTHomo sapiens 121Lys Ser Ser Ser Tyr
Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr Asn 1 5 10 15 Arg Gly Asp
Ser Thr Phe Glu Ser Lys Ser Tyr 20 25 12226PRTHomo sapiens 122Lys
Ser Ser Ser Tyr Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr Asn 1 5 10
15 Arg Gly Asp Ser Thr Phe Glu Ser Lys Ser 20 25 12324PRTHomo
sapiens 123Lys Ser Ser Ser Tyr Ser Lys Gln Phe Thr Ser Ser Thr Ser
Tyr Asn 1 5 10 15 Arg Gly Asp Ser Thr Phe Glu Ser 20 12429PRTHomo
sapiens 124Arg Gly Ser Glu Ser Gly Ile Phe Thr Asn Thr Lys Glu Ser
Ser Ser 1 5 10 15 His His Pro Gly Ile Ala Glu Phe Pro Ser Arg Gly
Lys 20 25 12531PRTHomo sapiens 125Ser Tyr Lys Met Ala Asp Glu Ala
Gly Ser Glu Ala Asp His Glu Gly 1 5 10 15 Thr His Ser Thr Lys Arg
Gly His Ala Lys Ser Arg Pro Val Arg 20 25 30 12626PRTHomo sapiens
126Asp Glu Ala Gly Ser Glu Ala Asp His Glu Gly Thr His Ser Thr Lys
1 5 10 15 Arg Gly His Ala Lys Ser Arg Pro Val Arg 20 25
12717PRTHomo sapiens 127Ser Ser Lys Ile Thr His Arg Ile His Trp Glu
Ser Ala Ser Leu Leu 1 5 10 15 Arg 12811PRTHomo sapiens 128His Arg
Ile His Trp Glu Ser Ala Ser Leu Leu 1 5 10 12918PRTHomo sapiens
129Ser Ser Lys Ile Thr His Arg Ile His Val Ile Glu Ser Ala Ser Leu
1 5 10 15 Leu Arg 13011PRTHomo sapiens 130Asn Gly Phe Lys Ser His
Ala Leu Gln Leu Asn 1 5 10 13114PRTHomo sapiens 131Gly Pro Pro Asp
Val Pro Asp His Ala Ala Tyr His Pro Phe 1 5 10 13215PRTHomo sapiens
132Pro Gly Pro Pro Asp Val Pro Asp His Ala Ala Tyr His Pro Phe 1 5
10 15 13329PRTHomo sapiens 133Met Phe Arg Pro Gly Val Leu Ser Ser
Arg Gln Leu Gly Leu Pro Gly 1 5 10 15 Pro Pro Asp Val Pro Asp His
Ala Ala Tyr His Pro Phe 20 25 13412PRTHomo sapiens 134Arg Pro His
Phe Phe Phe Pro Lys Ser Arg Ile Val 1 5 10 13530PRTHomo sapiens
135Ser Tyr Lys Met Ala Asp Glu Ala Gly Ser Glu Ala Asp His Glu Gly
1 5 10 15 Thr His Ser Thr Lys Arg Gly His Ala Lys Ser Arg Pro Val
20 25 30 13630PRTHomo sapiens 136Gly Leu Glu Glu Glu Leu Gln Phe
Ser Leu Gly Ser Lys Ile Asn Val 1 5 10 15 Lys Val Gly Gly Asn Ser
Lys Gly Thr Leu Lys Val Leu Arg 20 25 30 13727PRTHomo sapiens
137Pro Gly Val Leu Ser Ser Arg Gln Leu Gly Leu Pro Gly Pro Pro Asp
1 5 10 15 Val Pro Asp His Ala Ala Tyr His Pro Phe Arg 20 25
13826PRTHomo sapiens 138Gly Val Leu Ser Ser Arg Gln Leu Gly Leu Pro
Gly Pro Pro Asp Val 1 5 10 15 Pro Asp His Ala Ala Tyr His Pro Phe
Arg 20 25 13925PRTHomo sapiens 139Val Leu Ser Ser Arg Gln Leu Gly
Leu Pro Gly Pro Pro Asp Val Pro 1 5 10 15 Asp His Ala Ala Tyr His
Pro Phe Arg 20 25 14024PRTHomo sapiens 140Leu Ser Ser Arg Gln Leu
Gly Leu Pro Gly Pro Pro Asp Val Pro Asp 1 5 10 15 His Ala Ala Tyr
His Pro Phe Arg 20 14123PRTHomo sapiens 141Ser Ser Arg Gln Leu Gly
Leu Pro Gly Pro Pro Asp Val Pro Asp His 1 5 10 15 Ala Ala Tyr His
Pro Phe Arg 20 14222PRTHomo sapiens 142Leu Met Ile Asp Gln Asn Thr
Lys Ser Pro Leu Phe Met Gly Lys Val 1 5 10 15 Val Asn Pro Thr Gln
Lys 20
14322PRTHomo sapiens 143Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu
Phe Met Gly Lys Val 1 5 10 15 Val Asn Pro Thr Gln Lys 20
14424PRTHomo sapiens 144Arg Tyr Thr Ile Ala Ala Leu Leu Ser Pro Tyr
Ser Tyr Ser Thr Thr 1 5 10 15 Ala Val Val Thr Asn Pro Lys Glu 20
14523PRTHomo sapiens 145Tyr Thr Ile Ala Ala Leu Leu Ser Pro Tyr Ser
Tyr Ser Thr Thr Ala 1 5 10 15 Val Val Thr Asn Pro Lys Glu 20
14610PRTHomo sapiens 146Arg Ile His Trp Glu Ser Ala Ala Leu Leu 1 5
10 14713PRTHomo sapiens 147Ile Thr His Arg Ile His Trp Glu Ser Ala
Ala Leu Leu 1 5 10 14816PRTHomo sapiens 148Ser Ser Lys Ile Thr His
Arg Ile His Trp Glu Ser Ala Ala Leu Leu 1 5 10 15
* * * * *