WO2009126718A2

WO2009126718A2 - Tumor-associated biomarkers from the dark proteom

Info

Publication number: WO2009126718A2
Application number: PCT/US2009/039922
Authority: WO
Inventors: Douglas Lake; Kwasi Antwi; Phillip Stafford; Hojoon Lee; Michael Demeure; Stephen A. Johnston
Original assignee: Arizona Board Of Regents And On Behalf Of Arizona State University
Priority date: 2008-04-08
Filing date: 2009-04-08
Publication date: 2009-10-15
Also published as: WO2009126718A3

Abstract

Pancreas cancer often goes undetected until late stage disease. Unlike prostate and ovarian cancer, no markers are available to detect pancreas cancer in earlier stages of disease. Plasma is a convenient source to detect potential biomarkers for cancer because individuals provide peripheral blood for other routine clinically indicated tests. In one embodiment, the present invention identifies peptides that are present in pancreas cancer patient plasma samples that are not present in plasma from normal donors using nanospray ion trap LC- MS/MS methods.

Description

DESCRIPTION

TUMOR- ASSOCIATED BIOMARKERS FROM THE DARK PROTEOM

BACKGROUND OF THE INVENTION

This application claims priority to United States Provisional Patent Application Serial No. 61/043,359, filed on April 8, 2008. The entirety of the above-referenced disclosure is incorporated by reference.

1. Field of the Invention

The present invention relates generally to molecular diagnostics and therapeutics. More particularly, it concerns peptide biomarkers for diseases such as cancer.

2. Description of Related Art

Very few biomarkers and diagnostics are available for detecting cancer. As a result cancer is usually detected during late stage when it is more difficult to treat. Pancreas cancer, for example, is especially lethal when detected late, with 85% of cases detected with locally advanced, unresectable disease or metastatic disease. Median survival time from detection to death is less than 9 months. The majority of pancreas cancer is ductal adenocarcinoma with other forms of pancreas cancer having a much better prognosis. There remains a need for improved methods and compositions for identifying cancer in patients at an early stage. Additionally, there is a need for improved methods and compositions for the treatment of cancers.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an isolated peptide of between 8 to

40 amino acids comprising any of SEQ ID NOs: 1-214. In certain aspects, the present invention provides an isolated peptide of between 8 to 40 amino acids consisting of a sequence of any of SEQ ID NOs: 1-214. In another embodiment, the present invention provides an antibody that specifically binds to a peptide having a sequence of any one of SEQ ID NOs: 1-214. The antibody may be, for example, a polyclonal antibody, monoclonal antibody, antibody fragment, humanized antibody, or chimeric antibody. In another embodiment, the present invention provides a pharmaceutical composition comprising one or more peptides comprising the sequence of any of SEQ ID NOs:l-214. In certain aspects, the present invention provides a pharmaceutical composition comprising one or more peptides consisting of the sequence of any of SEQ ID NOs: 1-214. In some embodiments, the pharmaceutical composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, 17, 18, 19, or 20 or more peptides of SEQ ID NOs: SEQ ID NOs:l-214. The formulated composition can be compounded in any manner known to those of skill in the art.

The pharmaceutical composition may be formulated to stimulate an immune response in an animal, such as a human. In some embodiments, the pharmaceutical composition may be, for example, a vaccine.

In a further embodiment, the present invention provides a nucleic acid encoding any one of SEQ ID NOs: SEQ ID NOs:l-214 or a complementary sequence of such a nucleic acid sequence. In certain aspects, the nucleic acid is a DNA. In some aspects, the nucleic acid is an RNA.

In another embodiment, the present invention provides a method of diagnosing a disease in a subject comprising: obtaining a sample from a subject suspected of having or at risk for having the disease; and detecting whether one or more peptides with one or more of SEQ ID NOs: 1-214 is present in the sample; wherein the presence of such a peptide is indicative of the disease. The peptide may be detected by any manner known to those of skill in the art. In some aspects, detecting involves mass spectrometry. In certain aspects the method further comprises one or both of filtering the sample and/or chromatographically separating the sample.

The sample, in non-limiting embodiments may be a body fluid sample, for example, a plasma sample or a serum sample. In some embodiments, the disease is a cancer. In some embodiments the cancer is pancreatic cancer. In some embodiments the disease is a pancreatic disease. The disease of the pancreas may be, for example, a pancreatic cancer or a metabolic disease. In certain aspects of the invention, the pancreatic cancer is ductal adenocarcinoma, mucinous cystic neoplasm, Intraductal Papillary Mucinous Neoplasm (IPMN), ampullary adenoma, endocrine carcinoma, or islet cell carcinoma. In certain aspects of the invention, the metabolic disease is diabetes. In one embodiment, the present invention provides a method of diagnosing cancer in a subject comprising: obtaining a sample from a subject suspected of having or at risk for having cancer; detecting peptides in the sample; and diagnosing cancer if one or more of SEQ ID NOs: 1-214 are detected in the plasma sample. The peptide may be detected by any manner known to those of skill in the art. In certain embodiments, the detecting may be performed by mass spectrometry. The sample, in non-limiting embodiments may be a body fluid sample, for example, a plasma sample or a serum sample, hi certain aspects the method further comprises one or both of filtering the sample and/or chromatographically separating the sample. In some embodiments, the cancer is pancreatic cancer.

In yet another embodiment, the present invention provides a method of identifying a biomarker for a condition of interest comprising: obtaining a first sample from a first subject having a condition of interest and a second sample from a second subject who does not have the condition of interest; performing mass spectrometry to detect peptides in the first and the second samples; and comparing the detected peptides in the first and the second samples, wherein detecting a peptide in the first sample that is not detected in the second sample identifies a biomarker for the condition of interest. The sample, in non-limiting embodiments may be a body fluid sample, for example, a plasma sample or a serum sample. In certain aspects the method further comprises one or both of filtering the sample and/or chromatographically separating the sample. The condition of interest may be, for example, a disease, such a cancer or a metabolic disease.

The methods disclosed herein may be used in may be used to detect low molecular weight (LMW) peptides in body fluids. One or more filtration steps may be useful in this regard. For example, in certain embodiments the sample is through one or both of a 0.45 um membrane and/or a 3000 molecular weight cut-off filter.

In certain embodiments, the samples are separated by chromatography. This may be performed independently or in addition to one or more filtration steps. In certain aspects of the invention, the chromatography is HPLC or nano-HPLC.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. The use of the term "or" in the claims is used to mean " and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

Following long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the claims or specification, denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Flow chart outlining a method for obtaining biomarkers from plasma.

FIG. 2. Frameshift peptides detected in: (1) Established cell lines; (2) Tumor tissue elutions; and (3) Plasma from pancreas cancer patients.

FIGS. 3A-B. Peptides detected in plasma from pancreas cancer patients.

FIG. 4. Peptide ELISAs of plasma from pancreas cancer patients. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A. The Proteome

The term "proteome" refers to all the proteins expressed by a genome, and thus proteomics involves the identification of proteins in the body and the determination of their role in physiological and pathophysiological functions. The -30,000 genes defined by the

Human Genome Project translate into 300,000 to 1 million proteins when alternate splicing and post-translational modifications are considered. WO 2007/101227, herein incorporated by reference in its entirety, discloses a method of identification and use of novopeptides, which are a general class of antigens, for the treatment of cancer.

The present disclosure shows that the "Dark Proteome" provides a further source of peptides ("Dark Peptides"). The Dark Proteome peptides map to regions of the genome/transcriptome that are not currently believed to be translated into protein. In particular, over 200 peptides in plasma from pancreas cancer patients that do not appear to be present in normal plasma from over 40 healthy donors have been discovered. These peptides map to regions of the genome/transcriptome that are not known to be translated into protein. These peptides and/or their parent proteins provide biomarkers and/or diagnostics for pancreas neoplasms or metabolic disorders involving the pancreas.

Blood plasma is well-suited to proteomic analysis because it is easy to collect and provides a complex proteome. Of course, other body fluids or tissues, including serum, cerebrospinal fluid, synovial fluid, and urine may also be used in proteomic analysis.

B. Protein Analysis

The present invention employs methods of separating proteins from bodily fluids, such as plasma. Methods of separating proteins are well known to those of skill in the art and include, but are not limited to, various kinds of filtration, chromatography (e.g., anion exchange chromatography, affinity chromatography, sequential extraction, and high performance liquid chromatography) and mass spectrometry. The separation and detection of the proteins in a sample generates a protein spectra for that sample.

1. Mass Spectrometry Certain embodiments the present invention employ mass spectrometry. Mass spectrometry provides a means of "weighing" individual molecules by ionizing the molecules in vacuo and making them "fly" by volatilization. Under the influence of combinations of electric and magnetic fields, the ions follow trajectories depending on their individual mass (m) and charge (z). Mass spectrometry (MS), because of its extreme selectivity and sensitivity, has become a powerful tool for the quantification of a broad range of bioanalytes including pharmaceuticals, metabolites, peptides and proteins. Of particular interest in the present invention is electrospray ionization (nano-ESI) ion trap mass spectrometry (LC/MSD Trap XCT Ultra, Agilent Technologies). 2. Chromatography

Chromatography is used to separate organic compounds on the basis of their charge, size, shape, and solubilities. A chromatography consists of a mobile phase (solvent and the molecules to be separated) and a stationary phase either of paper (in paper chromatography) or glass beads, called resin, (in column chromatography) through which the mobile phase travels. Molecules travel through the stationary phase at different rates because of their chemistry. Types of chromatography that may be employed in the present invention include, but are not limited to, high performance liquid chromatography (HPLC), nano-HPLC, ion exchange chromatography (IEC), and reverse phase chromatography (RP). C. Examples The following examples are included to demonstrate certain embodiments of the invention. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 Over 200 peptides were discovered in plasma from pancreas cancer patients that do not appear to be present in normal plasma from over 40 healthy donors. These peptides map to regions of the genome/transcriptome that had not been believed to be translated into protein.

The methods employed are generally shown in FIG. 1 , which flowcharts that peptide biomarkers are identified by subtracting peptides in common from normal tissue and plasma from healthy donors from peptides in pancreas cancer patient plasma. Specifically, in the instant study, whole plasma was filtered through a 0.45 um membrane followed by ultrafiltration through a 3000 molecular weight (MW) cutoff filter. Nano-HPLC was performed on the filtered material followed by electrospray ionization (nano-ESI) ion trap mass spectrometry (LC/MSD Trap XCT Ultra, Agilent Technologies). Spectra obtained from the samples were matched with databases translated from tiling arrays of 1% of the genome (~30Mb) reported by the ENCODE consortium (Nature, June 14, 2007). Of course, those of skill in the art will understand that a variety of modifications to this precise method would be possible. Patients with pancreas cancer from whom plasma samples were obtained included the following pathological diagnoses: ductal adenocarcinoma, mucinous cystic neoplasm,

Intraductal Papillary Mucinous Neoplasm (IPMN), ampullar^ adenoma, endocrine carcinoma and islet cell carcinoma. Peptides were also identified in plasma from normal healthy donors, a subset of which were age and sex matched with pancreas cancer patients.

Peptides found in plasma from pancreas cancer patients are listed in Tables 1 and 2.

Peptides in common from normal tissue and plasma from healthy donors were subtracted from peptides in pancreas cancer patient plasma, a subset of which are age and sex-matched controls. False positive hit rate using random databases of same size and complexity was 23%. FIGs. 3 A and 3B detail some peptides that were determined in this manner.

Table 1

Peptide # of

Peptide SEQ ID NO. Gene Name Length Occurrence Patients

1. chr5_232123-2nd-1(53

HQNQCLEVQINFT a. a) 13 1 P1041_PLASMA_5.2999.3005.2,

SSEKLLPCVDWGPASTH 2. chr7 232926-3rd-1(27 a.a) 17 1 P1032_PLASMA_3.2000.2005.2,

3. chr11_227273-

AREPALVPAAKPGN Antisense_2nd-0(20 a. a) 14 1 P1040N_PLASMA_2.1725.1725.0,

PAVPARGRSCL 4. NM 021733.1 16(TSKS) 11 1 P4007_PLASMA_3.1129.1147.2,

5. chr14_228863-3rd-0(14

RECVARFKSK a.a) 10 1 P1034_PLASMA3.1922.1927.0,

6. P1015D_PLASMA_3.1777.1796.0,P 1015D_PLASMA_5.1803.1808.0.P1 015D_PLASMA_2.1783.1790.0.P10 27_PLASMA_2.1790.1795.0.P1029 C_PLASMA_5.1784.1792.0.P1029C _PLASMA_3.1778.1785.0, P1029C_ PLASMAJ .1806.1920.0.P1030D J³ LASMA_2.1764.1771.0.P1030D_PL ASMAJ .1770.1860.0.P1030D_PLA SMAJ .1957.2028.0, P1030D_PLAS MA_3.1911.2003.0, P1030D_PLASM A_3.1761.1766.0.P1031 E_PLASMA _5.1704.1716.0.P1031 E_PLASMA_ 4.1715.1721.0,P1032_PLASMA_5.1 832.183β.0,P1036J_PLASMA_1.166 chr11_227527-1st-1(18 4.1669.0,P1037_PLASMA2.1712.17

VVAGLGRAVTRL a.a) 12 17 72.0,

7. chr11_227169-2nd-0(55 P1032_PLASMA_1.2238.2251.2,P1

RGQTPAQQVMLP a.a) 12 2 041_PLASMA_3.2355.2368.2,

Q

O. P1033_PLASMA1.1689.1689.2.P10 chr7_233030- 41_PLASMA_5.1646.1653.2,P1041

ISKLPAHGL Antisense 1st-2(27 a.a) 9 3 _PLASMA_2.1627.1656.2,

LRDPTLHPYSTATSSSQS 9. XM 497989.1 3(LOC4421

HPCAS 24) 23 1 P4007_PLASMA_3.1281.1281.0,

10. chr22_230793-3rd-1(54

SQIPSLPR a.a) 8 1 P1043Q_PLASMA_3.1171.1179.2,

DSEGGSRGRR 11. chr22 230991- 10 1 P1036J PLASMA 3.1371.1378.0,

Peptide # of

Peptide SEQ ID NO. Gene Name Length Occurrence Patients

Antisense_3rd-0(50 a.a)

12. chr14_228990-2nd-0(23

KDRGWPHLE a.a) 9 1 P1027_PLASMA_4.2363.2375.2,

13. chr19_229869-3rd-0(35

CHSPAPESASRAQRLLP a.a) 17 1 P1037_PLASMA1.1992.2087.0,

14. chr11_227650-

RRALGGEGGCRRPQHP Antisense 2nd-0(87 a.a) 16 1 P1033_PLASMA4.1720.1752.2,

HPGSHRPGLSHIYSPRV 15. NM 001005178.1 0(OR52

AGC W1) 20 1 P4008_PLASMA_2.1395.1395.3,

QDHLSGECKTNSSSLGA 16. chr11_228174-

EL Antisense 1st-1(61 a.a) 19 1 P3004_PLASMA_1.1737.1743.0,

17. chii 9_229882-2nd-1 (131 P1033_PLASMA1.2150.2160.0.P10

REMGESLLPH a.a) 10 2 33_PLASMA4.2210.2216.0,

18. chii 6_229452-2nd-0(25

WLLSSARSLPQHDPF a.a) 15 1 P1015D_PLASMA_3.1342.1342.0,

19. chr5_231751-

MGCCPGGRAGVKEM Antisense 3rd-0(20 a.a) 14 1 P3004_PLASMA_1.2081.2085.2,

20. chr11_227446-2nd-0(118

GPQTPVPGARGAK a.a) 13 1 P1041_PLASMA_5.1825.1833.2,

21. >ENm007-Antisense 3rd-

AIGAISAHCNL 594 3(34 a.a) 11 1 P3001A_PLASMA_1.1410.1417.0,

22. chr7_233125-

SLRTQQPL Antisense_2nd-0(48 a.a) 8 1 P3004_PLASMA_3.1664.1671.2,

YSRLFPE 23. chr6 232486-1 st-1 (36 a.a) 7 1 P1041_PLASMA_5.2399.2417.2,

24. chr16_229482- P1027_PLASMA_3.1220.1234.2.P1

GDPLRTSLSVK Antisense 1st-0(54 a.a) 11 2 030D_PLASMA_3.1232.1239.2,

25. chr19_229690-3rd-0(39

LSMRLLPSSHQS a.a) 12 1 P1029C_PLASMA_5.1437.1437.0,

PNVSSLAGEKPKV 26. NM_000301.1_25(PLG) 13 1 P4007_PLASMA_5.1272.1280.0,

27. P1025_PLASMA5.1290.1290.0.P10 27_PLASMA_1.1248.1248.0,P1030 D_PLASMA_5.1256.1270.2.P1030D _PLASMA_4.1248.1254.0.P1032_P chr5_232244-2nd-0(17 LASMA_4.1274.1274.0, P1033_PLA

PAPPPLAHQSPG a.a) 12 SMA5.1304.1318.0,

PVHAAGCCLPVGGPAG 28. NM_014293.2_0(NPTXR) 18 1 P4008 PLASMA_3.2062.2062.2,

Peptide # of

Peptide SEQ ID NO. Gene Name Length Occurrence Patients GR

29. chr11_227767-

AASVQVGAPQQPRSK Antisense_1 st-1 (53 a. a) 15 1 P3004_PLASMA_2.1867.1867.0,

LHVSVLPGRAY 30. chr6_232648-3rd-2(19 a.a) 11 1 P1030D_PLASMA_2.2091.2098.0,

IDLTKTHYPMCILNI 31. chr7 233107-1 st-6(37 a.a) 15 1 P1041 _PLASMA_4.1827.1831.0,

32. chr22_230854-

MMQRTGQKTG Antisense 2nd-1(35 a.a) 10 1 P1029C_PLASMA_3.2889.2912.2, GSAVERASIPGRGSGCA 33. chr14_229085- RVASKLKE Antisense 2nd-1(55 a.a) 25 1 P1027_PLASMA_1.1510.1517.3, PIPSPHPRSPSRPELARP 34. chrX_234028- P1015D_PLASMA_2.1211.1239.0,P

GSLQ Antisense 2nd-0(59 a.a) 22 2 3004_PLASMA_4.1257.1257.2,

35. chr16_229465- P1031 E_PLASMA_4.2048.2063.3,P

EAARPLQLPSACHSP Antisense 2nd-0(66 a.a) 15 2 1031 E_PLASMA_1.2027.2055.3,

36. >ENm006-2nd-880_0(46 ARPPGLLPRPAPPS a.a) 14 1 P3001A_PLASMA_1.1102.1109.2,

37. chr2_231179-2nd-0(29 P4003_PLASMA_1.1170.1170.0.P4 RIGRPGPLFQPPP a.a) 13 2 003_PLASMA_5.1135.1135.0,

38. chr5_232115- P1034_PLASMA1.1340.1340.0.P10 SNGVGEGAALLENRI Antisense 2nd-3(36 a.a) 15 2 40N_PLASMA_5.1358.1358.0,

39. chr16_229418-3rd-0(64

GDPDHPQRHRALT a.a) 13 1 P4003_PLASMA_5.1651.1651.0,

LDPCSHGQREPGQCWG 40. chrX_233949-

QRAGRRGGEIARQSGR Antisense 1 st-0(35 a.a) 32 1 P1041_PLASMA_3.1539.1539.4,

41. chrX_233601-

PVPQVGRALM Antisense 3rd-0(27 a.a) 10 1 P1031 E_PLASMA_5.2051.2051.2,

42. XM 498866.1 1 (LOC4407

ELSFSRPAF 83) 9 1 P4007_PLASMA_4.1893.1899.2, LPSLPAGSASLQPSRRD 43. chr9_233475-

QLCS Antisense 3rd-4(47 a.a) 21 1 P1032_PLASMA_3.2004.2014.0,

44. chr11_227899-2nd-0(23 GGEREVPGRSADPTAAT a.a) 17 1 P1029C_PLASMA_4.1793.1793.2,

45. chrX_233620-

VCCSRPEGTGVR Antisense 1st-0(34 a.a) 12 1 P1032_PLASMA_4.1590.1597.2, GRVRGSADPLRFCVSTG 46. chr9_233363- RARQLDP Antisense_2nd-0(60 a.a) 24 1 P1034_PLASMA4.1910.1910.0, HPARALEPHA 47. NM_001036.2_75(RYR3) 10 1 P4007_PLASMA_1.1427.1455.0,

Peptide # of

Peptide SEQ ID NO. Gene Name Length Occurrence Patients

48. NM 017622.1 1 (FLJ2001 P4008_PLASMA_1.1977.1989.2.P4

WRGAGADGAERGL 4) 13 2 008_PLASMA_1.1997.1997.2,

49. chrX_233663-

GRAQGTPGR Antisense 1st-2(25 a.a) 9 1 P1025_PLASMA5.1327.1327.0,

50. chr21_230742-1st-1 (21 P1015D_PLASMA_3.2307.2314.0.P

QSWVYKVIE a.a) 9 2 1015D_PLASMA_4.2319.2326.0,

MGAPLPRPPSPEPLPPD 51.

PDPRTQAQPR chr7_233057-1st-0(46 a.a) 27 1 P1037_PLASMA2.1440.1440.0,

52. P1038L_PLASMA_3.1966.1973.0,P chr12_228327- 1038L_PLASMA_4.1946.2009.3.P1

CSEESKYLYLSVYASR Antisense 3rd-0(40 a.a) 16 3 038L_PLASMA_2.1935.1959.0,

53. chr15_229192-

GFGCSASAHLS Antisense 3rd-0(11 a.a) 11 1 P1030D_PLASMA_3.1459.1463.2,

54. chr16_229364-

KARAEGAPEQRGA Antisense_3rd-0(30 a.a) 13 1 P1015D_PLASMA_4.1358.1369.0,

DSTRGRQTR 55. chr7 232809-1 st-1 (23 a.a) 9 1 P1036J_PLASMA_2.1392.1392.0,

56. chr11_227598-3rd-0(27

LSREGGDRRS a.a) 10 1 P1029C_PLASMA_2.1338.1338.2,

57. chr13_228413-

PHLSAARGLHAGT Antisense_1st-1 (56 a.a) 13 1 P1041_PLASMA_4.2602.2621.0,

HPSVHPRQSRPF 58. chrδ 233278-3rd-0(23 a.a) 12 1 P1032_PLASMA_1.2138.2151.2,

VKPCVITSVI ELN N I LSTM I 59. chr18_229620- P1015D_PLASMA_2.1915.1930.0,P

IQRR Antisense 1st-6(73 a.a) 24 2 1031 E_PLASMA_5.1814.1814.0,

60. chr22_230771-1st-0(30

MKPAGKSMSPK a.a) 11 1 P1039M_PLASMA_5.1630.1638.0,

61. chr5_231894-2nd-7(37 P1015D_PLASMA_5.2263.2284.0,P

RDAILHSASLL a.a) 11 2 1015D_PLASMA_4.2242.2249.0,

LQRPGERSNNQTAKQR 62. chr19_229778-1st-0(20

M a.a) 17 1 P1025_PLASMA1.2279.2292.3,

63. P1039M_PLASMA_2.0961.0982.0,P 1039M_PLASMA_1.0988.1006.0.P1 039M_PLASMA_3.0987.1011.0,P10 chr11_227602-1st-1 (29 39M_PLASMA_4.0991.1010.2,P103

KCLWGPACPLGPIS a.a) 14 5 9M_PLASMA_5.0989.1006.0,

64. chr11_227638-

QDDQDPQRARC Antisense 3rd-0(45 a.a) 11 1 P1015D_PLASMA_5.2025.2032.2,

Peptide # of

Peptide SEQ ID NO. Gene Name Length Occurrence Patients

65. chr11_227951-

PLGWRGTGGIIP Antisense 2nd-0(29 a.a) 12 1 P1041_PLASMA_4.2048.2054.0,

66. chr20_230132-

GREVLGKPRSGLGIQGE Antisense_2nd-3(36 a.a) 17 1 P1036J_PLASMA_2.1321.1321.0,

LQGIPKYSPPSLI 67. chr6 232613-3rd-0(22 a.a) 13 1 P3004_PLASMA_2.1907.1913.2,

68. chr7_233196-

QSCGRSAEGAAAR Antisense 3rd-0(65 a.a) 13 1 P3004_PLASMA_3.1583.1592.0,

69. chr5_231849-

GSPAALSLVQRK Antisense 1st-1 (24 a.a) 12 1 P3004_PLASMA_4.1452.1457.2,

70. chrX_233967-

TPTLPGRPGCLALPSAP Antisense_1 st-0(60 a.a) 17 1 P1025_PLASMA1.1990.1990.0,

71. P1041_PLASMA_5.1769.1794.0.P1 041_PLASMA_1.1780.1801.0.P104 1_PLASMA_2.1763.1784.0,P1041_ chr21_230286- PLASMA_3.1767.1794.0,P1041_PL

GEQRRDPGSWRRGW Antisense_3rd-0(70 a.a) 14 ASMA_4.1768.1774.0,

72. P1043Q_PLASMA_4.1631.1638.0, P chr15_229120- 1043Q_PLASMA_5.1637.1637.0,P1

HCCEPIGFSVFS Antisense_2nd-0(14 a.a) 12 043Q_PLASMA_3.1636.1648.0,

Table 2

Peptides from cancer patient plasma (normal donor plasma subtracted) searchec on DarkDark database

Peptide Sequence Seq ID No. Length (# a.a) # Occurrences Patients

73. P1015, P1027, P1029, P1030, P1031 ,

LHLSDSASI 9 16 P1033, P1034, P3004

LWRWVLAMLPRMASNSWPQGILL 74. 23 8 P1027.P1029, P1031 , P1033, P3004

SQEAPLTPSRL 75. 11 7 P1034, P1040, P4006, P1035

CIPCRVISSR 76. 10 8 P1033, P3004, P1041

GLQEELNCNLTFRLQI 77. 16 4 P1029, P1036, P1043

GNTEGLQKSLAELGGHLDQQVEEFR 78. 25 4 P1025, P1032

SLAELGGHLDQQVEEFR 79. 17 8 P1025, P1027, P1030, P1032

SLAELGGHLDQQVEEFRR 80. 18 4 P1025, P1027, P1032

SLAELGGHLDQQVEEF 81. 16 2 P1025

AELGGHLDQQVEEFR 82. 15 1 P1025

AENRGGSRKSRGGRPGTVAHK 83. 21 2 P4003

AIPLPQGPPPQEMAPSHTSQE 84. 21 1 P1030

AKDVAYPKKK 85. 10 1 P4005

AQRRSDKIWR 86. 10 2 P4004, P4008

ASCPGLAGSHSIL 87. 13 2 P1043

ASDSGLPFLASNNLLRVTRKKSCSKDH 88. 27 1 P1032

ASSNPPTLASQSAGIMGRSQHTQPK 89. 25 2 P1025, P1030

ATQRASDKLGRE 90. 12 1 P1025

AVSRDGTIALHPG 91. 13 1 P3004

CLDHGSGYLM 92. 10 2 P1025, P1027

CPASGLGRECVCV 93. 13 1 P1025

DQEKKGLGILRCCC 94. 14 1 P4007

DQRGETPSLLN 95. 11 1 P1026

DQTIALQPGQRE 96. 12 2 P1025

EFKTRLANMAKPSL 97. 14 1 P1034

EKGLRTCLRIAPGLEPGCEE 98. 20 1 P3003

ELQLGFKTRMEIQNNL 99. 16 1 P1034

EQCLKLHCASGP 100. 12 1 P1034

ETNAELPVLSHSLEMTG 101. 17 1 P1031

^*Note - adjacent normal tissue peptides have not yet been subtracted from this list; also, does not contain patient tumor tissue peptides

EXAMPLE 2

Peptide ELISAs were performed using plasma from pancreas cancer patients. Plasma was serially diluted and added to peptide-coated ELISA plates. Sum absorbance was obtained by adding the optical density reading from each dilution of the plasma. Data is shown in FIG. 4.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of certain embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. An isolated peptide of between 8 to 40 amino acids comprising the sequence of any of SEQ ID NOs:l-214.

2. An isolated peptide of between 8 to 40 amino acids consisting of the sequence of any of SEQ ID NOs:l-214.

3. An antibody that specifically binds to a peptide having the sequence of any one of SEQ ID NOs:l-214.

4. A pharmaceutical composition comprising one or more peptides having the sequence of any of SEQ ID NOs:l-214.

5. A nucleic acid encoding a peptide having the sequence of any one of SEQ ID NOs: 1- 214.

6. A method of diagnosing a pancreatic disease in a subject comprising:

(a) obtaining a sample from a subject suspected of having or at risk for having the pancreatic disease;

(b) and detecting whether one or more peptides with the sequence one or more of

SEQ ID NOs: 1-214 is present in the sample; and

(c) diagnosing the pancreatic disease if the peptide is detected.

7. The method of claim 6, wherein the pancreatic disease is pancreatic cancer.

8. The method of claim 6, wherein the detecting involves mass spectrometry.

9. The method of claim 6, wherein the sample is a plasma or serum sample.

10. The method of claim 9, wherein the sample is a plasma sample.

11. The method of claim 9, wherein the sample is a serum sample.

12. The method of claim 7, wherein the pancreatic disease is a metabolic disease.

13. The method of claim 7, wherein the pancreatic cancer is ductal adenocarcinoma, mucinous cystic neoplasm, Intraductal Papillary Mucinous Neoplasm (IPMN), ampullary adenoma, endocrine carcinoma, or islet cell carcinoma.

14. The method of claim 12, wherein the metabolic disease is diabetes.

15. The method of claim 6 further comprising filtering the plasma or serum sample.

16. The method of claim 15, wherein filtering the plasma or serum sample comprises ultrafiltration through a 3000 molecular weight cut-off filter.

17. The method of claim 1 further comprising chromatographically separating the plasma or serum sample.

18. The method of claim 17, wherein chromatographically separating the plasma or serum sample comprises nano-HPLC.

19. The method of claim 1 , wherein the mass spectrometry is electrospray ionization (nano-ESI) ion trap mass spectrometry.