EP2097746A2 - Biomarker for diagnosing pancreatic cancer - Google Patents

Biomarker for diagnosing pancreatic cancer

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Publication number
EP2097746A2
EP2097746A2 EP07846381A EP07846381A EP2097746A2 EP 2097746 A2 EP2097746 A2 EP 2097746A2 EP 07846381 A EP07846381 A EP 07846381A EP 07846381 A EP07846381 A EP 07846381A EP 2097746 A2 EP2097746 A2 EP 2097746A2
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European Patent Office
Prior art keywords
seq
protein
pancreatic
diagnosis
precursor
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EP07846381A
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German (de)
French (fr)
Inventor
Barbara Sitek
Kai STÜHLER
Bence Sipos
Günter KLÖPPEL
Ibrahim Alkatout
Stephan Hahn
Helmut Meyer
Wolff Schmiegel
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4742—Keratin; Cytokeratin

Definitions

  • pancreatic cancer synonym: pancreatic cancer, pancreatic carcinoma
  • PDAC Pancreatic ductal adenocarcinoma
  • PanIN pancreatic intraepithelial
  • Neoplasms pancreatic lesions
  • CP chronic pancreatitis
  • endocrine tumors of the pancreas with determination by selected biomarkers.
  • the invention relates to suitable combinations of biomarkers, in particular for in vitro diagnostics.
  • the 5-year survival rate is about 1% the lowest among all cancers (Parkin, DM, F. Bray, et al. (2001). "Estimating the World Cancer: Globocan 2000.” Int J Cancer 94 (2): 153-6). Early diagnosis could increase the 5-year survival rate to 40% (Yeo, CJ and JL Cameron (1998). "Prognostic factors in ductal pancreatic cancer.”Langenbeck's Arch Surg 383 (2): 129-33).
  • pancreatic cancer should also be used for diagnosis, such as PDAC (pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasia), pancreatic lesions, CP (chronic pancreatitis), including pancreatic endocrine tumors.
  • PanIN concerns pancreatic lesions and subdivided these morphologically into Panln IA, IB, 2 and 3 (Kern, S., R. Hruban, et al., 2001). "A white paper: the product of a pancreatic cancer think tank.” Cancer Res 61 (12) : 4923-32).
  • Pancreatic lesions are also described for CP.
  • pancreatic cancer for the purpose of a suitable therapy of pancreatic cancer or its precursors and / or concomitant diseases, in particular PDAC (Pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasms), pancreatic lesions, CP (chronic pancreatitis), including endocrine tumors of the pancreas, an early diagnosis and Differentiation in connection with the need to make clinical decisions.
  • PDAC Pancreatic ductal adenocarcinoma
  • PanIN pancreatic intraepithelial neoplasms
  • pancreatic lesions for the purpose of a suitable therapy of pancreatic cancer or its precursors and / or concomitant diseases, in particular PDAC (Pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasms), pancreatic lesions, CP (chronic pancreatitis), including endocrine tumors of the pan
  • a disadvantage of known diagnostic methods using the previously known markers is that early and complete detection of high-risk patients fails and therefore a diagnosis is insufficient or even too late.
  • Serum biomarker for pancreatic cancer is C-19-9, with specificity only 60-90%, as this marker is also detectable in other diseases, especially in chronic pancreatitis, in the blood (Banfi et al. (1996) CA 19.9, CA. 242 and CEA in the diagnosis and follow-up of pancreatic cancer, Int J Biol Markers, 77-81, Banfi et al (1993) Behavior of tumor markers CA19.9, CA195, CAM43, CA242, and TPS in the diagnosis and follow -up ⁇ of pancreatic cancer, Clin Chem, 420-3).
  • the object is achieved by a method for the diagnosis of pancreatic cancer or its precursor and / or comorbidities, wherein a determination of at least one of the polypeptides / proteins selected from the group a.) Keratin 8 protein (SEQ ID No. 1), Vimentin (SEQ ID No. 2), mitochondrial malate dehydrogenase (SEQ ID No. 3), beta tropomyosin (SEQ ID No. 4), ACTG1 protein (SEQ ID No. 5), thioredoxin delta 3 (SEQ ID No. 6), B chain B triosephosphate isomerase (SEQ ID No. 7), annexin A2 (SEQ ID No.
  • TPM4-ALK fusion oncoprotein type 2 (SEQ ID No. 9), peptidylprolyl isomerase A (SEQ ID No. 10), smooth muscle mysoin light chain (SEQ ID No. 11), desmin (SEQ ID No. 12), major vault protein 1 (SEQ ID No. 13), heterogeneous nuclear ribonucleoprotein Al (SEQ ID No. 14), SlOOAlO (SEQ ID No. 15), EFla-like protein (SEQ ID No. 16), regulatory myosin light chain long version (SEQ ID No. 17), tropomyosin 1 ⁇ chain isoform 3 ( SEQ ID No. 18), tropomyosin 2 (beta) isoform 2 (SEQ ID No.
  • myosin regulatory light chain MRCL3 (SEQ ID No. 20), alpha-2 globin (SEQ ID No. 21), tropomyosin 4 (SEQ ID No. 22), transgulin (SEQ ID No. 23), keratin 7 (SEQ ID No. 24), ACTB protein (SEQ ID No. 25), M2-type pyruvate kinase (SEQ ID No. 26), Actin related protein 2/3 complex subunit 5 (SEQ ID No. 27), Anterior gradient 2 homologue (AGR 2) (SEQ ID No.28),
  • aldehyde dehydrogenase 1 (SEQ ID No. 41), aldehyde dehydrogenase IA1 (SEQ ID No. 42), T-complex protein 1 subunit beta (SEQ ID No. 43), apolipoprotein A4 (SEQ ID No. 44), malate dehydrogenase mitochondrial precursor ( SEQ ID No. 45), voltage-dependent anion selective channel protein 1 (SEQ ID No. 46), glyceraldehyde-3-phosphate dehydrogenase (SEQ ID No. 47), uracil DNA glycosylase (SEQ ID No.
  • the proteins according to the invention could be identified as potential biomarkers by means of a differential proteome analysis of diseased pancreatic ductal tissue - five progression stages - compared to normal (healthy) pancreatic ductal tissue.
  • Corresponding tissue samples were taken from diseased patients. The samples were homogenized in a hand homogenizer with lysis buffer and freed of DNA and other cell material to obtain a protein concentrate.
  • the proteins were with labeled with a dye and subjected to 2D gel electrophoresis with isoelectric focusing in the first and SDS gel electrophoresis in the second dimension.
  • the differential representation (ill / healthy) is presented in Tables (1 to 3), Examples and Figures, and shows different characteristic expression (up-regulated and down-regulated, read by the spots).
  • proteins according to the invention are as follows:
  • MVP Major vault protein
  • the invention also relates to such amino acid sequences (polypeptides, proteins) having a sequence identity or homology of 70% and more, preferably 80% and more, more preferably 90-95% and more with SEQ ID NO. 1 to SEQ ID no. 71 have. Also included are those analog amino acid sequences which, because of the replacement of one or more amino acids in these sequences, still provide the desired function of a biomarker for the diagnosis of pancreatic cancer.
  • amino acid sequences polypeptides, proteins having a sequence identity or homology of 70% and more, preferably 80% and more, more preferably 90-95% and more with SEQ ID NO. 1 to SEQ ID no. 71 have.
  • analog amino acid sequences which, because of the replacement of one or more amino acids in these sequences, still provide the desired function of a biomarker for the diagnosis of pancreatic cancer.
  • partial peptides or fragments of SEQ ID no. 1 to SEQ ID no. 71 are also included.
  • Combinations (sub-combination of the above totality of all biomarkers according to the invention) of the biomarkers according to the invention are advantageous for the purpose of diagnosis.
  • Those combinations which are particularly preferred are at least stratifin (14-3-3 sigma) (SEQ ID No. 29) and / or vimentin (SEQ ID No. 2) and / or major vault protein 1 (SEQ ID No. 13) and / or anterior gradient 2 homologous (AGR 2) (SEQ ID No.28), and / or SlOOAlO (SEQ ID No. 15 ) and / or EFIA-like protein (SEQ ID No. 16) and / or Annexin A2 (SEQ ID No. 8) and / or Annexin A4 (SEQ ID No. 38).
  • pancreatic cancer also encompasses its precursor and / or comorbidities, in particular PDAC (pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasms), pancreatic lesions, CP (chronic pancreatitis), including endocrine tumors of the pancreas, in particular pancreatic tumors and pancreatic tumors neoplasms.
  • PDAC pancreatic ductal adenocarcinoma
  • PanIN pancreatic intraepithelial neoplasms
  • pancreatic lesions pancreatic lesions
  • CP chronic pancreatitis
  • endocrine tumors of the pancreas in particular pancreatic tumors and pancreatic tumors neoplasms.
  • the invention also relates to the identification of patients at increased risk and / or unfavorable prognosis of pancreatic cancer, especially in symptomatic and / or asymptomatic patients.
  • the inventive method therefore allows clinical
  • the invention also relates to a method for diagnosing patients with pancreatic cancer for performing clinical decisions, such as advanced treatment and drug therapy.
  • the invention relates to a method for diagnosis for the early or differential diagnosis or prognosis of pancreatic cancer or a precursor disease, wherein a determination of the biomarker is carried out on a patient to be examined.
  • tissue samples or body fluid are taken from the patient to be examined, and the diagnosis is made in vitro / ex vivo, i. outside the human or animal body. Due to the determination of the markers according to the invention, a high significance for the pancreatic cancer or a precursor and / or concomitant disease is achieved, and the diagnosis can be made on the basis of the existing amount or its change (leveling: increase / decrease) in at least one patient sample.
  • the method according to the invention may be part of an in-vitro diagnosis by means of parallel or simultaneous determinations of the markers (eg multi-well plates with 96 or more wells), the determinations being carried out on at least one patient sample.
  • the markers eg multi-well plates with 96 or more wells
  • the method according to the invention can be carried out by means of 2D electrophoresis, in the first dimension an isoelectric focusing, in the second dimension a gel electrophoresis is carried out (in the broadest sense proteomics is to be used for this purpose) ,
  • the method according to the invention and its determinations can be carried out by means of a rapid test (for example, lateral-flow test), whether in single or multiparameter determination.
  • a rapid test for example, lateral-flow test
  • the method according to the invention can be carried out in vivo, wherein the biomarkers according to the invention are labeled with a probe, in particular an antibody which has a contrast agent and detected with a detector which is suitable for imaging (“Molecular Imaging”) (Ralph Weissleder , Molecular Imaging in Cancer, Science, Vol. 312, 1168 (2006)).
  • Molecular Imaging Molecular Imaging
  • the invention relates to the use of the biomarkers according to the invention for the diagnosis and / or prognosis and / or for the early or differential diagnosis of pancreatic cancer or its precursor and / or comorbidities.
  • Another object is to provide a corresponding diagnostic device for carrying out the method according to the invention.
  • such a diagnostic device in particular an array or assay (for example immunoassay, ELISA, etc.), in particular a protein biochip (US Pat.
  • the invention additionally relates to a kit for carrying out the methods according to the invention, in particular containing detection reagents and further auxiliaries.
  • detection reagents include e.g. Antibodies etc.
  • biomarkers according to the invention can likewise be carried out with the aid of further protein diagnostic methods familiar to the person skilled in the art, in particular using radioactively or fluorescently labeled antibodies.
  • suitable bioanalytical methods such as, for example, immunohistochemistry, antibody arrays, Luminex, ELISA, immunofluorescence, radioimmunoassays and other suitable bioanalytical methods, such as, for example, NMR spectroscopic methods, eg MRM (multi-reaction monitoring) or AQUA (absolute quantification), with the help of which the biomarkers can be measured quantitatively.
  • Tissue samples were obtained from patients who underwent surgery at the Department of General Surgery of the University Hospital Schleswig-Holstein, Campus Kiel (Germany). Tumor tissue of ductal pancreatic cancer and peritumoral parenchyma were immediately postoperatively in - 80 0 C flash-frozen and stored.
  • 5 ⁇ m-thick frozen sections of the peritumoral pancreatic sparchyma were prepared, briefly fixed in ethanol (Merck, Darmstadt, Germany), stained with hematoxylin-eosin and subsequently examined by a pathologist.
  • the PanINs were prepared according to the accepted criteria (Hruban, RH, NV Adsay, et al. (2001).
  • Pancreatic intraepithelial neoplasia a new nomenclature and classification system for pancreatic duct lesions. Am J Surg Pathol 25 (5): 579- 86). Tissue blocks containing the required PanIN lesions were cut in series (10 ⁇ m). For the 2-DE, the tissue sections were stained with hematoxylin and stored at -20 0 C immediately. The PanIN lesions were microdissected under a microscope using a sterile injection needle (size 0.65x25 mm, Braun, Melsoder, Germany) (BH2, Olympus, Wetzlar, Germany). Primarily, medium-sized interlobular ducts were selected to avoid contamination with periductal mesenchymal and acinar tissue.
  • microdissected cells were taken up in 100 ⁇ l of lysis buffer (TrisCl 30 mM, thiourea 2 M urea 7 M, CHAPS 4%, pH 8.0) and placed on ice in an ultrasonic bath immediately after the microdissection (6 ⁇ 10 sec pulses, ultrasonic cleaner, VWR Darmstadt, Darmstadt).
  • lysis buffer TrisCl 30 mM, thiourea 2 M urea 7 M, CHAPS 4%, pH 8.0
  • Adenocarcinoma were homogenized in 148 ⁇ l lysis buffer (TrisCl 30 mM, thiourea 2 M, urea 7 M, CHAPS 4%, pH 8).
  • the samples, each containing 1000 microdissected cells in 100 .mu.l lysis buffer were reduced by addition of 2 nmol TCEP and then incubated at 37 0 C for Ih in the dark.
  • the saturation dyes, Cy3 and Cy5 were first diluted with DMF (2 nmol / ⁇ l, Sigma) and then 4 nmol each to the added to reduced samples. The incubation was carried out at 37 ° C. for 30 minutes in the dark.
  • 10 ⁇ l of DTT (1.08 g / ml, Bio-Rad) were added. Subsequently, the samples were each provided with 10 ⁇ l of Ampholine 2-4 (GE Healthcare).
  • the carrier-ampholyte-based IEF tube gels 20 cm x 1.5 mm
  • Electrophore 16 (6): 1034-59 At the end of a 21, 25 hour program of stress, the ejected tube gels in equilibration buffer (125 mM
  • Tris 40% (w / v) glycerol, 3% (w / v) SDS, 65 mM DTT, pH 6.8) for 10 min.
  • the second dimension was performed in a Desaphor VA 300 system with polyacrylamide gels (15.2% acrylamide (total), 1.3% bisacrylamide) (Klose and Kobalt 1995 (supra)).
  • the tube gels were on the
  • the spots were manually punched out of a preparative gel. In order to determine the position of the spots in the gel, scale became more accurate after image acquisition Gel expression placed under the gel. The spots were subsequently digested in the gel with trypsin (Promega, Mannheim, Germany) and the peptides as described in Schulfer et al. Schaefer, H., JP Chervet, et al., (2004). "A peptide preconcentration approach for nano-high-performance liquid chromatography to diminish memory effects.” Proteomics 4 (9): 2541-4; Schaefer, H , K.
  • MS / MS spectra were searched for the NCBI protein sequence sub-database (human) using the SEQUEST TM algorithm, using the following search parameters (http://www.ncbi.nlm.nih.gov). Here were. following search parameters taken into account: mass tolerance + 1.5 Da for parent and fragment ions. Modification of all cysteines with Cy3. An Meinpsin interface. Proteins with a SequestMetaScore (Proteinscape TM) greater than 10 in at least 3 peptides were considered as identified.
  • SequestMetaScore Proteinscape TM
  • Tissue cylinders were punched from representative areas and embedded in recipient paraffin blocks, so that a total of 300 cylinders with pancreatic tissues (in a total of 6 tissue arrays) and two control cylinders each consisting of healthy tonsillar tissue were processed.
  • the work-up was carried out using MTAl tissue arrayer instrument (Beecher Instruments, Sun Prairie, WI, USA).
  • the normal pancreatic ducts as well as the ducts of the PanINs are derived from 12 pancreata of healthy suicided people who were autopsied at the Forensic Medicine Institute of the Semmelweis University in Budapest, Hungary
  • pancreata (Approval number: 140-1 / 1996) as well as 81 pancreata, which were taken as part of operations of gastrointestinal and pancreatic tumors of the surgical university hospitals in Kiel and Dresden, Germany.
  • pancreata was removed from tissue blocks of 48 pts at Kiel University Hospital.
  • the intensity of the staining was classified as mild, moderate and strong (corresponding to a score of 1, 2 or 3).
  • the stained areas were estimated in relation to the pancreatic ducts or the tumor areas in percent and again in
  • the final score was determined by the product of staining intensity and proportion of positively stained cells
  • a differential proteome analysis of microdissected cells from PanIN lesions, PDAC, and normal pancreatic ducts was performed to identify biomarker candidates for pancreatic tumor progression. For this approach, tumors from 9 pancreatic cancer patients were examined, providing a total of 4-9 lesions per lesion. The identified differential biomarkers were immunohistochemically validated on samples (tissue arrays) of 130 patients.
  • the reference proteome from the pancreatic tumor tissue was used, whose proteome pattern is in high agreement with the proteome of the microdissected material (> 91%).
  • LC-ESI-MS / MS a total of 38 non-redundant proteins could be identified (Table 1).
  • Proteins are confirmed the proteome data.
  • the comparison of proteome data and validation is presented on three proteins: 14-3-3 sigma, MVP, and AGR2 ( Figures 2, 3, and 4).
  • sequences according to the invention are as follows: SEQ ID no. 1
  • SEQ ID no. 45 Malate dehydrogenase, mitochondrial precursor MLSALARPVS AALRRSFSTS AQNNAKVAVL GASGGIGQPL SLLLKNSPLV SRLTLYDIAH TPGVAADLSH IETKAAVKGY LGPEQLPDCL KGCDVVVIPA GVPRKPGMTR DDLFNTNATI VATLTAACAQ HCPEAMICVI ANPVNSTIPI TAEVFKKHGV YNPNKIFGVT TLDIVRANTF VAELKGLDPA RVNVPVIGGH AGKTIIPLIS QCTPKVDFPQ DQLTALTGRI QEAGTEVVKA KAGAGSATLS MAYAGARFVF SLVDAMNGKE GVVECSFVKS 'QETECTYFST PLLLGKKGIE KNLGIGKVSS FEEKMISDAI PELKASIKKG EDFVKTLK

Abstract

The invention relates to a method for diagnosing pancreatic cancer (PaCa) or the precursor diseases and/or concomitant diseases thereof, in particular pancreatic ductal adenocarcinoma (PDAC), pancreatic intraepithelial neoplasia (PanIN), pancreatic lesions, chronic pancreatitis (CP), including endocrine pancreatic tumors. In said method, the diagnosis is performed using selected biomarkers. The invention further relates to biomarker combinations suitable for carrying out said method, particularly for in vitro diagnosis.

Description

Biomarker für die Diagnose von PankreaskrebsBiomarker for the diagnosis of pancreatic cancer
Beschreibungdescription
Die Erfindung betrifft ein Verfahren zur Diagnose von Pankreaskrebs (synonym: Bauspeicheldrüsenkrebs, Pankreaskarzinom) (PaCa) oder dessen Vorläufer- und/oder Begleiterkrankungen, insbesondere PDAC (Pancreatic ductal adenocarcinoma) , PanIN (Pankreatische intraepithelialeThe invention relates to a method for the diagnosis of pancreatic cancer (synonym: pancreatic cancer, pancreatic carcinoma) (PaCa) or its precursor and / or comorbidities, in particular PDAC (Pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial
Neoplasien) , Pankreasläsionen, CP (Chronische Pankreatitis) , einschließlich endokriner Tumoren der Pankreas, wobei eine Bestimmung mittels ausgewählten Biomarkern erfolgt. Ferner betrifft die Erfindung hierzu geeignete Kombinationen von Biomarkern, insbesondere zur in-vitro Diagnostik.Neoplasms), pancreatic lesions, CP (chronic pancreatitis), including endocrine tumors of the pancreas, with determination by selected biomarkers. Furthermore, the invention relates to suitable combinations of biomarkers, in particular for in vitro diagnostics.
Für Pankreaskrebs ist die 5 Jahr-Überlebensrate mit ca. 1 % die niedrigste Rate unter allen Krebsarten (Parkin, D. M., F. Bray, et al. (2001) . "Estimating the world Cancer bürden: Globocan 2000." Int J Cancer 94(2): 153-6). Eine frühzeitige Diagnose könnte die 5 Jahr-Überlebensrate auf 40 % erhöhen (Yeo, C. J. and J. L. Cameron (1998) . "Prognostic factors in ductal pancreatic Cancer." Langenbecks Arch Surg 383(2): 129- 33) . Zur Diagnose sind daher ebenfalls die Vorläufererkrankungen von Pankreaskrebs heranzuziehen, solche wie PDAC (Pancreatic ductal adenocarcinoma) , PanIN (Pankreatische intraepitheliale Neoplasien), Pankreasläsionen, CP (Chronische Pankreatitis) , einschließlich endokrine Tumore der Pankreas. Insbesondere PanIN betrifft Pankreasläsionen und unterteilt diese morphologisch in Panln IA, IB, 2 und 3 (Kern, S., R. Hruban, et al. (2001). "A white paper: the product of a pancreas Cancer think tank." Cancer Res 61(12): 4923-32). Pankreasläsionen sind ebenfalls für CP beschrieben. Ebenfalls relevant sind endokrine (benignen oder maligne) Tumoren der Pankreas, insbesondere neuroendokrine Tumore.For pancreatic cancer, the 5-year survival rate is about 1% the lowest among all cancers (Parkin, DM, F. Bray, et al. (2001). "Estimating the World Cancer: Globocan 2000." Int J Cancer 94 (2): 153-6). Early diagnosis could increase the 5-year survival rate to 40% (Yeo, CJ and JL Cameron (1998). "Prognostic factors in ductal pancreatic cancer."Langenbeck's Arch Surg 383 (2): 129-33). Therefore, the precursors of pancreatic cancer should also be used for diagnosis, such as PDAC (pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasia), pancreatic lesions, CP (chronic pancreatitis), including pancreatic endocrine tumors. In particular PanIN concerns pancreatic lesions and subdivided these morphologically into Panln IA, IB, 2 and 3 (Kern, S., R. Hruban, et al., 2001). "A white paper: the product of a pancreatic cancer think tank." Cancer Res 61 (12) : 4923-32). Pancreatic lesions are also described for CP. Also relevant are endocrine (benign or malignant) tumors of the pancreas, especially neuroendocrine tumors.
Zwecks einer geeigneten Therapie von Pankreaskrebs oder dessen Vorläufer- und/oder Begleiterkrankungen, insbesondere PDAC (Pancreatic ductal adenocarcinoma) , PanIN (Pankreatische intraepitheliale Neoplasien) , Pankreasläsionen, CP (Chronische Pankreatitis) , einschließlich endokriner Tumoren der Pankreas, bedarf es einer frühen Diagnose und Differenzierung in Verbindung mit der Notwendigkeit klinische Entscheidungen zu treffen.For the purpose of a suitable therapy of pancreatic cancer or its precursors and / or concomitant diseases, in particular PDAC (Pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasms), pancreatic lesions, CP (chronic pancreatitis), including endocrine tumors of the pancreas, an early diagnosis and Differentiation in connection with the need to make clinical decisions.
Nachteilig an bekannten Diagnoseverfahren unter Verwendung der bisher bekannten Markern ist jedoch, dass eine frühzeitige und vollständige Erfassung von Risikopatienten nicht gelingt und daher eine Diagnose nur ungenügend oder gar zu spät erfolgt.A disadvantage of known diagnostic methods using the previously known markers, however, is that early and complete detection of high-risk patients fails and therefore a diagnosis is insufficient or even too late.
Eine der Erfindung zugrunde liegende Aufgabe besteht daher darin, ein Verfahren zur Diagnose von Pankreaskrebs oder dessen Vorläufer- und/oder Begleiterkrankungen zu entwickeln, das eine verbesserte frühzeitige Diagnose sowie verbesserte Erfassung von Risikopatienten ermöglicht und den Therapieerfolg verbessert. Ferner ist nachteilig, dass im Stand der Technik zumeist keine hinreichende Sensitivität und/oder Spezifität der Marker erreicht wird. Zum Beispiel besteht eine wesentliche Schwierigkeit in der Frühdiagnose von PDAC im Fehlen eines spezifischen Biomarkers . Der am meisten verwendeteIt is therefore an object of the invention to develop a method for the diagnosis of pancreatic cancer or its precursor and / or concomitant diseases, which enables improved early diagnosis and improved detection of high-risk patients and improves therapeutic success. Furthermore, it is disadvantageous that in the prior art usually no sufficient sensitivity and / or specificity of the marker is achieved. For example, one major difficulty in the early diagnosis of PDAC is the lack of a specific biomarker. The most used
Serumbiomarker für Pankreaskrebs ist C-19-9, wobei die Spezifität nur 60-90% beträgt, da dieser Marker auch bei anderen Erkrankungen, besonders bei der chronischen Pankreatitis, im Blut nachweisbar ist (Banfi et al. (1996) CA 19.9, CA 242 and CEA in the diagnosis and follow-up of pancreatic Cancer, Int J Biol Markers, 77-81, Banfi et al (1993) Behavior of tumor markers CA19.9, CA195, CAM43, CA242, and TPS in the diagnosis and follow-up^ of pancreatic Cancer, Clin Chem, 420-3) .Serum biomarker for pancreatic cancer is C-19-9, with specificity only 60-90%, as this marker is also detectable in other diseases, especially in chronic pancreatitis, in the blood (Banfi et al. (1996) CA 19.9, CA. 242 and CEA in the diagnosis and follow-up of pancreatic cancer, Int J Biol Markers, 77-81, Banfi et al (1993) Behavior of tumor markers CA19.9, CA195, CAM43, CA242, and TPS in the diagnosis and follow -up ^ of pancreatic cancer, Clin Chem, 420-3).
Die Aufgabe wird durch ein Verfahren zur Diagnose von Pankreaskrebs oder dessen Vorläufer- und/oder Begleiterkrankungen gelöst, wobei eine Bestimmung mindestens eines der Polypeptide / Proteine ausgewählt aus der Gruppe a.) Keratin 8 protein (SEQ ID No. 1), Vimentin (SEQ ID No. 2), Mitochondrial malate dehydrogenase (SEQ ID No. 3), Beta tropomyosin (SEQ ID No. 4), ACTGl protein (SEQ ID No. 5), Thioredoxin delta 3 (SEQ ID No. 6), B Chain B Triosephosphate Isomerase (SEQ ID No. 7), Annexin A2 (SEQ ID No. 8), TPM4-ALK fusion oncoprotein type 2 (SEQ ID No. 9), Peptidylprolyl isomerase A (SEQ ID No. 10), Smooth muscle mysoin light chain (SEQ ID No. 11), Desmin (SEQ ID No. 12), Major vault protein 1 (SEQ ID No. 13), Heterogeneous nuclear ribonucleoprotein Al (SEQ ID No. 14), SlOOAlO (SEQ ID No. 15), EFla-like protein (SEQ ID No. 16), Regulatory myosin light chain long version (SEQ ID No. 17), Tropomyosin 1 alpha chain isoform 3 (SEQ ID No. 18), Tropomyosin 2 (beta) isoform 2 (SEQ ID No. 19), Myosin regulatory light chain MRCL3 (SEQ ID No. 20), Alpha-2- globin (SEQ ID No. 21), Tropomyosin 4 (SEQ ID No. 22), Transgelin (SEQ ID No. 23), Keratin 7 (SEQ ID No. 24), ACTB protein (SEQ ID No. 25), M2-type pyruvate kinase (SEQ ID No. 26) , Actin related protein 2/3 complex subunit 5 (SEQ ID No. 27), Anterior gradient 2 homolog (AGR 2) (SEQ ID No.28),The object is achieved by a method for the diagnosis of pancreatic cancer or its precursor and / or comorbidities, wherein a determination of at least one of the polypeptides / proteins selected from the group a.) Keratin 8 protein (SEQ ID No. 1), Vimentin (SEQ ID No. 2), mitochondrial malate dehydrogenase (SEQ ID No. 3), beta tropomyosin (SEQ ID No. 4), ACTG1 protein (SEQ ID No. 5), thioredoxin delta 3 (SEQ ID No. 6), B chain B triosephosphate isomerase (SEQ ID No. 7), annexin A2 (SEQ ID No. 8), TPM4-ALK fusion oncoprotein type 2 (SEQ ID No. 9), peptidylprolyl isomerase A (SEQ ID No. 10), smooth muscle mysoin light chain (SEQ ID No. 11), desmin (SEQ ID No. 12), major vault protein 1 (SEQ ID No. 13), heterogeneous nuclear ribonucleoprotein Al (SEQ ID No. 14), SlOOAlO (SEQ ID No. 15), EFla-like protein (SEQ ID No. 16), regulatory myosin light chain long version (SEQ ID No. 17), tropomyosin 1α chain isoform 3 ( SEQ ID No. 18), tropomyosin 2 (beta) isoform 2 (SEQ ID No. 19), myosin regulatory light chain MRCL3 (SEQ ID No. 20), alpha-2 globin (SEQ ID No. 21), tropomyosin 4 (SEQ ID No. 22), transgulin (SEQ ID No. 23), keratin 7 (SEQ ID No. 24), ACTB protein (SEQ ID No. 25), M2-type pyruvate kinase (SEQ ID No. 26), Actin related protein 2/3 complex subunit 5 (SEQ ID No. 27), Anterior gradient 2 homologue (AGR 2) (SEQ ID No.28),
Stratifin (14-3-3 sigma) (SEQ ID No. 29), Coactosin-like 1 (SEQ ID No. 30), Chaperonin heat shock 6OkD protein 1 (SEQ ID No. 31), Transgelin 2 (SEQ ID No. 32), Aldehyde dehydrogenase 1 (SEQ ID No. 33), Sarcomeric tropomyosin kappa (SEQ ID No. 34), Annexin A3 (SEQ ID No. 35), Delta-globin (SEQ ID No. 36), Serum albumin (SEQ ID No. 37), Protein PP4-X (Annexin A4) (SEQ ID No. 38), Crystallin (SEQ ID No. 39), Myosin regulatory light chain MRCL3 (SEQ ID No. 40) oder Gruppe b.) aldehyde dehydrogenase 1 (SEQ ID No. 41), Aldehyde dehydrogenase IAl (SEQ ID No. 42), T-complex protein 1 subunit beta (SEQ ID No. 43), Apolipoprotein A4 (SEQ ID No. 44), Malate dehydrogenase mitochondrial precursor (SEQ ID No. 45), Voltage-dependent anion selective Channel protein 1 (SEQ ID No. 46), glyceraldehydes-3-phosphate dehydrogenase (SEQ ID No. 47), uracil DNA glycosylase (SEQ ID No. 48), aging-associated- associated 9 protein (SEQ ID No. 49), Nipsnap homolog 3A (SEQ ID No. 50), peroxiredoxin 2 isoform b (SEQ ID No. 51), thiol- specific antioxidant protein (SEQ ID No. 52), enhancer protein (SEQ ID No. 53), Chromosome 17 open reading frame 25 (SEQ ID No. 54), hypothetical protein LOC51031 (SEQ ID No. 55), CGI- 150 protein (SEQ ID No. 56), Gelsolin isoform a (SEQ ID No. 57), Gelsolin precursor (SEQ ID No. 58), ATP-specific succinyl-CoA synthetase beta subunit (SEQ ID No. 59), TAR DNA binding protein (SEQ ID No. 60), 2, 4-dienoyl-CoA reductase mitochondrial precursor (SEQ ID No. 61), MDH2 (SEQ ID No. 62), heat shock protein beta-1 (SEQ ID No. 63), mitochondrial malate dehydrogenase precursor MDH-2 (SEQ ID No. 64), prostate and colon associated protein (SEQ ID No. 65) , secretagogin (SEQ ID No. 66), TPD 52 (SEQ ID No. 67), tumor protein D52 (SEQ ID No. 68), N8 protein long isoform (Fragment) variant (SEQ ID No. 69), tumor protein D52 isoform 2 (SEQ ID No. 70), triosephosphate isomerase 1 (SEQ ID No. 71) oder jeweils Fragmente oder Teilpeptide davon an einem zu untersuchenden Patienten erfolgt (nachstehend erfindungsgemäßes Verfahren) .Stratifin (14-3-3 sigma) (SEQ ID No. 29), Coactosin-like 1 (SEQ ID No. 30), Chaperonin heat shock 6OkD protein 1 (SEQ ID No. 31), Transgelin 2 (SEQ ID No. 1). 32), aldehyde dehydrogenase 1 (SEQ ID No. 33), sarcomeric tropomyosin kappa (SEQ ID No. 34), annexin A3 (SEQ ID No. 35), delta-globin (SEQ ID No. 36), serum albumin (SEQ ID No. 37), protein PP4-X (annexin A4) (SEQ ID No. 38), crystallin (SEQ ID No. 39), myosin regulatory light chain MRCL3 (SEQ ID No. 40) or group b.) Aldehyde dehydrogenase 1 (SEQ ID No. 41), aldehyde dehydrogenase IA1 (SEQ ID No. 42), T-complex protein 1 subunit beta (SEQ ID No. 43), apolipoprotein A4 (SEQ ID No. 44), malate dehydrogenase mitochondrial precursor ( SEQ ID No. 45), voltage-dependent anion selective channel protein 1 (SEQ ID No. 46), glyceraldehyde-3-phosphate dehydrogenase (SEQ ID No. 47), uracil DNA glycosylase (SEQ ID No. 48), aging associated-associated 9 protein (SEQ ID No. 49), Nipsnap homolog 3A (SEQ ID No. 50), peroxiredoxin 2 isoform b (SEQ ID No. 51), thiol specific antioxidant protein (SEQ ID No. 52), enhancer protein (SEQ ID No. 53), chromosomes 17 (SEQ ID No. 54), hypothetical protein LOC51031 (SEQ ID No. 55), CGI-150 protein (SEQ ID No. 56), gelsolin isoform a (SEQ ID No. 57), gelsolin precursor (SEQ ID No. 58), ATP-specific succinyl-CoA synthetase beta subunit (SEQ ID No. 59), TAR DNA binding protein (SEQ ID No. 60), 2, 4-dienoyl-CoA reductase mitochondrial precursor (SEQ ID No. 61), MDH2 (SEQ ID No. 62), heat shock protein beta-1 (SEQ ID No. 63), mitochondrial malate dehydrogenase precursor MDH-2 (SEQ ID No. 64), prostate and colon associated protein (SEQ ID No. 65), secretagogin (SEQ ID No. 66), TPD 52 (SEQ ID No. 67), tumor protein D52 ( SEQ ID No. 68), N8 protein long isoform (fragment) variant (SEQ ID No. 69), tumor protein D52 isoform 2 (SEQ ID No. 70), triosephosphate isomerase 1 (SEQ ID No. 71) or in each case fragments or Partial peptides thereof on a patient to be examined (hereinafter erfindu appropriate method).
Die erfindungsgemäßen Proteine konnten mittels einer differentiellen Proteomanalyse von krankem pankreatischen duktalen Gewebe - fünf Progressionsstadien - im Vergleich zu normalem (gesundem) pankreatischen duktalen Gewebe als potentielle Biomarker identifiziert werden. Hierzu wurden von erkrankten Patienten entsprechende Gewebeproben entnommen. Die Proben wurden in einem Handhomogenisator mit Lysispuffer homogenisiert und von DNA und sonstigem Zellmaterial befreit, um ein Proteinkonzentrat zu erhalten. Die Proteine wurden mit einem Farbstoff gelabelt und einer 2D-Gelelektrophorese unterworfen mit einer isoelektrischen Fokussierung in der ersten und einer SDS-Gelelektrophorese in der zweiten Dimension.The proteins according to the invention could be identified as potential biomarkers by means of a differential proteome analysis of diseased pancreatic ductal tissue - five progression stages - compared to normal (healthy) pancreatic ductal tissue. Corresponding tissue samples were taken from diseased patients. The samples were homogenized in a hand homogenizer with lysis buffer and freed of DNA and other cell material to obtain a protein concentrate. The proteins were with labeled with a dye and subjected to 2D gel electrophoresis with isoelectric focusing in the first and SDS gel electrophoresis in the second dimension.
Die differentielle Darstellung (krank/gesund) ist in den Tabellen (1 bis 3), Beispielen und Abbildungen dargelegt und zeigen unterschiedliche charakteristische Expression (hoch- (up-) und runter- (down-) reguliert, ausgelesen anhand der Spots) .The differential representation (ill / healthy) is presented in Tables (1 to 3), Examples and Figures, and shows different characteristic expression (up-regulated and down-regulated, read by the spots).
Die weitere Auswertung erfolgte mit Hilfe von LC-ESI-MS ( /MS) (Flüssigchomatographie-Elektrosprayionisations- Massenspektrometrie) . Dabei wurden zunächst die Proteine im Gel, in dem die Proben zuvor aufgetrennt wurden, mit Hilfe von Trypsin in einzelne Peptidfragmente zerlegt. Diese wurden mit Hilfe von Reversed-Phase HPLC voneinander separiert und massenspektrometrisch untersucht, um die einzelnen Proteine zu identifizieren. Selbstverständlich können hierbei auch andere geeignete massenspektrometrische Verfahren angewandt werden, beispielsweise MALDI-TOF-MS.Further evaluation was carried out using LC-ESI-MS (/ MS) (liquid chromatography-electrospray ionization mass spectrometry). Initially, the proteins in the gel, in which the samples were previously separated, were broken down into individual peptide fragments with the aid of trypsin. These were separated from each other with the aid of reversed-phase HPLC and mass spectrometrically analyzed to identify the individual proteins. Of course, other suitable mass spectrometric methods can also be used here, for example MALDI-TOF-MS.
Die erfindungsgemäßen Proteine (Biomarker) erweisen sich wie folgt:The proteins according to the invention (biomarkers) are as follows:
Tabelle 1: Gruppe a.) für PanINTable 1: group a.) For PanIN
2206 Beta tropomyosin 21 gι|6573280 48 344 47 299 280 gi|4022610 2206 Beta tropomyosin 21 gι | 6573280 48 344 47 299 280 gi | 4022610
1962 ACTG1 protein 29 57 395 54 294 110 11962 ACTG1 protein 29 57 395 54 294 110 1
2925 Thioredoxin delta 3 28 22 31 gι|3153859 5 168 57 93 2622925 thioredoxin delta 3 28 22 31 gι | 3153859 5 168 57 93 262
B Chain B, TπosephosphateB Chain B, Tπose Phosphates
2330 gι|999893 65 327 77 386 91 Isomerase gι|16306972330 gι | 999893 65 327 77 386 91 Isomerase gι | 1630697
2126 Annexiπ A2 23 57 360 55 298 191 82126 Annexiπ A2 23 57 360 55 298 191 8
TPM4-ALKfusιon gι|1044138TPM4-ALKfusιon gι | 1044138
2154 -21 -34 47 355 48 275 498 oncoprotein type 2 6 gι|6220534 2639 Peptidylprolyl isomerase A -23 73 271 79 114 419 92154 -21 -34 47 355 48 275 498 oncoprotein type 2 6 gι | 6220534 2639 peptidylprolyl isomerase A -23 73 271 79 114 419 9
Smooth muscle mysom lightSmooth muscle mysom light
2765 -41 gι|189022 44 225 47 129 216 chain2765 -41 gι | 189022 44 225 47 129 216 chain
821 Vimentin -22 gι|7576229 53 626 51 537 410821 Vimentin -22 gι | 7576229 53 626 51 537 410
Late up-Λtown- regulated spotsLate-uptown-regulated spots
999 Desmin 24 30 gι|1408188 53 583 52 535 242 gι|1599047999 Desmin 24 30 gι | 1408188 53 583 52 535 242 gι | 1599047
1243 Major vault protein (MVP) 51 59 536 53 993 31 81243 Major vault protein (MVP) 51 59 536 53 993 31 8
Heterogeneous πuclear gi|1404307Heterogeneous πuclear gi | 1404307
1836 30 81 426 92 387 91 πbonucleoprotein A1 01836 30 81 426 92 387 91 πbonucleoprotein A1 0
3022 S100A10 28 gι|4388970 72 142 75 111 17 gι|24210503022 S100A10 28 gι | 4388970 72 142 75 111 17 gι | 2421050
2697 EF1a-lιke protein 210 141 79 254 72 464 49 82697 EF1a-link protein 210 141 79 254 72 464 49 8
Regulatory myosin light gι|3333806Regulatory myosin light gι | 3333806
2711 -19 -21 47 249 46 199 174 chain long version 2 Tropomyosin 1 alpha chain gι|6325289 1926 -21 -26 47 403 46 327 225 isoform 3 62711 -19 -21 47 249 46 199 174 chain long version 2 Tropomyosin 1 alpha chain g | 6325289 1926 -21 -26 47 403 46 327 225 isoform 3 6
823 Vimentin -27 -32 gι|7576229 52 624 51 537 485823 Vimentin -27 -32 gι | 7576229 52 624 51 537 485
Tropomyosin 2 (beta) gι|5585970Tropomyosin 2 (beta) g | | 5585970
1738 -30 -26 43 300 46 330 676 isoform 2 31738 -30 -26 43 300 46 330 676 isoform 2 3
Myosin regulatory light chain gι|6289669Myosin regulatory light chain gι | 6289669
2649 -18 48 266 45 198 175 MRCL3 72649 -18 48 266 45 198 175 MRCL3 7
2946 Alpha-2-globιn -35 gι|1335076 79 164 87 151 397 gι|12803952946 alpha-2-globin-35 gm | 1335076 79 164 87 151 397 gι | 1280395
2085 Tropomyosin 4 -16 46 367 47 286 403 92085 tropomyosin 4 -16 46 367 47 286 403 9
2217 A25074 vimentin -69 gι|7576229 71 344 51 537 245 gι|62205322217 A25074 vimentin -69 gi | 7576229 71 344 51 537 245 gι | 6220532
2547 Transgelm -27 -18 32 75 285 89 226 647 62547 Transgelm -27 -18 32 75 285 89 226 647 6
Constant up-Jdown- regulated spots gι|6065572Constant up-Jdown- regulated spots gι | 6065572
738 Keratin 7 17 19 40 55 644 54 514 441 3 gι|1527750738 Keratin 7 17 19 40 55 644 54 514 441 3 gι | 1527750
1347 ACTB protein 31 42 32 24 59 515 56 402 200 31347 ACTB protein 31 42 32 24 59 515 56 402 200 3
2921 Thioredoxin delta 3 19 25 20 14 gι|3153859 50 169 57 93 369 gι|15277502921 thioredoxin delta 3 19 25 20 14 gι | 3153859 50 169 57 93 369 gι | 1527750
1276 ACTB protein 39 18 48 59 526 56 402 178 3 gι|33286421276 ACTB protein 39 18 48 59 526 56 402 178 3 gι | 3328642
1340 M2-type pyruvate kinase 23 31 60 515 87 580 87 21340 M2-type pyruvate kinase 23 31 60 515 87 580 87 2
Actin related protein 2/3 gι|5620452Actin related protein 2/3 gι | 5620452
2781 20 19 21 19 59 218 56 166 344 complex subunit 5 4 Anterior gradient 2 homolog gι|3718313 2793 60 113 86 38 81 215 95 200 143 (AGR 2) 62781 20 19 21 19 59 218 56 166 344 complex subunit 5 4 Anterior gradient 2 homologous gi | 3718313 2793 60 113 86 38 81 215 95 200 143 (AGR 2) 6
Anterior gradient 2 homolog gι|3718313Anterior gradient 2 homologous gι | 3718313
2799 33 58 57 54 67 81 211 95 200 40 (AGR 2) 6 gι|16306972799 33 58 57 54 67 81 211 95 200 40 (AGR 2) 6 gι | 1630697
2437 Annexin A2 33 101 69 37 33 55 305 77 386 112 82437 Annexin A2 33 101 69 37 33 55 305 77 386 112 8
2192 Stratifin (14-3-3 sigma) 29 20 40 gι|7981260 46 347 47 278 351 gi|27695622192 Stratifin (14-3-3 sigma) 29 20 40 gι | 7981260 46 347 47 278 351 gi | 2769562
2843 Coactosm-like 1 24 21 14 1 55 193 54 160 3102843 Coactosm-like 1 24 21 14 1 55 193 54 160 310
Chaperonin, heat shockChaperonin, heat shock
734 19 28 gι|6996447 54 645 57 611 222 6OkD protein 1 gι|5596037 2608 Transgelm 2 26 36 66 276 84 224 151 3734 19 28 gi | 6996447 54 645 57 611 222 6OkD protein 1 gι | 5596037 2608 Transgelm 2 26 36 66 276 84 224 151 3
791 Aldehyde dehydrogenase 1 -22 -33 -27 -30 -51 gι|2183299 64 634 63 548 78791 aldehyde dehydrogenase 1 -22 -33 -27 -30 -51 gι | 2183299 64 634 63 548 78
819 Vimentin -22 -35 -39 -56 -21 gι|7576229 51 625 51 537 345819 Vimentin -22 -35 -39 -56 -21 gι | 7576229 51 625 51 537 345
820 Vimentin -19 -21 -32 -49 gι|7576229 52 625 51 537 410820 Vimentin -19 -21 -32 -49 gι | 7576229 52 625 51 537 410
Sarcomeπc tropomyosin gι|4966001Sarcoma tropomyosin gι | 4966001
1828 -29 -32 -30 -24 54 425 45 326 461 kappa, TPM1-kappa 2 1852 gι|12654111828 -29 -32 -30 -24 54 425 45 326 461 kappa, TPM1-kappa 2 1852 gι | 1265411
Annexin A3 -17 -27 -43 58 420 56 364 127 5 gι|1846210Annexin A3 -17 -27 -43 58 420 56 364 127 5 gι | 1846210
2879 Delta-globin -31 -82 -31 76 181 80 161 204 923 Serum albumin -2 1 -2 1 -24 gι|28592 57 602 6 1 694 7 1 1811 Protein PP4-X (Annexin A4) -55 -4 8 -76 gι|189617 59 428 56 36 1 293 2022 Crystailin -80 -14 5 -103 gι|28634 6 1 38 3 55 124 2882879 Delta globin -31 -82 -31 76 181 80 161 204 923 Serum albumin -2 1 -2 1 -24 gι | 28592 57 602 6 1 694 7 1 1811 Protein PP4-X (Annexin A4) -55 -4 8 -76 gι | 189617 59 428 56 36 1 293 2022 Crystailin -80 -14 5 -103 gι | 28634 6 1 38 3 55 124 288
Myosm regulatory light chain 2660 -1 8 -2 0 -1 8 gi|2605594 4 9 26 3 46 197 174 MRCL3Myosm regulatory light chain 2660 -1 8 -2 0 -1 8 gi | 2605594 4 9 26 3 46 197 174 MRCL3
NCBI: National Centre for Biotechnology InformationNCBI: National Center for Biotechnology Information
Tabelle 2: Teil der Gruppe b.) für hochregulierte Proteine (Biomarker) in malignen Proben von Tumoren im PankreasTable 2: Part of group b.) For upregulated proteins (biomarkers) in malignant samples of tumors in the pancreas
Tabelle 3: Teil der Gruppe b.) für hochregulierte Proteine (Biomarker) in benignen Proben von Tumoren im Pankreas Table 3: Part of group b.) For upregulated proteins (biomarkers) in benign samples of tumors in the pancreas
Daher betrifft die Erfindung auch solche Aminosäure-Sequenzen (Polypeptide, Proteine), die eine Sequenzidentität oder Homologie von 70% und mehr, vorzugsweise von 80% und mehr, besonders bevorzugt von 90-95% und mehr mit SEQ ID No. 1 bis SEQ ID No. 71 aufweisen. Ebenfalls mit eingeschlossen sind ebenfalls solche analoge Aminosäure-Sequenzen, die aufgrund des Austausches von einer oder mehreren Aminosäure (n) in diesen Sequenzen, dennoch die gewünschte Funktion eines Biomarkers zur Diagnose von Pankreaskrebs gewährleisten. Erfindungsgemäß ausdrücklich eingeschlossen sind insbesondere Teilpeptide oder Fragmente von SEQ ID No. 1 bis SEQ ID No. 71,Therefore, the invention also relates to such amino acid sequences (polypeptides, proteins) having a sequence identity or homology of 70% and more, preferably 80% and more, more preferably 90-95% and more with SEQ ID NO. 1 to SEQ ID no. 71 have. Also included are those analog amino acid sequences which, because of the replacement of one or more amino acids in these sequences, still provide the desired function of a biomarker for the diagnosis of pancreatic cancer. In particular, partial peptides or fragments of SEQ ID no. 1 to SEQ ID no. 71,
In einer weiteren bevorzugten Ausfuhrungsform sindIn a further preferred embodiment
Kombinationen (Unterkombination aus der obigen Gesamtheit aller erfindungsgemäßen Biomarker) der erfindungsgemäßen Biomarker zur Diagnose vorteilhaft. In der Gruppe a.) sind insbesondere bevorzugt solche Kombination die zumindest Stratifin (14-3-3 sigma) (SEQ ID No. 29) und / oder Vimentin (SEQ ID No. 2) und / oder Major vault protein 1 (SEQ ID No. 13) und / oder Anterior gradient 2 homolog (AGR 2) (SEQ ID No.28), und / oder SlOOAlO (SEQ ID No. 15) und / oder EFIa- like protein (SEQ ID No. 16) und / oder Annexin A2 (SEQ ID No. 8) und / oder Annexin A4 (SEQ ID No. 38) enthalten.Combinations (sub-combination of the above totality of all biomarkers according to the invention) of the biomarkers according to the invention are advantageous for the purpose of diagnosis. In group a.), Those combinations which are particularly preferred are at least stratifin (14-3-3 sigma) (SEQ ID No. 29) and / or vimentin (SEQ ID No. 2) and / or major vault protein 1 (SEQ ID No. 13) and / or anterior gradient 2 homologous (AGR 2) (SEQ ID No.28), and / or SlOOAlO (SEQ ID No. 15 ) and / or EFIA-like protein (SEQ ID No. 16) and / or Annexin A2 (SEQ ID No. 8) and / or Annexin A4 (SEQ ID No. 38).
Der Begriff „Pankreaskrebs" umfasst erfindungsgemäß ebenfalls dessen Vorläufer- und/oder Begleiterkrankungen, insbesondere PDAC (Pancreatic ductal adenocarcinoma) , PanIN (Pankreatische intraepitheliale Neoplasien) , Pankreasläsionen, CP (Chronische Pankreatitis) , einschließlich endokriner Tumoren der Pankreas, insbesondere pankreatische Tumore und pankreatische Neoplasmen.According to the invention, the term "pancreatic cancer" also encompasses its precursor and / or comorbidities, in particular PDAC (pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasms), pancreatic lesions, CP (chronic pancreatitis), including endocrine tumors of the pancreas, in particular pancreatic tumors and pancreatic tumors neoplasms.
Die Erfindung betrifft ebenfalls die Identifizierung von Patienten mit erhöhtem Risiko oder/und einer ungünstigen Prognose eines Pankreaskrebs, insbesondere bei symptomatischen und / oder asymptomatischen Patienten.The invention also relates to the identification of patients at increased risk and / or unfavorable prognosis of pancreatic cancer, especially in symptomatic and / or asymptomatic patients.
Das erfindungsgemäße Verfahren ermöglicht daher klinischeThe inventive method therefore allows clinical
Entscheidungen, die zu einem schnellen Therapieerfolg und zur Vermeidung von Todesfällen führen. Solche klinische Entscheidungen umfassen ebenfalls weiterführende Behandlung mittels Arzneimitteln zur Behandlung oder Therapie von Pankreaskrebs.Decisions that lead to rapid therapy success and avoidance of death. Such clinical decisions also include continuing treatment with drugs for the treatment or therapy of pancreatic cancer.
Daher betrifft die Erfindung ebenfalls ein Verfahren zur Diagnose von Patienten mit Pankreaskrebs zur Durchführung von klinischen Entscheidungen, wie weiterführende Behandlung und Therapie mittels Arzneimitteln.Therefore, the invention also relates to a method for diagnosing patients with pancreatic cancer for performing clinical decisions, such as advanced treatment and drug therapy.
In einer weiteren bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens erfolgt die Diagnose zurIn a further preferred embodiment of the method according to the invention, the diagnosis for
Prognose, zur differentialdiagnostischen Früherkennung und Erkennung, zur Beurteilung des Schweregrades und zur therapiebegleitenden Verlaufsbeurteilung.Prognosis, for the differential diagnostic early detection and detection, for the evaluation of the severity grade and the therapy-accompanying course assessment.
In einer weiteren bevorzugten Ausführungsform betrifft die Erfindung ein Verfahren zur Diagnostik zur Früh- oder Differentialdiagnose oder Prognose von Pankreaskrebs oder einer Vorläuferkrankheit, wobei eine Bestimmung der Biomarker an einem zu untersuchenden Patienten durchgeführt wird.In a further preferred embodiment, the invention relates to a method for diagnosis for the early or differential diagnosis or prognosis of pancreatic cancer or a precursor disease, wherein a determination of the biomarker is carried out on a patient to be examined.
In einer Ausführungsform des erfindungsgemäßen Verfahrens wird dem zu untersuchenden Patienten Gewebeproben oder Körperflüssigkeit (Blut, Plasma, Pankreassekret ) entnommen, und die Diagnose erfolgt in vitro/ex vivo, d.h. außerhalb des menschlichen oder tierischen Körpers. Aufgrund der Bestimmung der erfindungsgemäßen Marker wird eine hohe Signifikanz für das Pankreaskrebs oder eine Vorläufer und / oder Begleiterkrankung erzielt und anhand der vorhandenen Menge oder deren Veränderung (Levelierung: Erhöhung / Erniedrigung) in mindestens einer Patientenprobe kann die Diagnose erfolgen.In one embodiment of the method according to the invention, tissue samples or body fluid (blood, plasma, pancreatic secretions) are taken from the patient to be examined, and the diagnosis is made in vitro / ex vivo, i. outside the human or animal body. Due to the determination of the markers according to the invention, a high significance for the pancreatic cancer or a precursor and / or concomitant disease is achieved, and the diagnosis can be made on the basis of the existing amount or its change (leveling: increase / decrease) in at least one patient sample.
In einer weiteren Ausführungsform der Erfindung kann das erfindungsgemäße Verfahren im Rahmen einer in-vitro Diagnose mittels parallelen oder simultanen Bestimmungen der Marker durchgeführt werden (z.B. Multititerplatten mit 96 und mehr Kavitäten) , wobei die Bestimmungen an mindestens einer Patientenprobe durchgeführt werden.In a further embodiment of the invention, the method according to the invention may be part of an in-vitro diagnosis by means of parallel or simultaneous determinations of the markers (eg multi-well plates with 96 or more wells), the determinations being carried out on at least one patient sample.
In einer weiteren Ausführungsform der Erfindung kann das erfindungsgemäße Verfahren mit Hilfe einer 2D-Elektrophorese erfolgen, wobei in der ersten Dimension eine isoelektrische Fokussierung, in der zweiten Dimension eine Gelelektrophorese durchgeführt wird (im weitesten Sinne ist hierzu die Proteomforschung („Proteomics") anzuwenden).In a further embodiment of the invention, the method according to the invention can be carried out by means of 2D electrophoresis, in the first dimension an isoelectric focusing, in the second dimension a gel electrophoresis is carried out (in the broadest sense proteomics is to be used for this purpose) ,
In einer weiteren Ausführungsform kann das erfindungsgemäße Verfahren und dessen Bestimmungen mittels einem Schnelltest durchgeführt werden (z.B. lateral-flow Test), sei es in Einzel- oder Multiparameterbestimmung.In a further embodiment, the method according to the invention and its determinations can be carried out by means of a rapid test (for example, lateral-flow test), whether in single or multiparameter determination.
In einer weiteren Ausführungsform kann das erfindungsgemäße Verfahren in-vivo durchgeführt werden, wobei die erfindungsgemäßen Biomarker mit einer Sonde, insbesondere ein Antikörper, die ein Kontrastmittel aufweisen markiert und mit einem in der Bildgebung geeignetem Detektor („Molecular Imaging") nachgewiesen werden (Ralph Weissleder, Molecular Imaging in Cancer, Science, Vol. 312, 1168 (2006)).In a further embodiment, the method according to the invention can be carried out in vivo, wherein the biomarkers according to the invention are labeled with a probe, in particular an antibody which has a contrast agent and detected with a detector which is suitable for imaging ("Molecular Imaging") (Ralph Weissleder , Molecular Imaging in Cancer, Science, Vol. 312, 1168 (2006)).
Ferner betrifft die Erfindung die Verwendung der erfindungsgemäßen Biomarker zur Diagnose und / oder Prognose und/oder zur Früh- oder Differentialdiagnose von Pankreaskrebs oder dessen Vorläufer- und / oder Begleiterkrankungen.Furthermore, the invention relates to the use of the biomarkers according to the invention for the diagnosis and / or prognosis and / or for the early or differential diagnosis of pancreatic cancer or its precursor and / or comorbidities.
Eine weitere Aufgabe ist die Bereitstellung einer entsprechenden diagnostischen Vorrichtung zur Durchführung der erfindungsgemäßen Verfahren.Another object is to provide a corresponding diagnostic device for carrying out the method according to the invention.
Im Rahmen dieser Erfindung wird unter einer solchen diagnostischen Vorrichtung, insbesondere ein Array oder Assay verstanden (z.B. Immunoassay, ELISA etc.), insbesondere ein Proteinbiochip (US6346413B1. US20050014292) im weitesten Sinne eine Vorrichtung zur Durchführung der erfindungsgemäßen Verfahren.In the context of this invention, such a diagnostic device, in particular an array or assay (for example immunoassay, ELISA, etc.), in particular a protein biochip (US Pat.
Die Erfindung betrifft zudem ein Kit zur Durchführung der erfindungsgemäßen Verfahren, insbesondere enthaltend Nachweisreagenzien und weitere Hilfsmittel. Solche Nachweisreagenzien umfassen z.B. Antikörper etc.The invention additionally relates to a kit for carrying out the methods according to the invention, in particular containing detection reagents and further auxiliaries. Such detection reagents include e.g. Antibodies etc.
Der Nachweis und die Quantifizierung der erfindungsgemäßen Biomarker kann ebenfalls mit Hilfe weiterer, dem Fachmann geläufiger Protein-Diagnoseverfahren durchgeführt werden, insbesondere unter Verwendung radioaktiv oder fluoreszenzmarkierter Antikörper. Zu nennen sind hier insbesondere dazu geeignete bioanalytische Verfahren, wie zum Beispiel Immunhistochemie, Antikörperarrays, Luminex, ELISA, Immunfluoreszenz, Radioimmunoassays sowie weiteren geeigneten bioanalytischen Verfahren, wie zum Beispiel rnassenspektrometrischen Verfahren, z.B. MRM (Multi reaction monitoring) oder AQUA (absolute Quantification) , mit deren Hilfe die Biomarker quantitativ gemessen werden können, durchgeführt werden.The detection and quantification of the biomarkers according to the invention can likewise be carried out with the aid of further protein diagnostic methods familiar to the person skilled in the art, in particular using radioactively or fluorescently labeled antibodies. These include, in particular, suitable bioanalytical methods, such as, for example, immunohistochemistry, antibody arrays, Luminex, ELISA, immunofluorescence, radioimmunoassays and other suitable bioanalytical methods, such as, for example, NMR spectroscopic methods, eg MRM (multi-reaction monitoring) or AQUA (absolute quantification), with the help of which the biomarkers can be measured quantitatively.
Nachfolgende Beispiele und Abbildungen dienen zur näherenThe following examples and illustrations are for closer
Erläuterung der Erfindung, jedoch ohne die Erfindung auf diese Beispiele und Abbildungen zu beschränken.Explanation of the invention, but without limiting the invention to these examples and illustrations.
Beispiele und Abbildungen:Examples and illustrations:
MikrodisSektionmicrodissection
Die Gewebeproben wurden von Patienten gewonnen, die in der Klinik für Allgemeine Chirurgie des Universitätsklinikums Schleswig-Holstein, Campus Kiel (Deutschland) , operiert wurden. Tumorgewebe von duktalen Pankreaskarzinomen und peritumoralem Parenchym wurden unverzüglich postoperativ bei - 800C schockgefroren und gelagert. Für die Darstellung normaler Pankreasgänge und PanINs wurden 5 μm dicke Gefrierschnitte des peritumoralen Pankreasparenchyms angefertigt, diese kurz in Ethanol fixiert (Merck, Darmstadt, Germany) , mit Hämatoxylin- Eosin angefärbt und anschließend von einem Pathologen begutachtet. Die PanINs wurden nach den anerkannten Kriterien (Hruban, R. H., N. V. Adsay, et al. (2001). "Pancreatic intraepithelial neoplasia: a new nomenclature and Classification System for pancreatic duct lesions." Am J Surg Pathol 25(5): 579-86) klassifiziert. Gewebeblöcke, die die erforderten PanIN-Läsionen enthielten wurden in Serien geschnitten (10 μm) . Für die 2-DE wurden die Gewebeschnitte nur mit Hämatoxilin gefärbt und umgehend bei -200C gelagert. Die PanIN-Läsionen wurden unter einem Mikroskop mit Hilfe einer sterilen Injektionsnadel (size 0.65x25 mm, Fa. Braun, Melsungen, Germany) mikrodisseziert (BH2, Olympus, Wetzlar, Germany) . Vornehmlich wurden mittelgroße interlobuläre Gänge ausgewählt, um eine Kontamination mit periduktalem mesenchymalem und Azinusgewebe zu vermeiden. Die mikrodissezierten Zellen wurden in 100 μl Lysepuffer (TrisCl 30 mM; thiourea 2 M; urea 7 M; CHAPS 4%, pH 8.0) aufgenommen und im Ultraschallbad unmittelbar nach der Mikrodissektion auf Eis gelegt (6 x 10 s pulses; ultrasonic cleaner, VWR Darmstadt, Darmstadt) .Tissue samples were obtained from patients who underwent surgery at the Department of General Surgery of the University Hospital Schleswig-Holstein, Campus Kiel (Germany). Tumor tissue of ductal pancreatic cancer and peritumoral parenchyma were immediately postoperatively in - 80 0 C flash-frozen and stored. For the preparation of normal pancreatic ducts and PanINs, 5 μm-thick frozen sections of the peritumoral pancreatic sparchyma were prepared, briefly fixed in ethanol (Merck, Darmstadt, Germany), stained with hematoxylin-eosin and subsequently examined by a pathologist. The PanINs were prepared according to the accepted criteria (Hruban, RH, NV Adsay, et al. (2001). "Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions." Am J Surg Pathol 25 (5): 579- 86). Tissue blocks containing the required PanIN lesions were cut in series (10 μm). For the 2-DE, the tissue sections were stained with hematoxylin and stored at -20 0 C immediately. The PanIN lesions were microdissected under a microscope using a sterile injection needle (size 0.65x25 mm, Braun, Melsungen, Germany) (BH2, Olympus, Wetzlar, Germany). Primarily, medium-sized interlobular ducts were selected to avoid contamination with periductal mesenchymal and acinar tissue. The microdissected cells were taken up in 100 μl of lysis buffer (TrisCl 30 mM, thiourea 2 M urea 7 M, CHAPS 4%, pH 8.0) and placed on ice in an ultrasonic bath immediately after the microdissection (6 × 10 sec pulses, ultrasonic cleaner, VWR Darmstadt, Darmstadt).
Herstellung des Referenzproteoms Für die Generierung des Referenzproteoms 100 mg Gewebes vomPreparation of the reference proteome For the generation of the reference proteome 100 mg tissue from
Adenokarzinom wurden in 148 μl Lysepuffer (TrisCl 30 mM; thiourea 2 M; urea 7 M; CHAPS 4%, pH 8) homogenisiert.Adenocarcinoma were homogenized in 148 μl lysis buffer (TrisCl 30 mM, thiourea 2 M, urea 7 M, CHAPS 4%, pH 8).
Anschließend wurden die Proben sonifiziert (6 x 10 Pulse, aufSubsequently, the samples were sonicated (6 x 10 pulses, on
Eis) und zentrifugiert (12.000 x g für 5 min). Die Proteinbestimmung wurde mittels eines Proteinassays durchgeführt (Bio-Rad) .Ice) and centrifuged (12,000 x g for 5 min). The protein determination was carried out by means of a protein assay (Bio-Rad).
ProteinlabellingProtein Labeling
Die Proben mit jeweils 1000 mikrodissezierter Zellen in 100 μl Lysepuffer wurden durch Zugabe von 2 nmol TCEP reduziert und anschließend bei 370C für Ih im dunkeln inkubiert. Die Sättigungsfarbstoffe, Cy3 und Cy5 wurden zunächst mit DMF (2 nmol/μl; Sigma) verdünnt und dann jeweils 4 nmol zu den reduzierten Proben zugefügt. Die Inkubation wurde bei 370C für 30 min im dunkeln durchgeführt. Um die Labellingreaktion zu beenden, 10 μl DTT (1.08 g/ml; Bio-Rad) wurden zugefügt. Anschließend wurden die Proben mit jeweils 10 μl Ampholine 2-4 (GE Healthcare) versehen.The samples, each containing 1000 microdissected cells in 100 .mu.l lysis buffer were reduced by addition of 2 nmol TCEP and then incubated at 37 0 C for Ih in the dark. The saturation dyes, Cy3 and Cy5, were first diluted with DMF (2 nmol / μl, Sigma) and then 4 nmol each to the added to reduced samples. The incubation was carried out at 37 ° C. for 30 minutes in the dark. To terminate the labeling reaction, 10 μl of DTT (1.08 g / ml, Bio-Rad) were added. Subsequently, the samples were each provided with 10 μl of Ampholine 2-4 (GE Healthcare).
Zweidimensionale GelelektrophoreseTwo-dimensional gel electrophoresis
Für die Separation der Proteine in der ersten Dimension wurde die Trägerampholyt-basierte IEF (Röhrchengele 20 cm x 1.5 mm) nach Klose und Kobalz eingesetzt (Klose, J. and U. Kobalz (1995). "Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome." Electrophoresis 16(6): 1034-59). Nach Ablauf eines 21, 25-stündigen Spannungsprogramms wurden die ausgestoßenen Röhrchengele in Äquillibrierungspuffer (125 mMFor the separation of the proteins in the first dimension, the carrier-ampholyte-based IEF (tube gels 20 cm x 1.5 mm) according to Klose and Kobalz was used (Klose, J. and U. Kobalz (1995) "Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. "Electrophoresis 16 (6): 1034-59). At the end of a 21, 25 hour program of stress, the ejected tube gels in equilibration buffer (125 mM
Tris, 40% (w/v) Glycerin, 3% (w/v) SDS, 65 mM DTT, pH 6.8) für 10 min inkubiert. Die zweite Dimension wurde in einem Desaphor VA 300 System mit Polyacrylamidgelen (15.2% Acrylamid (total), 1.3% Bisacrylamid) durchgeführt (Klose und Kobalz 1995 (supra) ) . Die Röhrchengele wurden auf dieTris, 40% (w / v) glycerol, 3% (w / v) SDS, 65 mM DTT, pH 6.8) for 10 min. The second dimension was performed in a Desaphor VA 300 system with polyacrylamide gels (15.2% acrylamide (total), 1.3% bisacrylamide) (Klose and Kobalt 1995 (supra)). The tube gels were on the
Polyacrylamidgele (20 cm x 30 cm x 1.5 mm) appliziert und mit 1%-iger Agarose, die 0.01% (w/v) Bromophenolblau Farbstoff enthielt (Riedel deHaen, Seelze, Deutschland) fixiert. Das für die Proteinidentifizierung verwendete Gelsystem (IEF: 20 cm x 1.5 mm, SDS-PAGE: 20 cm x 30 cm x 1.5 mm) wurde unter den gleichen Bedingungen prozessiert. Hierbei wurde das MS- kompatible Silberfärbungsprotokoll nach Nesterenko et al. verwendet (Nesterenko, M. V., M. Tilley, et al. (1994). "A simple modification of Blum's silver stain method allows for 30 minute detection of proteins in Polyacrylamide gels." J Biochem Biophys Methods 28(3): 239-42).Polyacrylamide gels (20 cm x 30 cm x 1.5 mm) and fixed with 1% agarose containing 0.01% (w / v) bromophenol blue dye (Riedel de Haen, Seelze, Germany). The gel system used for protein identification (IEF: 20 cm x 1.5 mm, SDS-PAGE: 20 cm x 30 cm x 1.5 mm) was processed under the same conditions. Here, the MS-compatible silver staining protocol according to Nesterenko et al. (Nesterenko, MV, M. Tilley, et al. (1994). "A simple modification of Blum's silver stain method allows for 30 minute detection of proteins in polyacrylamide gels. "J Biochem Biophys Methods 28 (3): 239-42).
Bildakquisition und -analyseImage acquisition and analysis
Für die Bildakquisition mit Typhoon 9400 Fluoreszenzscanner (Amersham Biosciences/GE Healthcare) verblieben die Gele zwischen den Glasplatten. Die Anregungswellenlänge und die Emissionsfilter wurden spezifisch für die jeweiligen Fluoreszenzfarbstoffe gemäß dem Handbuch ausgewählt. Vor der Bildanalyse mit der DeCyder Software (Amersham Biosciences/GE Healthcare) wurden die Bilder mit der ImageQuant TM Software (Amersham Biosciences/GE Healthcare) zu Recht geschnitten. Die intra-gel Spotdetektion und Quantifizierung wurde mithilfe des Differential In-gel Analysis (DIA) Modus der DeCyder Software durchgeführt. Die geschätzte Spotanzahl wurde auf 3000 gesetzt. Als Ausschlussfilter wurde der Anstieg der Spotflanke (slope) größer als 1,6 gewählt. Für die Bestimmung des Referenzproteoms wurden die Matchingraten zwischen mikrodissektierten PDAC Zellen, einem pankreatischen Zelllinien-Pool und PDAC Tumorgewebe für verschiedene Gelflächen bestimmt.For image acquisition with Typhoon 9400 fluorescence scanners (Amersham Biosciences / GE Healthcare), the gels remained between the glass plates. The excitation wavelength and the emission filters were selected specifically for the respective fluorescent dyes according to the manual. Prior to image analysis using the DeCyder software (Amersham Biosciences / GE Healthcare), the images were cut with the ImageQuant ™ software (Amersham Biosciences / GE Healthcare). Intra-gel spot detection and quantification was performed using the DeCyder Software's Differential In-gel Analysis (DIA) mode. The estimated number of spots has been set at 3000. As an exclusion filter, the slope of the slope was chosen to be greater than 1.6. For the determination of the reference proteome, the matching rates between microdissected PDAC cells, a pancreatic cell line pool and PDAC tumor tissue for different gel areas were determined.
In-gel Verdau and Proteinidentifizierung mittels nanoLC-ESI- MS/MSIn-gel digestion and protein identification by nanoLC-ESI-MS / MS
Die Spots wurden aus einem präparativen Gel manuell ausgestochen. Um die Position der Spots im Gel zu bestimmen, wurde nach der Bildakqusition ein maßstabsgerechter Gelausdruck unter das Gel platziert. Die Spots wurden anschließend im Gel mir Trypsin verdaut (Promega, Mannheim, Deutschland) und die Peptide wie in Schäfer et al. beschrieben extrahiert (Schaefer, H., J. P. Chervet, et al. (2004). "A peptide preconcentration approach for nano-high-performance liquid chromatography to diminish memory effects." Proteomics 4(9): 2541-4; Schaefer, H., K. Marcus, et al. (2003). "Identification of phosphorylation and acetylation sites in alphaA-crystallin of the eye lens (mus musculus) after two- dimensional gel electrophoresis . " Anal Bioanal Chem 376(7):The spots were manually punched out of a preparative gel. In order to determine the position of the spots in the gel, scale became more accurate after image acquisition Gel expression placed under the gel. The spots were subsequently digested in the gel with trypsin (Promega, Mannheim, Germany) and the peptides as described in Schäfer et al. Schaefer, H., JP Chervet, et al., (2004). "A peptide preconcentration approach for nano-high-performance liquid chromatography to diminish memory effects." Proteomics 4 (9): 2541-4; Schaefer, H , K. Marcus, et al., (2003) "Identification of phosphorylation and acetylation sites in alphaA-crystallin of the eye lens (musculus) after two-dimensional gel electrophoresis." Anal Bioanal Chem 376 (7):
966-72) . Für die Peptidanalytik wurde ein System bestehend aus FAMOSTM (automatischer Probensammler) , SwitchosTM (Ladepumpe und Schaltventiele) , und UltimateTM (Separationspumpe and UV- detektor) (LC Packings Dionex, Amsterdam, Niederlande) online gekoppelt mit dem Ionenfallen-Massespektrometer LCQ Deca XP (Thermo Electron, San Jose, CA, USA) ausgerüstet mit einer nano-Elektrospray Ionquellen (PicoView™ 100, New Objective Inc., Woburn, MA, USA) und SilicaTips™ (FS360-20-10-D, New Objective Inc.) verwendet. ^Für die Proteinidentifizierung wurden die MS/MS-Spektren mithilfe des SEQUEST™ Algorithmus unter Berücksichtigung der folgenden Suchparameter gegen die NCBI-Proteinsequenz Subdatenbank (human) gesucht (http://www.ncbi.nlm.nih.gov). Hierbei wurden. folgende Suchparameter berücksichtigt: Massentoleranz + 1.5 Da für Eltern- und Fragmentionen. Modifizierung aller Cysteine mit Cy3. Eine überlesene Trypsinschnittstelle. Proteine mit einem SequestMetaScore (ProteinscapeTM) größer als 10 bei mindestens 3 Peptiden wurden als identifiziert berücksichtigt.966-72). For peptide analysis, a system consisting of FAMOSTM (automatic sampler), SwitchosTM (charge pump and switching valves), and UltimateTM (separation pump and UV detector) (LC Packings Dionex, Amsterdam, Netherlands) was coupled online with the LCQ Deca XP ion trap mass spectrometer ( Thermo Electron, San Jose, Calif.) Equipped with nano-electrospray ion sources (PicoView ™ 100, New Objective Inc., Woburn, MA, USA) and SilicaTips ™ (FS360-20-10-D, New Objective Inc.). used. ^ For protein identification, the MS / MS spectra were searched for the NCBI protein sequence sub-database (human) using the SEQUEST ™ algorithm, using the following search parameters (http://www.ncbi.nlm.nih.gov). Here were. following search parameters taken into account: mass tolerance + 1.5 Da for parent and fragment ions. Modification of all cysteines with Cy3. An exquisite trypsin interface. Proteins with a SequestMetaScore (Proteinscape ™) greater than 10 in at least 3 peptides were considered as identified.
Herstellung von Tissue ArraysProduction of tissue arrays
Für normale Pankreasgänge sowie für PanINs wurde jeweils einer, für duktale Adenokarzinome jeweils zwei 1.5-mm dickeFor normal pancreatic ducts as well as for PanINs in each case one, for ductal adenocarcinomas two 1.5-mm thick
Gewebszylinder aus repräsentativen Arealen ausgestanzt und in Empfängerparaffinblöcke eingebettet, sodass insgesamt 300 Zylinder mit Pankreasgeweben (in insgesamt 6 Tissue Arrays) sowie jeweils zwei Kontrollzylinder bestehend aus gesundem Tonsillengewebe verarbeitet wurden. Die Aufarbeitung erfolgte mithilfe von MTAl tissue arrayer Instrument (Beecher Instruments, Sun Prairie, WI, USA) . Die normalen Pankreasgänge sowie die Gänge der PanINs entstammen 12 Pankreata gesunder suizidierter Menschen, die am Rechtsmedizinischen Institut der Semmelweis Universität in Budapest, Ungarn obduziert wurdenTissue cylinders were punched from representative areas and embedded in recipient paraffin blocks, so that a total of 300 cylinders with pancreatic tissues (in a total of 6 tissue arrays) and two control cylinders each consisting of healthy tonsillar tissue were processed. The work-up was carried out using MTAl tissue arrayer instrument (Beecher Instruments, Sun Prairie, WI, USA). The normal pancreatic ducts as well as the ducts of the PanINs are derived from 12 pancreata of healthy suicided people who were autopsied at the Forensic Medicine Institute of the Semmelweis University in Budapest, Hungary
(Genehmigungsnummer: 140-1/1996) als auch 81 Pankreata, die im Rahmen von Operationen gastrointestinaler und pankreatischer Tumoren der chirurgischen Universitätskliniken in Kiel und Dresden, Deutschland, entnommen wurden. Für die Tissue Arrays der Pankreaskarzinome wurden von Gewebsblöcken von 48 in der chirurgischen Universitätsklinik Kiel entfernten Pankreata verwendet .(Approval number: 140-1 / 1996) as well as 81 pancreata, which were taken as part of operations of gastrointestinal and pancreatic tumors of the surgical university hospitals in Kiel and Dresden, Germany. For the tissue arrays of pancreatic carcinomas, pancreata was removed from tissue blocks of 48 pts at Kiel University Hospital.
Immunhistochemie Alle Untersuchungen wurden auf formalinfixierten paraffineingebetteten Geweben durchgeführt. 3 μm-dünne Schnitte wurden entparaffiniert und rehydriert. Anschließend wurden immunhistochemische Färbungen nach der etablierten Methode angefertigt. Vor der Applikation des primären Antikörpers wurde ein Serum-Blocking für 20 Minuten durchgeführt. Der murine anti-14-3-3-sigma Antikörper (Acris, I.N.6., 2.5 μg/μl, 1:40), der anti-LRP/MVP-Antikörper (Kamiya Biomedical Company, 1032, 0.5 μg/μl, 1:400) sowie der Kaninchen anti-AGR2-Antikörper (Imgenex, 10 μg/μl, 1:50) wurden als primäre Antikörper eingesetzt. Die Signalentwicklung erfolgte durch ein Maus oder Kaninchen Färbekit (Vectastain Elite Peroxidase kit, PK-6102, Vector Laboratories, Burmingame, USA) . Für die Negativkontrolle wurde der primäre Antikörper weggelassen.Immunohistochemistry All studies were performed on formalin-fixed paraffin-embedded tissues. 3 μm thin sections were dewaxed and rehydrated. Subsequently, immunohistochemical stains were established after the Method made. Prior to application of the primary antibody, serum blocking was performed for 20 minutes. The murine anti-14-3-3 sigma antibody (Acris, IN6., 2.5 μg / μl, 1:40), the anti-LRP / MVP antibody (Kamiya Biomedical Company, 1032, 0.5 μg / μl, 1: 400) and rabbit anti-AGR2 antibodies (Imgenex, 10 μg / μl, 1:50) were used as primary antibodies. Signaling was by a mouse or rabbit staining kit (Vectastain Elite Peroxidase kit, PK-6102, Vector Laboratories, Burmingame, USA). For the negative control, the primary antibody was omitted.
Auswertung der immunhistochemischen FärbungenEvaluation of immunohistochemical staining
Die Intensität der Färbung wurde in mild, mäßig und stark eingeteilt (entsprechend einem Punktewert 1, 2 oder 3) . Die angefärbten Areale wurden in Bezug auf die Pankreasgänge bzw. der Tumorareale in Prozent geschätzt und wiederum inThe intensity of the staining was classified as mild, moderate and strong (corresponding to a score of 1, 2 or 3). The stained areas were estimated in relation to the pancreatic ducts or the tumor areas in percent and again in
Punktewerte unterteilt (<10%=l, 10-50%=2, 51-80%03, >80%=4) .Scores are divided (<10% = l, 10-50% = 2, 51-80% 03,> 80% = 4).
Die endgültige Punktzahl (score) wurde vom Produkt der Färbeintesität und Anteil positiv gefärbter Zellen bestimmtThe final score was determined by the product of staining intensity and proportion of positively stained cells
(Minimum 0, Maximum 12) (Remmele, Hildebrand et al. 1986) .(Minimum 0, maximum 12) (Remmele, Hildebrand et al., 1986).
Statistikstatistics
Die Mittelwerte der immunhistochemisch bestimmten Punktewerte der normalen Pankreasgänge, der verschiedenen PanIN-Läsionen sowie des duktalen Adenokarzinoms wurden anhand des Mann- Whitney U und Kruskal-Wallis-H Testes verglichen. Allen angewendeten statistischen Tests wurde ein Signifikanzniveau von 0,05 zugrunde gelegt. Bei multiplen Vergleichen wurde der p-Wert gemäß Bonferroni modifiziert. Alle statistischen Berechnungen wurden mithilfe der SPSS 10.1-Software angefertigt .The mean values of the immunohistochemically determined scores of the normal pancreatic ducts, the different PanIN lesions and the ductal adenocarcinoma were compared on the basis of the Mann-Whitney U and Kruskal-Wallis-H test. All applied statistical tests became a level of significance of 0.05. For multiple comparisons, the p-value was modified according to Bonferroni. All statistical calculations were made using the SPSS 10.1 software.
Für die Identifizierung der Biomarkerkandidaten für pankreatische Tumorprogression eine differenzielle Proteomanalyse mikrodissezierter Zellen aus PanIN-Läsionen, PDAC und normalen Pankreasgänge wurde durchgeführt. Für diesen Ansatz wurden Tumore von 9 Pankreaskrebspatienten untersucht, die insgesamt 4-9 Proben per Läsion lieferten. Die identifizierten differenziellen Biomarker wurden an Proben (Gewebearrays) von 130 Patienten immunhistochemisch validiert.A differential proteome analysis of microdissected cells from PanIN lesions, PDAC, and normal pancreatic ducts was performed to identify biomarker candidates for pancreatic tumor progression. For this approach, tumors from 9 pancreatic cancer patients were examined, providing a total of 4-9 lesions per lesion. The identified differential biomarkers were immunohistochemically validated on samples (tissue arrays) of 130 patients.
Expressionsprofile der differenziellen Proteine In der differenziellen Proteomanalyse mittels 2D Elektrophorese wurden insgesamt 86 unterschiedliche Proteinspots detektiert, die eine differenzielle Expression zeigten. Davon waren 19 Spots in der PanIN 1A-Läsion, 37 in der PanIN 1B-Läsion, 40 in der PanIN 2-Läsion, 39 in der PanIN 3-Läsion und 32 in PDAC differenziell gegenüber der normalen Pankreasgänge reguliert (p < 0.05, Regulationsfaktor > 1.6). Jeweils ein repräsentatives Gel für jedes Tumorstadium inklusive der regulierten Proteinspots ist in der Abbildung 1 dargestellt.Expression Profiles of the Differential Proteins In differential proteome analysis by means of 2D electrophoresis, a total of 86 different protein spots were detected which showed a differential expression. Of these, 19 spots in the PanIN 1A lesion, 37 in the PanIN 1B lesion, 40 in the PanIN 2 lesion, 39 in the PanIN 3 lesion and 32 in PDAC were differentially regulated from the normal pancreatic ducts (p <0.05, regulation factor > 1.6). One representative gel for each tumor stage including the regulated protein spots is shown in Figure 1.
Für die Identifizierung der differenziellen Proteinspots wurde das Referenzproteom aus dem pankreatischen Tumorgewebe verwendet, dessen Proteommuster eine Hoche Übereinstimmung mit dem Proteom des mikrodissezierten Materials zeigte (> 91 %). Mit Hilfe von LC-ESI-MS/MS konnten insgesamt 38 nicht redundante Proteine identifiziert werden (Tabelle 1).For the identification of the differential protein spots, the reference proteome from the pancreatic tumor tissue was used, whose proteome pattern is in high agreement with the proteome of the microdissected material (> 91%). Using LC-ESI-MS / MS, a total of 38 non-redundant proteins could be identified (Table 1).
Validierung der Proteomdaten mittels Immunhistochemie Für die Wahl der Proteine für die immunhistochemische Validierung wurden deren Expressionsprofile während der Tumorprogression berücksichtigt. Deshalb wurden die differenziellen Proteinspots in 3 Gruppen eingeteilt: 1) Proteinspots, die eine frühe Regulation in den Läsionen PanIN IA und PanIN IB zeigen, 2) Konstant veränderte Proteinspots während der gesamten Tumorprogression, 3) Proteinspots, die im fortgeschrittenen Tumorstadium (PanIN 2 bis PDAC) differentiell exprimiert sind (siehe Tabelle 1) . Des Weiteren wurde auch die potentielle Rolle der Proteine bei der Tumorbiologie als Kriterium für immunhistochemische Validierung in Betracht gezogen. Aus den 38 nicht redundanten Proteinen wurden zunächst sieben für die Validierung an 130 Patienten ausgewählt: AGR2, MVP, stratifin, annexin A2, EFIa- like protein, annexin A4 und SlOOAlO. Davon konnten an sechsValidation of proteomic data by immunohistochemistry For the selection of proteins for immunohistochemical validation, their expression profiles were taken into account during tumor progression. Therefore, the differential protein spots were divided into 3 groups: 1) protein spots showing early regulation in the lesions PanIN IA and PanIN IB, 2) constantly changing protein spots during the entire tumor progression, 3) protein spots occurring in the advanced tumor stage (PanIN 2 bis PDAC) are differentially expressed (see Table 1). Furthermore, the potential role of proteins in tumor biology as a criterion for immunohistochemical validation was also considered. Of the 38 non-redundant proteins, seven were selected for validation in 130 patients: AGR2, MVP, stratifin, annexin A2, EFI-like protein, annexin A4, and SlOOAlO. Of these, six could
Proteinen die Proteomdaten bestätigt werden. Der Vergleich der Proteomdaten und der Validierung wird an drei Proteinen dargestellt: 14-3-3 sigma, MVP und AGR2 (Abbildungen 2, 3 und 4) .Proteins are confirmed the proteome data. The comparison of proteome data and validation is presented on three proteins: 14-3-3 sigma, MVP, and AGR2 (Figures 2, 3, and 4).
Immunhistochemisches Expressionsmuster von MVP Die Färbungen mit dem MVP-Antikörper zeigten eine intrazytoplasmatische Färbereaktion. Die mittleren Punktewerte für die MVP-Färbung waren: normale Gänge 3.70 (Standardabweichung 3.0, Spannweite 0-9); PanIN-la 4.60 (Standardabweichung 3.2, Spannweite 0-12); PanIN-lb 7.82 (Standardabweichung 3.2, .Spannweite 0-12); PanIN-2 7.93 (Standardabweichung 3.8, Spannweite 2-12); PanIN-3 10.00 (Standardabweichung 2.8, Spannweite 3-12) sowie duktale Adenokarzinome 8.32 (Standardabweichung 3.0, Spannweite 1-12) (Abbildung 2). Die Punktwerte der verschiedenen Krankheitsgruppen zeigten signifikante Unterschiede untereinander (Kruskal-Wallis-Test, p< 0.001). PanIN-lB, PanIN-2, PanIN-3 und PDAC zeigen eine signifikant höhere Expression von MVP als normale Pankreasgänge (Mann-Whitney U- Test, p< 0.001). Zwischen PanIN-lB, PanIN-2, PanIN-3 und PDAC ließen sich keine statistischen Unterschiede feststellen (Kruskal-Wallis-Test, p=0.110). Die erhöhte Expression von MVP in PanIN-3 konnte sowohl in der Proteomanalyse als auch immunhistochemisch festgestellt werden (Abbildung 3 A, B)Immunohistochemical expression pattern of MVP The staining with the MVP antibody showed an intracytoplasmic staining reaction. The mean scores for MVP staining were: normal ducts 3.70 (standard deviation 3.0, range 0-9); PanIN-la 4.60 (standard deviation 3.2, span 0-12); PanIN-lb 7.82 (standard deviation 3.2, span 0-12); PanIN-2 7.93 (standard deviation 3.8, span 2-12); PanIN-3 10.00 (standard deviation 2.8, range 3-12) and ductal adenocarcinomas 8.32 (standard deviation 3.0, range 1-12) (Figure 2). The scores of the various disease groups showed significant differences between them (Kruskal-Wallis test, p <0.001). PanIN-1B, PanIN-2, PanIN-3 and PDAC show a significantly higher expression of MVP than normal pancreatic ducts (Mann-Whitney U-test, p <0.001). There were no statistical differences between PanIN-1B, PanIN-2, PanIN-3 and PDAC (Kruskal-Wallis test, p = 0.110). The increased expression of MVP in PanIN-3 was detected both in proteome analysis and immunohistochemistry (Figure 3 A, B).
Immunhistochemisches Expressionsmuster von 14-3-3 sigma Die Färbung der Tissue Arrays mit 14-3-3-sigma Antikörper zeigte eine hauptsächlich intrazytoplasmatische und weniger membranständige Färbereaktion. Die mittleren Punktwerte für die 14-3-3 sigma-Färbung waren: normale Pankreasgänge 2.04 (Standardabweichung 3.1, Spannweite 0-12); PanIN-lA 2.80 (Standardabweichung 2.6, Spannweite 0-8); PanIN-lB 5.30 (Standardabweichung 3.8, Spannweite 0-12); PanIN-2 8.34 (Standardabweichung 3.1, Spannweite 2-12); PanIN-3 10.61 (Standardabweichung 1.9, Spannweite 6-12) und PDAC 9.61 (Standardabweichung 2.8, Spannweite 2-12) (Abbildung 2). Die 14-3-3-sigma Expression war signifikant unterschiedlich im Vergleich der verschiedenen Gruppen (Kruskal-Wallis-Test, p< 0.001). 14-3-3-sigma Protein wurde in PanIN-lB signifikant stärker exprimiert als in normalen Gängen und in PanIN-lAImmunohistochemical expression pattern of 14-3-3 sigma The staining of the tissue arrays with 14-3-3-sigma antibodies revealed a mainly intracytoplasmic and less membrane-bound staining reaction. The mean scores for the 14-3-3 sigma stain were: normal pancreatic ducts 2.04 (standard deviation 3.1, range 0-12); PanIN-lA 2.80 (standard deviation 2.6, span 0-8); PanIN-lB 5.30 (standard deviation 3.8, span 0-12); PanIN-2 8.34 (standard deviation 3.1, span 2-12); PanIN-3 10.61 (standard deviation 1.9, span 6-12) and PDAC 9.61 (Standard deviation 2.8, span 2-12) (Figure 2). The 14-3-3 sigma expression was significantly different in the comparison of the different groups (Kruskal-Wallis test, p <0.001). 14-3-3 sigma protein was significantly more expressed in PanIN-lB than in normal ducts and PanIN-lA
(Mann-Whitney U-Test, p< 0.001). 14-3-3-sigma Protein wurde außerdem in PanIN-2, PanIN-3 und PDAC signifikant stärker exprimiert als in PanIN-lB (Mann-Whitney U-Test, p<0.001). Die Ergebnisse der Proteomanalyse und die Ergebnisse der immunohistochemischen Auswertung zeigten ein ähnliches 14-3-3- sigma Expressionsmuster bei PanIN-lB - PanIN-3 Läsionen(Mann-Whitney U test, p <0.001). 14-3-3 sigma protein was also significantly more expressed in PanIN-2, PanIN-3 and PDAC than in PanIN-1B (Mann-Whitney U test, p <0.001). The results of the proteome analysis and the results of the immunohistochemical evaluation showed a similar 14-3-3 sigma expression pattern in PanIN-1B-PanIN-3 lesions
(Abbildung 3 A, B) .(Figure 3 A, B).
Immunhistochemisches Expressionsmuster von AGR 2 Die Färbung der Tissue Arrays mit AGR2-Antikörper zeigte ein hauptsächlich intrazytoplasmatisches und weniger membranständiges Expressionsmuster. Die mittleren Punktwerte der AGR 2-Färbung waren: normale Pankreasgänge 7.59 (Standardabweichung 3.5, Spannweite 2-12); PanIN-lA 10.97 (Standardabweichung 2.0, Spannweite 6-12); PanIN-lB 10.16 (Standardabweichung 2.6, Spannweite 3-12); PanIN-2 8.96 (Standardabweichung 2.9, Spannweite 3-12); PanIN-3 8.47 (Standardabweichung 3.3, Spannweite 3-12) und PDAC 6.53 (Standardabweichung 2.6, Spannweite 1-12) (Abbildung 2). Im Vergleich der verschiedenen Gruppen untereinander ließen sich signifikante Unterschiede der Punktwerte feststellen (Kruskal- Wallis-Test, p< 0.001). Außerdem konnte gezeigt werden, dass AGR2 in PanIN-lA, PanIN-lB, PanIN-2 und PanIN-3 signifikant stärker exprimiert wird als in normalen Pankreasgängen (Mann- Whitney U-Test, p= 0.002). Im Vergleich mit den PanIN-Läsionen zeigte AGR2 der PDAC eine signifikant schwächere Expression (Mann-Whitney U-Test, p<0.001). Die Ergebnisse der Proteomanalyse entsprachen den immunhistochemischen Reaktionen für PanIN-lA - PanIN-3.Immunohistochemical expression pattern of AGR 2 The staining of the tissue arrays with AGR2 antibody showed a mainly intracytoplasmic and less membrane-bound expression pattern. The mean scores of AGR 2 staining were: normal pancreatic ducts 7.59 (standard deviation 3.5, span 2-12); PanIN-lA 10.97 (standard deviation 2.0, span 6-12); PanIN-lb 10.16 (standard deviation 2.6, span 3-12); PanIN-2 8.96 (standard deviation 2.9, span 3-12); PanIN-3 8.47 (standard deviation 3.3, span 3-12) and PDAC 6.53 (standard deviation 2.6, span 1-12) (Figure 2). Comparing the different groups, significant differences in scores were found (Kruskal-Wallis test, p <0.001). In addition, it could be shown that AGR2 in PanIN-IA, PanIN-IB, PanIN-2 and PanIN-3 significantly is more strongly expressed than in normal pancreatic ducts (Mann-Whitney U test, p = 0.002). In comparison with the PanIN lesions, AGR2 of the PDAC showed a significantly weaker expression (Mann-Whitney U test, p <0.001). The results of the proteome analysis corresponded to the immunohistochemical reactions for PanIN-lA - PanIN-3.
Differentialdiagnose von Pankreaskrebs, PDAC zur Pankreatitis: Bei den Proteinen AGR 2, 14-3-3 sigma und MVP wurde in der vorliegenden Studie eine erhöhte Expression während derDifferential diagnosis of pancreatic cancer, PDAC for pancreatitis: In the present study, an increased expression during the proteins AGR 2, 14-3-3 sigma and MVP during the
Progression von PDAC sowohl in der Proteomstudie als auch in der immunhistochemischen Analyse nachgewiesen. Um die Anwendung dieser Proteine zu Differenzierung zwischen Pankreaskrebs und Pankreatitis zu beurteilen, wurde ihre Expression auch bei Gewebearrays von 40 Pankreatitispatienten untersucht. Im Gegensatz zu Pankreaskrebspatienten wurde bei den Pankreatitispatienten keine bzw. eine geringere Konzentration detektiert. Der Expressionslevel dieser Proteine im Gewebe der Pankreatitispatienten ist vergleichbar mit dem Expressionslevel im gesunden Gewebe. Somit zeigen AGR 2, 14-3- 3 sigma und MVP ein hohes Potential für die Verwendung als nicht invasive oder in-vivo Biomarker für die Unterscheidung (Differentialdiagnose) zwischen PDAC bzw. Pankreaskrebs und Pankreatitis (siehe Abbildung 4).Progression of PDAC has been demonstrated in both proteomic and immunohistochemical analysis. To evaluate the application of these proteins to differentiation between pancreatic cancer and pancreatitis, their expression was also examined in tissue arrays of 40 pancreatitis patients. In contrast to pancreatic cancer patients no or a lower concentration was detected in pancreatitis patients. The expression level of these proteins in the tissue of pancreatitis patients is comparable to the expression level in healthy tissue. Thus, AGR 2, 14-3- 3 sigma and MVP show a high potential for use as noninvasive or in vivo biomarkers for distinguishing (differential diagnosis) between PDAC / pancreatic cancer and pancreatitis (see Figure 4).
Die erfindungsgemäßen Sequenzen (SEQ ID No. 1 - 71) lauten wie folgt: SEQ ID No. 1The sequences according to the invention (SEQ ID No. 1-71) are as follows: SEQ ID no. 1
>gi I 33875698 I gb|AAH00654.2 I KRT8 protein [Homo sapiens]> gi I 33875698 I gb | AAH00654.2 I KRT8 protein [Homo sapiens]
FSAPSRISAWFGPPASTPASTMSIRVTQKSYKVSTSGPRAFSSRSYTSGPGSRISSSSFSRVGSSNFRGG LGGGYGGASGMGGITAVTVNQSLLSPLVLEVDPNIQAVRTQEKEQIKTLNNKFASFIDKVRFLEQQNKML ETKWSLLQQQKTARSNMDNMFESYINNLRRQLETLGQEKLKLEAELGNMQGLVEDFKNKYEDEINKRTEM ENEFVLIKKDVDEAYMNKVELESRLEGLTDEINFLRQLYEEEIRELQSQISDTSVVLSMDNSRSLDMDSI IAEVKAQYEDIANRSRAEAESMYQIKYEELQSLAGKHGDDLRRTKTEISEMNRNISRLQAEIEGLKGQRA SLEAAIADAEQRGELAIKDANAKLSELEAALQRAKQDMARQLREYQELMNVKLALDIEIATYRKLLEGEE SRLESGMQNMSIHTKTTSGYAGGLSSAYGGLTSPGLSYSLGSSFGSGAGSSSFSRTSSSRAVVVKKIETR DGKLVSESSDVLPKFSAPSRISAWFGPPASTPASTMSIRVTQKSYKVSTSGPRAFSSRSYTSGPGSRISSSSFSRVGSSNFRGG LGGGYGGASGMGGITAVTVNQSLLSPLVLEVDPNIQAVRTQEKEQIKTLNNKFASFIDKVRFLEQQNKML ETKWSLLQQQKTARSNMDNMFESYINNLRRQLETLGQEKLKLEAELGNMQGLVEDFKNKYEDEINKRTEM ENEFVLIKKDVDEAYMNKVELESRLEGLTDEINFLRQLYEEEIRELQSQISDTSVVLSMDNSRSLDMDSI IAEVKAQYEDIANRSRAEAESMYQIKYEELQSLAGKHGDDLRRTKTEISEMNRNISRLQAEIEGLKGQRA SLEAAIADAEQRGELAIKDANAKLSELEAALQRAKQDMARQLREYQELMNVKLALDIEIATYRKLLEGEE SRLESGMQNMSIHTKTTSGYAGGLSSAYGGLTSPGLSYSLGSSFGSGAGSSSFSRTSSSRAVVVKKIETR DGKLVSESSDVLPK
SEQ ID No. 2SEQ ID no. 2
>gi I 7576229 | emb | CAB87963.1 | vimentin [Homo sapiens]> gi I 7576229 | emb | CAB87963.1 | vimentin [Homo sapiens]
MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRL RSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKG QGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDV DNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVA AKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEEN FAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSS LNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEMSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRL RSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKG QGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDV DNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVA AKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEEN FAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSS LNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLE
SEQ ID No. 3SEQ ID no. 3
>gi I 12804929 I gb| AAH01917.1 I Malate dehydrogenase 2, NAD (mitochondrial) [Homo sapiens]> gi I 12804929 I gb | AAH01917.1 I Malate dehydrogenase 2, NAD (mitochondrial) [Homo sapiens]
MLSALARPVSAALRRSFSTSAQNNAKVAVLGASGGIGQPLSLLLKNSPLVSRLTLYDIAHTPGVAADLSH IETKAAVKGYLGPEQLPDCLKGCDVVVIPAGVPRKPGMTRDDLFNTNATIVATLTAACAQHCPEAMICVI ANPVNSTIPITAEVFKKHGVYNPNKIFGVTTLDIVRANTFVAELKGLDPARVNVPVIGGHAGKTIIPLIS QCTPKVDFPQDQLTALTGRIQEAGTEVVKAKAGAGSATLSMAYAGARFVFSLVDAMNGKEGVVECSFVKS QETECTYFSTPLLLGKKGIEKNLGIGKVSSFEEKMISDAIPELKASIKKGEDFVKTLKMLSALARPVSAALRRSFSTSAQNNAKVAVLGASGGIGQPLSLLLKNSPLVSRLTLYDIAHTPGVAADLSH IETKAAVKGYLGPEQLPDCLKGCDVVVIPAGVPRKPGMTRDDLFNTNATIVATLTAACAQHCPEAMICVI ANPVNSTIPITAEVFKKHGVYNPNKIFGVTTLDIVRANTFVAELKGLDPARVNVPVIGGHAGKTIIPLIS QCTPKVDFPQDQLTALTGRIQEAGTEVVKAKAGAGSATLSMAYAGARFVFSLVDAMNGKEGVVECSFVKS QETECTYFSTPLLLGKKGIEKNLGIGKVSSFEEKMISDAIPELKASIKKGEDFVKTLK
SEQ ID No. 4SEQ ID no. 4
>gi I 6573280 I gb I AAF17621.il beta tropomyosin [Homo sapiens]> gi I 6573280 I gb I AAF17621.il beta tropomyosin [Homo sapiens]
MDAIKKKMQMLKLDKENAIDRAEQAEADKKQAEDRCKQLEEEQQALQKKLKGTEDEVEKYSESVKEAQEKMDAIKKKMQMLKLDKENAIDRAEQAEADKKQAEDRCKQLEEEQQALQKKLKGTEDEVEKYSESVKEAQEK
LEQAEKKATDAEADVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAADESERGMKVIENRAMKDEEKLEQAEKKATDAEADVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAADESERGMKVIENRAMKDEEK
MELQEMQLKEAKHIAEDSDRKYEEVARKLVILEGELERSEERAEVAESRARQLEEELRTMDQALKSLMASMELQEMQLKEAKHIAEDSDRKYEEVARKLVILEGELERSEERAEVAESRARQLEEELRTMDQALKSLMAS
EEEYSTKEDKYEEEIKLLEEKLKEAETRAEFAERSVAKLEKTIDDLEEEEYSTKEDKYEEEIKLLEEKLKEAETRAEFAERSVAKLEKTIDDLE
SEQ ID No. 5SEQ ID no. 5
>gi I 402261011 gb I AAH23548.il ACTGl protein [Homo sapiens]> gi I 402261011 gb I AAH23548.il ACTGl protein [Homo sapiens]
KANREKMTQIMFETFNTPAMYVAIQAVLSLYASGRTTGIVMDSGDGVTHTVPIYEGYALPHAILRLDLAGKANREKMTQIMFETFNTPAMYVAIQAVLSLYASGRTTGIVMDSGDGVTHTVPIYEGYALPHAILRLDLAG
RDLTDYLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQEMATAASSSSLEKSYELPDGQVITIGNRDLTDYLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQEMATAASSSSLEKSYELPDGQVITIGN
ERFRCPEALFQPSFLGMESCGIHETTFNSIMKCDVDIRKDLYANTVLSGGTTMYPGIADRMQKEITALAPERFRCPEALFQPSFLGMESCGIHETTFNSIMKCDVDIRKDLYANTVLSGGTTMYPGIADRMQKEITALAP
STMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISKQEYDESGPSIVHRKCFSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISKQEYDESGPSIVHRKCF
SEQ ID No. 6SEQ ID no. 6
>gi| 3153859|gb|AAC17430.1| thioredoxin delta 3 [Homo sapiens]> Gi | 3153859 | gb | AAC17430.1 | thioredoxin delta 3 [Homo sapiens]
VKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHDVASECEVKCMPTFQFFKKGQKVGEFSG ANKEKLEATINELVVKQIESKTAFQEALDAAGDKLVVVSFSATWCGPCKMIKPFFHDVASECEVKCMPTFQFFKKGQKVGEFSG ANKEKLEATINELV
SEQ ID No. 7SEQ ID no. 7
>gi I 999893 |pdb| IHTI |B Chain B, Triosephosphate Isomerase (Tim)> gi I 999893 | pdb | IHTI | B Chain B, Triosephosphate Isomerase (Tim)
(E. C.5.3.1.1) Complexed With 2-Phosphoglycolic Acid(E.C.5.3.1.1) Complexed With 2-Phosphoglycolic Acid
APSRKFFVGGNWKMNGRKQSLGELIGTLNAAKVPADTEVVCAPPTAYIDFARQKLDPKIAVAAQNCYKVT NGAFTGEISPGMIKDCGATWVVLGHSERRHVFGESDELIGQKVAHALAEGLGVIACIGEKLDEREAGITE KVVFEQTKVIADNVKDWSKVVLAYEPVWAIGTGKTATPQQAQEVHEKLRGWLKSNVSDAVAQSTRIIYGG SVTGATCKELASQPDVDGFLVGGASLKPEFVDIINAKQAPSRKFFVGGNWKMNGRKQSLGELIGTLNAAKVPADTEVVCAPPTAYIDFARQKLDPKIAVAAQNCYKVT NGAFTGEISPGMIKDCGATWVVLGHSERRHVFGESDELIGQKVAHALAEGLGVIACIGEKLDEREAGITE KVVFEQTKVIADNVKDWSKVVLAYEPVWAIGTGKTATPQQAQEVHEKLRGWLKSNVSDAVAQSTRIIYGG SVTGATCKELASQPDVDGFLVGGASLKPEFVDIINAKQ
SEQ ID No. 8SEQ ID no. 8th
>gi I 16306978 I gb I AAH09564.il Annexin A2 [Homo sapiens]> gi I 16306978 I gb I AAH09564.il Annexin A2 [Homo sapiens]
MSTVHEILCKLSLEGDHSTPPSAYGSVKAYTNFDAERDALNIETAIKTKGVDEVTIVNILTNRSNAQRQD IAFAYQRRTKKELASALKSALSGHLETLILGLLKTPAQYDASELKASMKGLGTDEDSLIEIICSRTNQEL QEINRVYKEMYKTDLEKDIISDTSGDFRKLMVALAKGRRAEDGSVIDYELIDQDARDLYDAGVKRKGTDV PKWISIMTERSVPHLQKVFDRYKSYSPYDMLESIRKEVKGDLENAFLNLVQCIQNKPLYFADRLYDSMKG KGTRDKVLIRIMVSRSEVDMLKIRSEFKRKYGKSLYYYIQQDTKGDYQKALLYLCGGDD SEQ ID No. 9MSTVHEILCKLSLEGDHSTPPSAYGSVKAYTNFDAERDALNIETAIKTKGVDEVTIVNILTNRSNAQRQD IAFAYQRRTKKELASALKSALSGHLETLILGLLKTPAQYDASELKASMKGLGTDEDSLIEIICSRTNQEL QEINRVYKEMYKTDLEKDIISDTSGDFRKLMVALAKGRRAEDGSVIDYELIDQDARDLYDAGVKRKGTDV PKWISIMTERSVPHLQKVFDRYKSYSPYDMLESIRKEVKGDLENAFLNLVQCIQNKPLYFADRLYDSMKG KGTRDKVLIRIMVSRSEVDMLKIRSEFKRKYGKSLYYYIQQDTKGDYQKALLYLCGGDD SEQ ID no. 9
>gi|10441386|gb|AAG17014.1 |AF186109_l TPM4-ALK fusion oncoprotein type 2> gi | 10441386 | gb | AAG17014.1 | AF186109_l TPM4-ALK fusion oncoprotein type 2
[Homo sapiens][Homo sapiens]
MAGLNSLEAVKRKIQALQQQADEAEDRAQGLQRELDGERERREKAEGDVAALNRRIQLVEEELDRAQERLMAGLNSLEAVKRKIQALQQQADEAEDRAQGLQRELDGERERREKAEGDVAALNRRIQLVEEELDRAQERL
ATALQKLEEAEKAADESERGMKVIENRAMKDEEKMEIQEMQLKEAKHIAEEADRKYEEVARKLVILEGELATALQKLEEAEKAADESERGMKVIENRAMKDEEKMEIQEMQLKEAKHIAEEADRKYEEVARKLVILEGEL
ERAEERAEVSELKCGDLEEELKNVTNNLKSLEAASEKYSEKEDKYEEEIKLLSDKLKEAETRAEFAERTVERAEERAEVSELKCGDLEEELKNVTNNLKSLEAASEKYSEKEDKYEEEIKLLSDKLKEAETRAEFAERTV
AKLEKTIDDLEVYRRKHQELQAMQMELAKLEKTIDDLEVYRRKHQELQAMQMEL
SEQ ID No. 10SEQ ID no. 10
>gi| 62205349|gb|AAH93076.1| PPIA protein [Homo sapiens]> Gi | 62205349 | gb | AAH93076.1 | PPIA protein [Homo sapiens]
MCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLDGKHVVFGMCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLDGKHVVFG
KVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLEKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE
SEQ ID No. 11SEQ ID no. 11
>gi I 189022 I gb| AAA36348.1 I smooth muscle mysoin light chain> gi I 189022 I gb | AAA36348.1 I smooth muscle mysoin light chain
MRALGQNPTNAEVLKVLGNPKSDEMNVKVLDFEHFLPMLQTVAKNKDQGTYEDYVEGLRVFDKEGNGTVMMRALGQNPTNAEVLKVLGNPKSDEMNVKVLDFEHFLPMLQTVAKNKDQGTYEDYVEGLRVFDKEGNGTVM
GAEIRHVLVTLGEKMTEEEVEMLVAGHEDSNGCINYEAFVRHILSGGAEIRHVLVTLGEKMTEEEVEMLVAGHEDSNGCINYEAFVRHILSG
SEQ ID No. 12SEQ ID no. 12
>gi| 1408188|gb|AAC50680.1| desmin> Gi | 1408188 | gb | AAC50680.1 | desmin
MSQAYSSSQRVSSYRRTFGGAPGFPLGSPLSSPVFPRAGFGSKGSSSSVTSRVYQVSRTSGGAGGLGSLR ASRLGTTRTPSSYGAGELLDFSLADAVNQEFLTTRTNEKVELQELNDRFANYIEKVRFLEQQNALAAEVN RLKGREPTRVAELYEEELRELRRQVEVLTNQRARVDVERDNLLDDLQRLKAKLQEEIQLKEEAENNLAAF RADVDAATLARIDLERRIESLNEEIAFLKKVHEEEIRELQAQLQEQQVQVEMDMSKPDLTAALRDIRAQY ETIAAKNISEAEEWYKSKVSDLTQAANKNNDALRQAKQEMMEYRHQIQSYTCEIDALKGTNDSLMRQMRE LEDRFASEASGYQDNIARLEEEIRHLKDEMARHLREYQDLLNVKMALDVEIATYRKLLEGEESRINLPIQ TYSALNFRETSPEQRGSEVHTKKTVMIKTIETRDGEVVSEATQQQHEVLMSQAYSSSQRVSSYRRTFGGAPGFPLGSPLSSPVFPRAGFGSKGSSSSVTSRVYQVSRTSGGAGGLGSLR ASRLGTTRTPSSYGAGELLDFSLADAVNQEFLTTRTNEKVELQELNDRFANYIEKVRFLEQQNALAAEVN RLKGREPTRVAELYEEELRELRRQVEVLTNQRARVDVERDNLLDDLQRLKAKLQEEIQLKEEAENNLAAF RADVDAATLARIDLERRIESLNEEIAFLKKVHEEEIRELQAQLQEQQVQVEMDMSKPDLTAALRDIRAQY ETIAAKNISEAEEWYKSKVSDLTQAANKNNDALRQAKQEMMEYRHQIQSYTCEIDALKGTNDSLMRQMRE LEDRFASEASGYQDNIARLEEEIRHLKDEMARHLREYQDLLNVKMALDVEIATYRKLLEGEESRINLPIQ TYSALNFRETSPEQRGSEVHTKKTVMIKTIETRDGEVVSEATQQQHEVL
SEQ ID No. 13SEQ ID no. 13
>gi I 15990478 I gb| AAH15623.1 I Major vault protein [Homo sapiens]> gi I 15990478 I gb | AAH15623.1 I Major vault protein [Homo sapiens]
MATEEFIIRIPPYHYIHVLDQNSNVSRVEVGPKTYIRQDNERVLFAPMRMVTVPPRHYCTVANPVSRDAQMATEEFIIRIPPYHYIHVLDQNSNVSRVEVGPKTYIRQDNERVLFAPMRMVTVPPRHYCTVANPVSRDAQ
GLVLFDVTGQVRLRHADLEIRLAQDPFPLYPGEVLEKDITPLQVVLPNTALHLKALLDFEDKDGDKVVAGGLVLFDVTGQVRLRHADLEIRLAQDPFPLYPGEVLEKDITPLQVVLPNTALHLKALLDFEDKDGDKVVAG
DEWLFEGPGTYIPRKEVEVVEIIQATIIRQNQALRLRARKECWDRDGKERVTGEEWLVTTVGAYLPAVFEDEWLFEGPGTYIPRKEVEVVEIIQATIIRQNQALRLRARKECWDRDGKERVTGEEWLVTTVGAYLPAVFE
EVLDLVDAVILTEKTALHLRARRNFRDFRGVSRRTGEEWLVTVQDTEAHVPDVHEEVLGVVPITTLGPHNEVLDLVDAVILTEKTALHLRARRNFRDFRGVSRRTGEEWLVTVQDTEAHVPDVHEEVLGVVPITTLGPHN
YCVILDPVGPDGKNQLGQKRVVKGEKSFFLQPGEQLEQGIQDVYVLSEQQGLLLRALQPLEEGEDEEKVSYCVILDPVGPDGKNQLGQKRVVKGEKSFFLQPGEQLEQGIQDVYVLSEQQGLLLRALQPLEEGEDEEKVS
HQAGDHWLIRGPLEYVPSAKVEVVEERQAIPLDENEGIYVQDVKTGKVRAVIGSTYMLTQDEVLWEKELPHQAGDHWLIRGPLEYVPSAKVEVVEERQAIPLDENEGIYVQDVKTGKVRAVIGSTYMLTQDEVLWEKELP
PGVEELLNKGQDPLADRGEKDTAKSLQPLAPRNKTRVVSYRVPHNAAVQVYDYREKRARVVFGPELVSLGPGVEELLNKGQDPLADRGEKDTAKSLQPLAPRNKTRVVSYRVPHNAAVQVYDYREKRARVVFGPELVSLG
PEEQFTVLSLSAGRPKRPHARRALCLLLGPDFFTDVITIETADHARLQLQLAYNWHFEVNDRKDPQETAKPEEQFTVLSLSAGRPKRPHARRALCLLLGPDFFTDVITIETADHARLQLQLAYNWHFEVNDRKDPQETAK
LFSVPDFVGDACKAIASRVRGAVASVTFDDFHKNSARIIRTAVFGFETSEAKGPDGMALPRPRDQAVFPQLFSVPDFVGDACKAIASRVRGAVASVTFDDFHKNSARIIRTAVFGFETSEAKGPDGMALPRPRDQAVFPQ
NGLVVSSVDVQSVEPVDQRTRDALQRSVQLAIEITTNSQEAAAKHEAQRLEQEARGRLERQKILDQSEAENGLVVSSVDVQSVEPVDQRTRDALQRSVQLAIEITTNSQEAAAKHEAQRLEQEARGRLERQKILDQSEAE
KARKELLELEALSMAVESTGTAKAEAESRAEAARIEGEGSVLQAKLKAQALAIETEAELQRVQKVRELELKARKELLELEALSMAVESTGTAKAEAESRAEAARIEGEGSVLQAKLKAQALAIETEAELQRVQKVRELEL
VYARAQLELEVSKAQQLAEVEVKKFKQMTEAIGPSTIRDLAVAGPEMQVKLLQSLGLKSTLITDGSTPINVYARAQLELEVSKAQQLAEVEVKKFKQMTEAIGPSTIRDLAVAGPEMQVKLLQSLGLKSTLITDGSTPIN
LFNTAFGLLGMGPEGQPLGRRVASGPSPGEGISPQSAQAPQAPGDNHVVPVLRLFNTAFGLLGMGPEGQPLGRRVASGPSPGEGISPQSAQAPQAPGDNHVVPVLR
SEQ ID No. 14SEQ ID no. 14
>gi I 14043070 I ref |NP_112420.1 I heterogeneous nuclear ribonucleoprotein Al isoform b [Homo sapiens]I gi I 14043070 I ref | NP_112420.1 I heterogeneous nuclear ribonucleoprotein Al isoform b [Homo sapiens]
MSKSESPKEPEQLRKLFIGGLSFETTDESLRSHFEQWGTLTDCVVMRDPNTKRSRGFGFVTYATVEEVDAMSKSESPKEPEQLRKLFIGGLSFETTDESLRSHFEQWGTLTDCVVMRDPNTKRSRGFGFVTYATVEEVDA
AMNARPHKVDGRVVEPKRAVSREDSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDR GSGKKRGFAFVTFDDHDSVDKIVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGRSGSGNFGGGRGGGF GGNDNFGRGGNFSGRGGFGGSRGGGGYGGSGDGYNGFGNDGGYGGGGPGYSGGSRGYGSGGQGYGNQGSG YGGSGSYDSYNNGGGGGFGGGSGSNFGGGGSYNDFGNYNNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAK PRNQGGYGGSSSSSSYGSGRRFAMNARPHKVDGRVVEPKRAVSREDSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDR GSGKKRGFAFVTFDDHDSVDKIVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGRSGSGNFGGGRGGGF GGNDNFGRGGNFSGRGGFGGSRGGGGYGGSGDGYNGFGNDGGYGGGGPGYSGGSRGYGSGGQGYGNQGSG YGGSGSYDSYNNGGGGGFGGGSGSNFGGGGSYNDFGNYNNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAK PRNQGGYGGSSSSSSYGSGRRF
SEQ ID No. 15SEQ ID no. 15
>gi|4388970|pdb| 1BT6|B Chain B, PIl (SlOOaIO), Ligand Of Annexin Ii In> Gi | 4388970 | pdb | 1BT6 | B Chain B, PII (SlOOaIO), Ligand Of Annexin Ii In
Complex With Annexin Ii N-TerminusComplex With Annexin Ii N-terminus
PSQMEHAMETMMFTFHKFAGDKGYLTKEDLRVLMEKEFPGFLENQKDPLAVDKIMKDLDQCRDGKVGFQSPSQMEHAMETMMFTFHKFAGDKGYLTKEDLRVLMEKEFPGFLENQKDPLAVDKIMKDLDQCRDGKVGFQS
FFSLIAGLTIACNDYFVVHMKQKGKKFFSLIAGLTIACNDYFVVHMKQKGKK
SEQ ID No. 16SEQ ID no. 16
>gi|24210508|gb|AAN51932.1|AF322220_l cervical Cancer suppressor 3 (EFIa- like protein> gi | 24210508 | gb | AAN51932.1 | AF322220_l cervical cancer suppressor 3 (EFI-like protein
MITGTSQADCAVLIVAAGVGEFEAGISKNGQTREHALLAYTLGVKQLIVGVNKMDSTEPPYSQKRYEEIVMITGTSQADCAVLIVAAGVGEFEAGISKNGQTREHALLAYTLGVKQLIVGVNKMDSTEPPYSQKRYEEIV
KEVSTYIKKIGYNPDTVAFVPISGWNGDNMLEPSANMPWFKGWKVTRKDGNASGTTLLEALDCILPPTRPKEVSTYIKKIGYNPDTVAFVPISGWNGDNMLEPSANMPWFKGWKVTRKDGNASGTTLLEALDCILPPTRP
TDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPVNVTTEVKSVEMHHEALSEALPGDNVGFNTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPVNVTTEVKSVEMHHEALSEALPGDNVGFN
VKNVSVKDVRRGNVAGDSKNDPPMEAAGFTAQVIILNHPGQISAGYAPVLDCHTAHIACKFAELKEKIDR RSGKKLEDGPKFLKSGDAAIVDMVPGKPMCVESFSDYPPLGRFAVRDMRQTVAVGVIKAVDKKAAGAGKV TKSAQKAQKAKVKNVSVKDVRRGNVAGDSKNDPPMEAAGFTAQVIILNHPGQISAGYAPVLDCHTAHIACKFAELKEKIDR RSGKKLEDGPKFLKSGDAAIVDMVPGKPMCVESFSDYPPLGRFAVRDMRQTVAVGVIKAVDKKAAGAGKV TKSAQKAQKAK
SEQ ID No. 17SEQ ID no. 17
>gi I 33338062 I gb| AAQ13653.1 I regulatory myosin light chain long Version [Homo sapiens]> gi I 33338062 I gb | AAQ13653.1 I regulatory myosin light chain long version [Homo sapiens]
MSSKRAKAKTTKKRPQRATSNVFAMFDQSQIQEFKEAFNMIDQNRDGFIDKEDLHDMLASLGKNPTDEYL EGMMSEAPGPYNFTMFLTMFGEKLNGTDPEDVIRNAFACFDEESSGFIHEDHLRELLTTMGDRFTDEEVD EMYREAPIDKKGNFNYVEFTRILKHGAKDKDDMSSKRAKAKTTKKRPQRATSNVFAMFDQSQIQEFKEAFNMIDQNRDGFIDKEDLHDMLASLGKNPTDEYL EGMMSEAPGPYNFTMFLTMFGEKLNGTDPEDVIRNAFACFDEESSGFIHEDHLRELLTTMGDRFTDEEVD EMYREAPIDKKGNFNYVEFTRILKHGAKDKDD
SEQ ID No. 18SEQ ID no. 18
>gi| 63252896 I ref | NP_001018004.1 | tropomyosin 1 alpha chain isoform 3 [Homo sapiens]> Gi | 63252896 I ref | NP_001018004.1 | tropomyosin 1 alpha chain isoform 3 [Homo sapiens]
MDAIKKKMQMLKLDKENALDRAEQAEADKKAAEDRSKQLEDELVSLQKKLKGTEDELDKYSEALKDAQEKMDAIKKKMQMLKLDKENALDRAEQAEADKKAAEDRSKQLEDELVSLQKKLKGTEDELDKYSEALKDAQEK
LELAEKKATDAEADVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAADESERGMKVIESRAQKDEEKLELAEKKATDAEADVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAADESERGMKVIESRAQKDEEK
MEIQEIQLKEAKHIAEDADRKYEEVARKLVIIESDLERAEERAELSEGKCAELEEELKTVTNNLKSLEAQMEIQEIQLKEAKHIAEDADRKYEEVARKLVIIESDLERAEERAELSEGKCAELEEELKTVTNNLKSLEAQ
AEKYSQKEDRYEEEIKVLSDKLKEAETRAEFAERSVTKLEKSIDDLEEKVAHAKEENLSMHQMLDQTLLEAEKYSQKEDRYEEEIKVLSDKLKEAETRAEFAERSVTKLEKSIDDLEEKVAHAKEENLSMHQMLDQTLLE
LNNMLNNM
SEQ ID No. 19SEQ ID no. 19
>gi I 55859703 I emb I CAI10974.il tropomyosin 2 (beta) [Homo sapiens]> gi I 55859703 I emb I CAI10974.il tropomyosin 2 (beta) [Homo sapiens]
MDAIKKKMQMLKLDKENAIDRAEQAEADKKQAEDRCKQLEEEQQALQKKLKGTEDEVEKYSESVKEAQEKMDAIKKKMQMLKLDKENAIDRAEQAEADKKQAEDRCKQLEEEQQALQKKLKGTEDEVEKYSESVKEAQEK
LEQAEKKATDAEADVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAADESERGMKVIENRAMKDEEKLEQAEKKATDAEADVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAADESERGMKVIENRAMKDEEK
MELQEMQLKEAKHIAEDSDRKYEEVARKLVILEGELERSEERAEVAESRARQLEEELRTMDQALKSLMASMELQEMQLKEAKHIAEDSDRKYEEVARKLVILEGELERSEERAEVAESRARQLEEELRTMDQALKSLMAS
EEEYSTKEDKYEEEIKLLEEKLKEAETRAEFAERSVAKLEKTIDDLEETLASAKEENVEIHQTLDQTLLEEEEYSTKEDKYEEEIKLLEEKLKEAETRAEFAERSVAKLEKTIDDLEETLASAKEENVEIHQTLDQTLLE
LNNLLNNL
SEQ ID No. 20SEQ ID no. 20
>gi I 62896697 |dbj IBAD96289.1 I myosin regulatory light chain MRCL3 variant [Homo sapiens]> gi I 62896697 | dbj IBAD96289.1 I myosin regulatory light chain MRCL3 variant [Homo sapiens]
MSSKRTKTKTKKRPQRATSNVFAMFDQSQIQEFKEAFNMIDQNRNGFIDKEDLHDMLASLGKNPTDEYLD AMMNEÄPGPINFTMFLTMFGEKLNGTDPEDVIRNAFACFDEEATGTIQEDYLRELLTTMGDRFTDEEVDE LYREAPIDKKGNFNYIEFTRILKHGAKDKDDMSSKRTKTKTKKRPQRATSNVFAMFDQSQIQEFKEAFNMIDQNRNGFIDKEDLHDMLASLGKNPTDEYLD AMMNEÄPGPINFTMFLTMFGEKLNGTDPEDVIRNAFACFDEEATGTIQEDYLRELLTTMGDRFTDEEVDE LYREAPIDKKGNFNYIEFTRILKHGAKDKDD
SEQ ID No. 21SEQ ID no. 21
>gi I 13350761 emb ICAA23774.1 I alpha-2-globin [Homo sapiens]> gi I 13350761 emb ICAA23774.1 I alpha-2-globin [Homo sapiens]
VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAV AHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAV AHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKY
RR
SEQ ID No. 22SEQ ID no. 22
>gi I 12803959 I gb I AAH02827.il Tropomyosin 4 [Homo sapiens]> gi I 12803959 I gb I AAH02827.il Tropomyosin 4 [Homo sapiens]
MAGLNSLEAVKRKIQALQQQADEAEDRAQGLQRELDGERERREKAEGDVAALNRRIQLFEEELDRAQERL ATALQKLEEAEKAADESERGMKVIENRAMKDEEKMEIQEMQLKEAKHIAEEADRKYEEVARKLVILEGEL ERAEERAEVSELKCGDLEEELKNVTNNLKSLEAASEKYSEKEDKYEEEIKLLSDKLKEAETRAEFAERTV AKLEKTIDDLEEKLAQAKEENVGLHQTLDQTLNELNCIMAGLNSLEAVKRKIQALQQQADEAEDRAQGLQRELDGERERREKAEGDVAALNRRIQLFEEELDRAQERL ATALQKLEEAEKAADESERGMKVIENRAMKDEEKMEIQEMQLKEAKHIAEEADRKYEEVARKLVILEGEL ERAEERAEVSELKCGDLEEELKNVTNNLKSLEAASEKYSEKEDKYEEEIKLLSDKLKEAETRAEFAERTV AKLEKTIDDLEEKLAQAKEENVGLHQTLDQTLNELNCI
SEQ ID No. 23SEQ ID no. 23
>gi I 62205326 | gb | AAH93050.il Transgelin [Homo sapiens]> gi I 62205326 | gb | AAH93050.il Transgelin [Homo sapiens]
MANKGPSYGMSREVQSKIEKKYDEELEERLVEWIIVQCGPDVGRPDRGRLGFQVWLKNGVILSKLVNSLYMANKGPSYGMSREVQSKIEKKYDEELEERLVEWIIVQCGPDVGRPDRGRLGFQVWLKNGVILSKLVNSLY
PDGSKPVKVPENPPSMVFKQMEQVAQFLKAAEDYGVIKTDMFQTVDLFEGKDMAAVQRTLMALGSLAVTKPDGSKPVKVPENPPSMVFKQMEQVAQFLKAAEDYGVIKTDMFQTVDLFEGKDMAAVQRTLMALGSLAVTK
NDGHYRGDPNWFMKKAQEHKREFTESQLQEGKHVIGLQMGSNRGASQAGMTGYGRPRQIISNDGHYRGDPNWFMKKAQEHKREFTESQLQEGKHVIGLQMGSNRGASQAGMTGYGRPRQIIS
SEQ ID No. 24SEQ ID no. 24
>gi| 60655723|gb|AAX32425.1| keratin 7 [synthetic construct]> Gi | 60655723 | gb | AAX32425.1 | keratin 7 [synthetic construct]
MSIHFSSPVFTSRSAAFSGRGAQVRLSSARPGGLGSSSLYGLGASRPRVAVRSAYGGPVGAGIREVTINQ SLLAPLRLDADPSLQRVRQEESEQIKTLNNKFASFIDKVRFLEQQNKLLETKWTLLQEQKSAKSSRLPDI FEAQIAGLRGQLEALQVDGGRLEAELRSMQDVVEDFKNKYEDEINRRTAAENEFVVLKKDVDAAYMSKVE LEAKVDALNDEINFLRTLNETELTELQSQISDTSVVLSMDNSRSLDLDGIIAEVKAQYEEMAKCSRAEAE AWYQTKFETLQAQAGKHGDDLRNTRNEISEMNRAIQRLQAEIDNIKNQRAKLEAAIAEAEERGELALKDA RAKQEELEAALQRAKQDMARQLREYQELMSVKLALDIEIATYRKLLEGEESRLAGDGVGAVNISVMNSTG GSSSGGGIGLTLGGTMGSNALSFSSSAGPGLLKAYSIRTASASRRSARDMSIHFSSPVFTSRSAAFSGRGAQVRLSSARPGGLGSSSLYGLGASRPRVAVRSAYGGPVGAGIREVTINQ SLLAPLRLDADPSLQRVRQEESEQIKTLNNKFASFIDKVRFLEQQNKLLETKWTLLQEQKSAKSSRLPDI FEAQIAGLRGQLEALQVDGGRLEAELRSMQDVVEDFKNKYEDEINRRTAAENEFVVLKKDVDAAYMSKVE LEAKVDALNDEINFLRTLNETELTELQSQISDTSVVLSMDNSRSLDLDGIIAEVKAQYEEMAKCSRAEAE AWYQTKFETLQAQAGKHGDDLRNTRNEISEMNRAIQRLQAEIDNIKNQRAKLEAAIAEAEERGELALKDA RAKQEELEAALQRAKQDMARQLREYQELMSVKLALDIEIATYRKLLEGEESRLAGDGVGAVNISVMNSTG GSSSGGGIGLTLGGTMGSNALSFSSSAGPGLLKAYSIRTASASRRSARD
SEQ ID No. 25SEQ ID no. 25
>gi| 15277503|gb|AAH12854.1| ACTB protein [Homo sapiens]> Gi | 15277503 | gb | AAH12854.1 | ACTB protein [Homo sapiens]
MCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIVTNWDDMEKI WHHTFYNELRVAPEEHPVLLTEAPLNPKANLEKMTQIMFETFNTPAMYVAIQAVLSLYASGRTTGIVMDS GDGVTHTVPIYEGYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQ EMATAASSSSLEKSYELPDGQVITIGNERFRCPEALFQPSFLGMESCGIHETTFNSIMKCDVDIRKDLYA NTVLSGGTTMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISKQEYDES GPSIVHRKCFMCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIVTNWDDMEKI WHHTFYNELRVAPEEHPVLLTEAPLNPKANLEKMTQIMFETFNTPAMYVAIQAVLSLYASGRTTGIVMDS GDGVTHTVPIYEGYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQ EMATAASSSSLEKSYELPDGQVITIGNERFRCPEALFQPSFLGMESCGIHETTFNSIMKCDVDIRKDLYA NTVLSGGTTMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISKQEYDES GPSIVHRKCF
SEQ ID No. 26SEQ ID no. 26
>gi 33286422 I ref | NP_872271.1 | pyruvate kinase 3 isoform 2 [Homo sapiens]> gi 33286422 I ref | NP_872271.1 | pyruvate kinase 3 isoform 2 [Homo sapiens]
MSKPHSEAGTAFIQTQQLHAAMADTFLEHMCRLDIDSPPITARNTGIICTIGPASRSVETLKEMIKSGMN VARLNFSHGTHEYHAETIKNVRTATESFASDPILYRPVAVALDTKGPEIRTGLIKGSGTAEVELKKGATL KITLDNAYMEKCDENILWLDYKNICKVVEVGSKIYVDDGLISLQVKQKGADFLVTEVENGGSLGSKKGVN LPGAAVDLPAVSEKDIQDLKFGVEQDVDMVFASFIRKASDVHEVRKVLGEKGKNIKIISKIENHEGVRRF DEILEASDGIMVARGDLGIEIPAEKVFLAQKMMIGRCNRAGKPVICATQMLESMIKKPRPTRAEGSDVAN AVLDGADCIMLSGETAKGDYPLEAVRMQHLIAREAEAAMFHRKLFEELVRASSHSTDLMEAMAMGSVEAS YKCLAAALIVLTESGRSAHQVARYRPRAPIIAVTRNPQTARQAHLYRGIFPVLCKDPVQEAWAEDVDLRV NFAMNVGKARGFFKKGDVVIVLTGWRPGSGFTNTMRVVPVPMSKPHSEAGTAFIQTQQLHAAMADTFLEHMCRLDIDSPPITARNTGIICTIGPASRSVETLKEMIKSGMN VARLNFSHGTHEYHAETIKNVRTATESFASDPILYRPVAVALDTKGPEIRTGLIKGSGTAEVELKKGATL KITLDNAYMEKCDENILWLDYKNICKVVEVGSKIYVDDGLISLQVKQKGADFLVTEVENGGSLGSKKGVN LPGAAVDLPAVSEKDIQDLKFGVEQDVDMVFASFIRKASDVHEVRKVLGEKGKNIKIISKIENHEGVRRF DEILEASDGIMVARGDLGIEIPAEKVFLAQKMMIGRCNRAGKPVICATQMLESMIKKPRPTRAEGSDVAN AVLDGADCIMLSGETAKGDYPLEAVRMQHLIAREAEAAMFHRKLFEELVRASSHSTDLMEAMAMGSVEAS YKCLAAALIVLTESGRSAHQVARYRPRAPIIAVTRNPQTARQAHLYRGIFPVLCKDPVQEAWAEDVDLRV NFAMNVGKARGFFKKGDVVIVLTGWRPGSGFTNTMRVVPVP
SEQ ID No. 27SEQ ID no. 27
>gi I 56204524 | emb | CAI19482.1 | actin related protein 2/3 complex, subunit 5,> gi I 56204524 | emb | CAI19482.1 | actin related protein 2/3 complex, subunit 5,
16kDa [Homo sapiens]16kDa [Homo sapiens]
MSKNTVSSARFRKVDVDEYDENKFVDEEDGGDGQAGPDEGEVDSCLRHSITGNMTAALQAALKNPPINTKMSKNTVSSARFRKVDVDEYDENKFVDEEDGGDGQAGPDEGEVDSCLRHSITGNMTAALQAALKNPPINTK
SQAVKDRAGSIVLKVLISFKANDIEKAVQSLDKNGVDLLMKYIYKGFESPSDNSSAMLLQWHEKALAAGGSQAVKDRAGSIVLKVLISFKANDIEKAVQSLDKNGVDLLMKYIYKGFESPSDNSSAMLLQWHEKALAAGG
VGSIVRVLTARKTVVGSIVRVLTARKTV
SEQ ID No. 28SEQ ID no. 28
>gi I 37183136 I gb I AAQ89368.il AGR2 [Homo sapiens]> gi I 37183136 I gb I AAQ89368.il AGR2 [Homo sapiens]
MEKIPVSAFLLLVALSYTLARDTTVKPGAKKDTKDSRPKLPQTLSRGWGDQLIWTQTYEEALYKSKTSNK PLMIIHHLDECPHSQALKKVFAENKEIQKLAEQFVLLNLVYETTDKHLSPDGQYVPRIMFVDPSLTVRAD ITGRYSNRLYAYEPADTALLLDNMKKALKLLKTELMEKIPVSAFLLLVALSYTLARDTTVKPGAKKDTKDSRPKLPQTLSRGWGDQLIWTQTYEEALYKSKTSNK PLMIIHHLDECPHSQALKKVFAENKEIQKLAEQFVLLNLVYETTDKHLSPDGQYVPRIMFVDPSLTVRAD ITGRYSNRLYAYEPADTALLLDNMKKALKLLKTEL
SEQ ID No. 29SEQ ID no. 29
>gi|7981260|emb|CAB92118.1| stratifin [Homo sapiens]> Gi | 7981260 | emb | CAB92118.1 | stratifin [Homo sapiens]
MERASLIQKAKLAEQAERYEDMAAFMKGAVEKGEELSCEERNLLSVAYKNVVGGQRAAWRVLSSIEQKSNMERASLIQKAKLAEQAERYEDMAAFMKGAVEKGEELSCEERNLLSVAYKNVVGGQRAAWRVLSSIEQKSN
EEGSEEKGPEVREYREKVETELQGVCDTVLGLLDSHLIKEAGDAESRVFYLKMKGDYYRYLAEVATGDDKEEGSEEKGPEVREYREKVETELQGVCDTVLGLLDSHLIKEAGDAESRVFYLKMKGDYYRYLAEVATGDDK
KRIIDSARSAYQEAMDISKKEMPPTNPIRLGLALNFSVFHYEIANSPEEAISLAKTTFDEAMADLHTLSEKRIIDSARSAYQEAMDISKKEMPPTNPIRLGLALNFSVFHYEIANSPEEAISLAKTTFDEAMADLHTLSE
DSYKDSTLIMQLLRDNLTLWTADNAGEEGGEAPQEPQSDSYKDSTLIMQLLRDNLTLWTADNAGEEGGEAPQEPQS
SEQ I D No . 30SEQ ID NO. 30
>gi | 27695621 | gb | AAH42970 . 1 | Coactosin-li ke 1 ( Dictyostelium) [ Homo sapiens ]> gi | 27695621 | gb | AAH42970. 1 | Coactosin-1 (Dictyostelium) [Homo sapiens]
MATKIDKEACRAAYNLVRDDGSAVIWVT FKYDGSTIVHGEQGAEYQHFIQQCTDDVRLFAFVRFTTGDAMMATKIDKEACRAAYNLVRDDGSAVIWVT FKYDGSTIVHGEQGAEYQHFIQQCTDDVRLFAFVRFTTGDAM
SKRSKFALI TWIGENVSGLQRAKTGTDKTLVKEVVQNFAKEFVISDRKELEEDFIKSELKKAGGANYDAQSKRSKFALI TWIGENVSGLQRAKTGTDKTLVKEVVQNFAKEFVISDRKELEEDFIKSELKKAGGANYDAQ
TETE
SEQ ID No . 31SEQ ID No. 31
>gi| 6996447 | emb | CAB75426.1 | chaperonin 60, Hsp60 [Homo sapiens]> Gi | 6996447 | emb | CAB75426.1 | chaperonin 60, Hsp60 [Homo sapiens]
MLRLPTVFRQMRPVSRVLAPHLTRAYAKDVKFGADARALMLQGVDLLADAVAVTMGPKGRTVIIEQSWGS PKVTKDGVTVAKSIDLKDKYKNIGAKLVQDVANNTNEEAGDGTTTATVLARSIAKEGFEKISKGANPVEI RRGVMLAVDAVIAELKKQSKPVTTPEEIAQVATISANGDKEIGNIISDAMKKVGRKGVITVKDGKTLNDE LEIIEGMKFDRGYISPYFINTSKGQKCEFQDAYVLLSEKKISSIQSIVPALEIANAHRKPLVIIAEDVDG EALSTLVLNRLKVGLQVVAVKAPGFGDNRKNQLKDMAIATGGAVFGEEGLTLNLEDVQPHDLGKVGEVIV TKDDAMLLKGKGDKAQIEKRIQEIIEQLDVTTSEYEKEKLNERLAKLSDGVAVLKVGGTSDVEVNEKKDR VTDALNATRAAVEEGIVLGGGCALLRCIPALDSLTPANEDQKIGIEIIKRTLKIPAMTIAKNAGVEGSLI VEKIMQSSSEVGYDAMAGDFVNMVEKGIIDPTKVVRTALLDAAGVASLLTTAEVVVTEIPKEEKDPGMGA MGGMGGGMGGGMFMLRLPTVFRQMRPVSRVLAPHLTRAYAKDVKFGADARALMLQGVDLLADAVAVTMGPKGRTVIIEQSWGS PKVTKDGVTVAKSIDLKDKYKNIGAKLVQDVANNTNEEAGDGTTTATVLARSIAKEGFEKISKGANPVEI RRGVMLAVDAVIAELKKQSKPVTTPEEIAQVATISANGDKEIGNIISDAMKKVGRKGVITVKDGKTLNDE LEIIEGMKFDRGYISPYFINTSKGQKCEFQDAYVLLSEKKISSIQSIVPALEIANAHRKPLVIIAEDVDG EALSTLVLNRLKVGLQVVAVKAPGFGDNRKNQLKDMAIATGGAVFGEEGLTLNLEDVQPHDLGKVGEVIV TKDDAMLLKGKGDKAQIEKRIQEIIEQLDVTTSEYEKEKLNERLAKLSDGVAVLKVGGTSDVEVNEKKDR VTDALNATRAAVEEGIVLGGGCALLRCIPALDSLTPANEDQKIGIEIIKRTLKIPAMTIAKNAGVEGSLI VEKIMQSSSEVGYDAMAGDFVNMVEKGIIDPTKVVRTALLDAAGVASLLTTAEVVVTEIPKEEKDPGMGA MGGMGGGMGGGMF
SEQ ID No. 32SEQ ID no. 32
>gi|55960373|emb|CAI14602.1| transgelin 2 [Homo sapiens]> Gi | 55960373 | emb | CAI14602.1 | transgelin 2 [Homo sapiens]
MANRGPAYGLSREVQQKIEKQYDADLEQILIQWITTQCRKDVGRPQPGRENFQNWLKDGTVLCELINALYMANRGPAYGLSREVQQKIEKQYDADLEQILIQWITTQCRKDVGRPQPGRENFQNWLKDGTVLCELINALY
PEGQAPVKKIQASTMAFKQMEQISQFLQAAERYGINTTDIFQTVDLWEGKNMACVQRTLMNLGGLAVARDPEGQAPVKKIQASTMAFKQMEQISQFLQAAERYGINTTDIFQTVDLWEGKNMACVQRTLMNLGGLAVARD
DGLFSGDPNWFPKKSKENPRNFSDNQLQEGKNVIGLQMGTNRGASQAGMTGYGMPRQILDGLFSGDPNWFPKKSKENPRNFSDNQLQEGKNVIGLQMGTNRGASQAGMTGYGMPRQIL
SEQ ID No. 33SEQ ID no. 33
>gi I 2183299 I gb I AAC51652.il aldehyde dehydrogenase 1 [Homo sapiens]> gi I 2183299 I gb I AAC51652.il aldehyde dehydrogenase 1 [Homo sapiens]
MSSSGTPDLPVLLTDLKIQYTKIFINNEWHDSVSGKKFPVFNPATEEELCQVEEGDKEDVDKAVKAARQA FQIGSPWRTMDASERGRLLYKLADLIERDRLLLATMESMNGGKLYSNAYLSDLAGCIKTLRYCAGWADKI QGRTIPIDGNFFTYTRHEPIGVCGQIIPWNFPLVMLIWKIGPALSCGNTVVVKPAEQTPLTALHVASLIK EAGFPPGVVNIVPGYGPTAGAAISSHMDIDKVAFTGSTEVGKLIKEAAGKSNLKRVTLELGGKSPCIVLA DADLDNAVEFAHHGVFYHQGQCCIAASRIFVEESIYDEFVRRSVERAKKYILGNPLTPGVTQGPQIDKEQ YDKILDLIESGKKEGAKLECGGGPWGNKGYFVQPTVFSNVTDEMRIAKEEIFGPVQQIMKFKSLDDVIKR ANNTFYGLSAGVFTKDIDKAITISSALQAGTVWVNCYGVVSAQCPFGGFKMSGNGRELGEYGFHEYTEVK TVTVKISQKNSMSSSGTPDLPVLLTDLKIQYTKIFINNEWHDSVSGKKFPVFNPATEEELCQVEEGDKEDVDKAVKAARQA FQIGSPWRTMDASERGRLLYKLADLIERDRLLLATMESMNGGKLYSNAYLSDLAGCIKTLRYCAGWADKI QGRTIPIDGNFFTYTRHEPIGVCGQIIPWNFPLVMLIWKIGPALSCGNTVVVKPAEQTPLTALHVASLIK EAGFPPGVVNIVPGYGPTAGAAISSHMDIDKVAFTGSTEVGKLIKEAAGKSNLKRVTLELGGKSPCIVLA DADLDNAVEFAHHGVFYHQGQCCIAASRIFVEESIYDEFVRRSVERAKKYILGNPLTPGVTQGPQIDKEQ YDKILDLIESGKKEGAKLECGGGPWGNKGYFVQPTVFSNVTDEMRIAKEEIFGPVQQIMKFKSLDDVIKR ANNTFYGLSAGVFTKDIDKAITISSALQAGTVWVNCYGVVSAQCPFGGFKMSGNGRELGEYGFHEYTEVK TVTVKISQKNS
SEQ ID No. 34SEQ ID no. 34
>gi I 49660012 I gb| AAT68294.1 I sarcomeric tropomyosin kappa; TPMl-kappa [Homo sapiens]> gi I 49660012 I gb | AAT68294.1 I sarcomeric tropomyosin kappa; TPMl-kappa [Homo sapiens]
MDAIKKKMQMLKLDKENALDRAEQAEADKKAAEDRSKQLEEDIAAKEKLLRVSEDERDRVLEELHKAEDSMDAIKKKMQMLKLDKENALDRAEQAEADKKAAEDRSKQLEEDIAAKEKLLRVSEDERDRVLEELHKAEDS
LLAAEEAAAKAEADVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAADESERGMKVIESRAQKDEEKLLAAEEAAAKAEADVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAADESERGMKVIESRAQKDEEK
MEIQEIQLKEAKHIAEDADRKYEEVARKLVIIESDLERAEERAELSEGKCAELEEELKTVTNDLKSLEAQMEIQEIQLKEAKHIAEDADRKYEEVARKLVIIESDLERAEERAELSEGKCAELEEELKTVTNDLKSLEAQ
AEKYSQKEDRYEEEIKVLSDKLKEAETRAEFAERSVTKLEKSIDDLEDELYAQKLKYKAISEELDHALNDAEKYSQKEDRYEEEIKVLSDKLKEAETRAEFAERSVTKLEKSIDDLEDELYAQKLKYKAISEELDHALND
MTSIMTSI
SEQ ID No. 35SEQ ID no. 35
>gi| 12654115|gb|AAH00871.1| Annexin A3 [Homo sapiens]> Gi | 12654115 | gb | AAH00871.1 | Annexin A3 [Homo sapiens]
MASIWVGHRGTVRDYPDFSPSVDAEAIQKAIRGIGTDEKMLISILTERSNAQRQLIVKEYQAAYGKELKDMASIWVGHRGTVRDYPDFSPSVDAEAIQKAIRGIGTDEKMLISILTERSNAQRQLIVKEYQAAYGKELKD
DLKGDLSGHFEHLMVALVTPPAVFDAKQLKKSMKGAGTNEDALIEILTTRTSRQMKDISQAYYTVYKKSLDLKGDLSGHFEHLMVALVTPPAVFDAKQLKKSMKGAGTNEDALIEILTTRTSRQMKDISQAYYTVYKKSL
GDDISSETSGDFRKALLTLADGRRDESLKVDEHLAKQDAQILYKAGENRWGTDEDKFTEILCLRSFPQLKGDDISSETSGDFRKALLTLADGRRDESLKVDEHLAKQDAQILYKAGENRWGTDEDKFTEILCLRSFPQLK
LTFDEYRNISQKDIVDSIKGELSGHFEDLLLAIVNCVRNTPAFLAERLHRALKGIGTDEFTLNRIMVSRSLTFDEYRNISQKDIVDSIKGELSGHFEDLLLAIVNCVRNTPAFLAERLHRALKGIGTDEFTLNRIMVSRS
EIDLLDIRTEFKKHYGYSLYSAIKSDTSGDYEITLLKICGGDDEIDLLDIRTEFKKHYGYSLYSAIKSDTSGDYEITLLKICGGDD
SEQ ID No. 36SEQ ID no. 36
>gi I 18462107 I gb I AAL72118.il delta-globin [Homo sapiens]> gi I 18462107 I gb I AAL72118.il delta-globin [Homo sapiens]
MVHLTPEEKTAVNALWGKVNVDAVGGEALGRLLVVYPWTQRFFESFGDLSSPDAVMGNPKVKAHGKKVLGMVHLTPEEKTAVNALWGKVNVDAVGGEALGRLLVVYPWTQRFFESFGDLSSPDAVMGNPKVKAHGKKVLG
AFSDGLAHLDNLKGTFSQLSELHCDKLHVDPENFRLLGNVLVCVLARNFGKEFTPQMQAAYQKVVAGVANAFSDGLAHLDNLKGTFSQLSELHCDKLHVDPENFRLLGNVLVCVLARNFGKEFTPQMQAAYQKVVAGVAN
ALAHKYHALAHKYH
SEQ ID No. 37SEQ ID no. 37
>gi I 28592 | emb | CAA23754.1 | serum albumin [Homo sapiens]> gi I 28592 | emb | CAA23754.1 | serum albumin [Homo sapiens]
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEV TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADD RADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFK QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEK TPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHK PKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLMKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEV TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELR DEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADD RADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFK QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEK TPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHK PKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
SEQ ID No. 38SEQ ID no. 38
>gi| 189617 | gb | AAC41689.1| protein PP4-X> Gi | 189617 | gb | AAC41689.1 | protein PP4-X
MAMATKGGTVKAASGFNAMEDAQTLRKAMKGLGTDEDAIISVLAYRNTAQRQEIRTAYKSTIGRDLIDDLMAMATKGGTVKAASGFNAMEDAQTLRKAMKGLGTDEDAIISVLAYRNTAQRQEIRTAYKSTIGRDLIDDL
KSELSGNFEQVIVGMMTPTVLYDVQELQRAMKGAGTDEGCLIEILASRTPEEIRRISQTYQQQYGRSLEDKSELSGNFEQVIVGMMTPTVLYDVQELQRAMKGAGTDEGCLIEILASRTPEEIRRISQTYQQQYGRSLED
DIRSDTSFMFQRVLVSLSAGGRDEGNYLDDALVRQDAQDLYEAGEKKWGTDEVKFLTVLCSRNRNHLLHVDIRSDTSFMFQRVLVSLSAGGRDEGNYLDDALVRQDAQDLYEAGEKKWGTDEVKFLTVLCSRNRNHLLHV
FDEYKRISQKDIEQSIKSETSGSFEDALLAIVKCMRNKSAYFAEKLYKSMKGLGTDDNTLIRVMVSRAEIFDEYKRISQKDIEQSIKSETSGSFEDALLAIVKCMRNKSAYFAEKLYKSMKGLGTDDNTLIRVMVSRAEI
DMLDIRAHFKRLYGKSLYSFIKGDTSGDYRKVLLVLCGGDDDMLDIRAHFKRLYGKSLYSFIKGDTSGDYRKVLLVLCGGDD
SEQ ID No. 39SEQ ID no. 39
>gi I 28634 I emb I CAA32891.il crystallin [Homo sapiens]> gi I 28634 I emb I CAA32891.il crystallin [Homo sapiens]
MDVTIQHPWFKRTLGPFYPSRLFDQFFGEGLFEYDLLPFLSSTISPYYRQSLFRTVLDSGISEVRSDRDKMDVTIQHPWFKRTLGPFYPSRLFDQFFGEGLFEYDLLPFLSSTISPYYRQSLFRTVLDSGISEVRSDRDK
FVIFLDVKHFSPEDLTVKVQDDFVEIHGKHNERQFVIFLDVKHFSPEDLTVKVQDDFVEIHGKHNERQ
SEQ ID No. 40SEQ ID no. 40
>gi I 2605594 |dbj IBAA23323.1 I myosin regulatory light chain [Homo sapiens]> gi I 2605594 | dbj IBAA23323.1 I myosin regulatory light chain [Homo sapiens]
MSSKKAKTKTTKKRPQRATSNVFAMFDQSQIQEFKEAFNMIDQNRDGFIDKEDLHDMLASLGKNPTDAYLMSSKKAKTKTTKKRPQRATSNVFAMFDQSQIQEFKEAFNMIDQNRDGFIDKEDLHDMLASLGKNPTDAYL
DAMMNEAPGPINFTMFLTMFGEKLNGTDPEDVIRNAFACFDEEATGTIQEDYLRELLTTMGDRFTDEGVDDAMMNEAPGPINFTMFLTMFGEKLNGTDPEDVIRNAFACFDEEATGTIQEDYLRELLTTMGDRFTDEGVD
ELYREAPIDKKGNFNYIEFTRILKHGAKDKDDELYREAPIDKKGNFNYIEFTRILKHGAKDKDD
SEQ ID No. 41 ■SEQ ID no. 41 ■
>gi I 2183299 |gb| AAC51652.1 I aldehyde dehydrogenase 1 [Homo sapiens] MSSSGTPDLPVLLTDLKIQYTKIFINNEWHDSVSGKKFPVFNPATEEELCQVEEGDKEDVDKAVKAARQA FQIGSPWRTMDASERGRLLYKLADLIERDRLLLATMESMNGGKLYSNAYLSDLAGCIKTLRYCAGWADKI QGRTIPIDGNFFTYTRHEPIGVCGQIIPWNFPLVMLIWKIGPALSCGNTVVVKPAEQTPLTALHVASLIK EAGFPPGVVNIVPGYGPTAGAAISSHMDIDKVAFTGSTEVGKLIKEAAGKSNLKRVTLELGGKSPCIVLA DADLDNAVEFAHHGVFYHQGQCCIAASRIFVEESIYDEFVRRSVERAKKYILGNPLTPGVTQGPQIDKEQ YDKILDLIESGKKEGAKLECGGGPWGNKGYFVQPTVFSNVTDEMRIAKEEIFGPVQQIMKFKSLDDVIKR ANNTFYGLSAGVFTKDIDKAITISSALQAGTVWVNCYGVVSAQCPFGGFKMSGNGRELGEYGFHEYTEVK TVTVKISQKNS> gi I 2183299 | gb | AAC51652.1 I aldehyde dehydrogenase 1 [Homo sapiens] MSSSGTPDLPVLLTDLKIQYTKIFINNEWHDSVSGKKFPVFNPATEEELCQVEEGDKEDVDKAVKAARQA FQIGSPWRTMDASERGRLLYKLADLIERDRLLLATMESMNGGKLYSNAYLSDLAGCIKTLRYCAGWADKI QGRTIPIDGNFFTYTRHEPIGVCGQIIPWNFPLVMLIWKIGPALSCGNTVVVKPAEQTPLTALHVASLIK EAGFPPGVVNIVPGYGPTAGAAISSHMDIDKVAFTGSTEVGKLIKEAAGKSNLKRVTLELGGKSPCIVLA DADLDNAVEFAHHGVFYHQGQCCIAASRIFVEESIYDEFVRRSVERAKKYILGNPLTPGVTQGPQIDKEQ YDKILDLIESGKKEGAKLECGGGPWGNKGYFVQPTVFSNVTDEMRIAKEEIFGPVQQIMKFKSLDDVIKR ANNTFYGLSAGVFTKDIDKAITISSALQAGTVWVNCYGVVSAQCPFGGFKMSGNGRELGEYGFHEYTEVK TVTVKISQKNS
SEQ ID No. 42SEQ ID no. 42
>gi I 21361176 I ref | NP_000680.2 | aldehyde dehydrogenase IAl [Homo sapiens]> gi I 21361176 I ref | NP_000680.2 | aldehydes dehydrogenase IAl [Homo sapiens]
MSSSGTPDLPVLLTDLKIQYTKIFINNEWHDSVSGKKFPVFNPATEEELCQVEEGDKEDVDKAVKAARQAMSSSGTPDLPVLLTDLKIQYTKIFINNEWHDSVSGKKFPVFNPATEEELCQVEEGDKEDVDKAVKAARQA
FQIGSPWRTMDASERGRLLYKLADLIERDRLLLATMESMNGGKLYSNAYLNDLAGCIKTLRYCAGWADKIFQIGSPWRTMDASERGRLLYKLADLIERDRLLLATMESMNGGKLYSNAYLNDLAGCIKTLRYCAGWADKI
QGRTIPIDGNFFTYTRHEPIGVCGQIIPWNFPLVMLIWKIGPALSCGNTVVVKPAEQTPLTALHVASLIKQGRTIPIDGNFFTYTRHEPIGVCGQIIPWNFPLVMLIWKIGPALSCGNTVVVKPAEQTPLTALHVASLIK
EAGFPPGVVNIVPGYGPTAGAAISSHMDIDKVAFTGSTEVGKLIKEAAGKSNLKRVTLELGGKSPCIVLAEAGFPPGVVNIVPGYGPTAGAAISSHMDIDKVAFTGSTEVGKLIKEAAGKSNLKRVTLELGGKSPCIVLA
DADLDNAVEFAHHGVFYHQGQCCIAASRIFVEESIYDEFVRRSVERAKKYILGNPLTPGVTQGPQIDKEQDADLDNAVEFAHHGVFYHQGQCCIAASRIFVEESIYDEFVRRSVERAKKYILGNPLTPGVTQGPQIDKEQ
YDKILDLIESGKKEGAKLECGGGPWGNKGYFVQPTVFSNVTDEMRIAKEEIFGPVQQIMKFKSLDDVIKRYDKILDLIESGKKEGAKLECGGGPWGNKGYFVQPTVFSNVTDEMRIAKEEIFGPVQQIMKFKSLDDVIKR
ANNTFYGLSAGVFTKDIDKAITISSALQAGTVWVNCYGVVSAQCPFGGFKMSGNGRELGEYGFHEYTEVKANNTFYGLSAGVFTKDIDKAITISSALQAGTVWVNCYGVVSAQCPFGGFKMSGNGRELGEYGFHEYTEVK
TVTVKISQKNSTVTVKISQKNS
SEQ ID No. 43SEQ ID no. 43
T-complex protein 1 subunit betaT-complex protein 1 subunit beta
ASLSLAPVNI FKAGADEERA ETARLTSFIG AIAIGDLVKS TLGPKGMDKI LLSSGRDASL MVTNDGATIL KNIGVDNPAA KVLVDMSRVQ DDEVGDGTTS VTVLAAELLR EAESLIAKKI HPQTIIAGWR EATKAAREAL LSSAVDHGSD EVKFRQDLMN IAGTTLSSKL LTHHKDHFTK LAVEAVLRLK GSGNLEAIHI IKKLGGSLAD SYLDEGFLLD KKIGVNQPKR IENAKILIAN TGMDTDKIKI FGSRVRVDST AKVAEIEHAE KEKMKEKVER ILKHGINCFI NRQLIYNYPE QLFGAAGVMA IEHADFAGVE RLALVTGGEI ASTFDHPELV KLGSCKLIEE VMIGEDKLIH FSGVALGEAC TIVLRGATQQ ILDEAERSLH DALCVLAQTV KDSRTVYGGG CSEMLMAHAV TQLANRTPGK EAVAMESYAK ALRMLPTIIA DNAGYDSADL VAQLRAAHSE GNTTAGLDMR EGTIGDMAIL GITESFQVKR QVLLSAAEAA EVILRVDNII KAAPRKRVPD HHPCASLSLAPVNI FKAGADEERA ETARLTSFIG AIAIGDLVKS TLGPKGMDKI LLSSGRDASL MVTNDGATIL KNIGVDNPAA KVLVDMSRVQ DDEVGDGTTS VTVLAAELLR EAESLIAKKI HPQTIIAGWR EATKAAREAL LSSAVDHGSD EVKFRQDLMN IAGTTLSSKL LTHHKDHFTK LAVEAVLRLK GSGNLEAIHI IKKLGGSLAD SYLDEGFLLD KKIGVNQPKR IENAKILIAN TGMDTDKIKI FGSRVRVDST AKVAEIEHAE KEKMKEKVER ILKHGINCFI NRQLIYNYPE QLFGAAGVMA IEHADFAGVE RLALVTGGEI ASTFDHPELV KLGSCKLIEE VMIGEDKLIH FSGVALGEAC TIVLRGATQQ ILDEAERSLH DALCVLAQTV KDSRTVYGGG CSEMLMAHAV TQLANRTPGK EAVAMESYAK ALRMLPTIIA DNAGYDSADL VAQLRAAHSE GNTTAGLDMR EGTIGDMAIL GITESFQVKR QVLLSAAEAA EVILRVDNII KAAPRKRVPD HHPC
SEQ ID No. 44 Apolipoprotein A-IV mflkavvltl alvavagara evsadqvatv mwdyfsqlsn nakeavehlq kseltqqlna Ifqdklgevn tyagdlqkkl vpfatelher lakdseklke eigkeleelr arllphanev sqkigdnlre lqqrlepyad qlrtqvntqa eqlrrqldpl aqrmervlre nadslqaslr phadelkaki dqnveelkgr ltpyadefkv kidqtveelr rslapyaqdt qeklnhqleg ltfqmkknae elkarisasa eelrqrlapl aedvrgnlkg nteglqksla elgghldqqv eefrrrvepy genfnkalvq qmeqlrqklg phagdveghl sflekdlrdk vnsffstfke kesqdktlsl peleqqqeqq qeqqqeqvqm laplesSEQ ID no. 44 apolipoprotein A-IV mflkavvltl alvavagara evsadqvatv mwdyfsqlsn nakeavehlq kseltqqlna Ifqdklgevn tyagdlqkkl vpfatelher lakdseklke eigkeleelr arllphanev sqkigdnlre lqqrlepyad qlrtqvntqa eqlrrqldpl aqrmervlre nadslqaslr phadelkaki dqnveelkgr ltpyadefkv kidqtveelr rslapyaqdt qeklnhqleg ltfqmkknae elkarisasa eelrqrlapl aedvrgnlkg nteglqksla elgghldqqv eefrrrvepy genfnkalvq qmeqlrqklg phagdveghl sflekdlrdk vnsffstfke kesqdktlsl peleqqqeqq qeqqqeqvqm laples
SEQ ID No. 45 Malate dehydrogenase, mitochondrial precursor MLSALARPVS AALRRSFSTS AQNNAKVAVL GASGGIGQPL SLLLKNSPLV SRLTLYDIAH TPGVAADLSH IETKAAVKGY LGPEQLPDCL KGCDVVVIPA GVPRKPGMTR DDLFNTNATI VATLTAACAQ HCPEAMICVI ANPVNSTIPI TAEVFKKHGV YNPNKIFGVT TLDIVRANTF VAELKGLDPA RVNVPVIGGH AGKTIIPLIS QCTPKVDFPQ DQLTALTGRI QEAGTEVVKA KAGAGSATLS MAYAGARFVF SLVDAMNGKE GVVECSFVKS 'QETECTYFST PLLLGKKGIE KNLGIGKVSS FEEKMISDAI PELKASIKKG EDFVKTLKSEQ ID no. 45 Malate dehydrogenase, mitochondrial precursor MLSALARPVS AALRRSFSTS AQNNAKVAVL GASGGIGQPL SLLLKNSPLV SRLTLYDIAH TPGVAADLSH IETKAAVKGY LGPEQLPDCL KGCDVVVIPA GVPRKPGMTR DDLFNTNATI VATLTAACAQ HCPEAMICVI ANPVNSTIPI TAEVFKKHGV YNPNKIFGVT TLDIVRANTF VAELKGLDPA RVNVPVIGGH AGKTIIPLIS QCTPKVDFPQ DQLTALTGRI QEAGTEVVKA KAGAGSATLS MAYAGARFVF SLVDAMNGKE GVVECSFVKS 'QETECTYFST PLLLGKKGIE KNLGIGKVSS FEEKMISDAI PELKASIKKG EDFVKTLK
SEQ ID No. 46SEQ ID no. 46
Voltage-dependent anion-selective Channel protein 1Voltage-dependent anion-selective channel protein 1
AVPPTYADLG KSARDVFTKG YGFGLIKLDL KTKSENGLEF TSSGSANTET TKVTGSLETK YRWTEYGLTF TEKWNTDNTL GTEITVEDQL ARGLKLTFDS SFSPNTGKKN AKIKTGYKRE HINLGCDMDF DIAGPSIRGA LVLGYEGWLA GYQMNFETAK SRVTQSNFAV GYKTDEFQLH TNVNDGTEFG GSIYQKVNKK LETAVNLAWT AGNSNTRFGI AAKYQIDPDA CFSAKVNNSS LIGLGYTQTL KPGIKLTLSA LLDGKNVNAG GHKLGLGLEF QAAVPPTYADLG KSARDVFTKG YGFGLIKLDL KTKSENGLEF TSSGSANTET TKVTGSLETK YRWTEYGLTF TEKWNTDNTL GTEITVEDQL ARGLKLTFDS SFSPNTGKKN AKIKTGYKRE HINLGCDMDF DIAGPSIRGA LVLGYEGWLA GYQMNFETAK SRVTQSNFAV GYKTDEFQLH TNVNDGTEFG GSIYQKVNKK LETAVNLAWT AGNSNTRFGI AAKYQIDPDA CFSAKVNNSS LIGLGYTQTL KPGIKLTLSA LLDGKNVNAG GHKLGLGLEF QA
SEQ ID No. 47SEQ ID no. 47
>gi I 31645 | emb | CAA25833.1 | glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens]> gi I 31645 | emb | CAA25833.1 | Glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens]
MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVIN GNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKY DNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPAS TGAAKAVGKVIPELDGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQ VVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKEMGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVIN GNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKY DNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPAS TGAAKAVGKVIPELDGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQ VVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE
SEQ ID No. 48SEQ ID no. 48
>gi I 35053 I emb|CAA37794.1 I uracil DNA glycosylase [Homo sapiens]> gi I 35053 I emb | CAA37794.1 I uracil DNA glycosylase [Homo sapiens]
MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVIN GNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKY DNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGNCGVMAAGLSRTSSLPL LALKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQV VSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMASKEMGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVIN GNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKY DNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGNCGVMAAGLSRTSSLPL LALKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQV VSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMASKE
SEQ ID No. 49SEQ ID no. 49
>gi I 54303910 I gb| AAV33305.1 I aging-associated gene 9 protein [Homo sapiens]> gi I 54303910 I gb | AAV33305.1 I aging-associated gene 9 protein [Homo sapiens]
MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVINMGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVIN
GNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISTPSADAPMLVMGVNHEKYGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISTPSADAPMLVMGVNHEKY
DNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPAS
TGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQ
VVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKEVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE
SEQ ID No. 50SEQ ID no. 50
>gi I 13543557 I gb I AAH05935.il Nipsnap homolog 3A (C. elegans) [Homo sapiens]> gi I 13543557 I gb I AAH05935.il Nipsnap homolog 3A (C. elegans) [Homo sapiens]
MLVLRSALTRALASRTLAPQMCSSFATGPRQYDGIFYEFRSYYLKPSKMNEFLENFEKNAHLRTAHSELV GYWSVEFGGRMNTVFHIWKYDNFAHRTEVRKALAKDKEWQEQFLIPNLALIDKQESEITYLVPWCKLEKP PKEGVYELATFQMKPGGPALWGDAFKRAVHAHVNLGYTKLVGVFHTEYGALNRVHVLWWNESADSRAAGR HKSHEDPRVVAAVRESVNYLVSQQNMLLIPTSFSPLKMLVLRSALTRALASRTLAPQMCSSFATGPRQYDGIFYEFRSYYLKPSKMNEFLENFEKNAHLRTAHSELV GYWSVEFGGRMNTVFHIWKYDNFAHRTEVRKALAKDKEWQEQFLIPNLALIDKQESEITYLVPWCKLEKP PKEGVYELATFQMKPGGPALWGDAFKRAVHAHVNLGYTKLVGVFHTEYGALNRVHVLWWNESADSRAAGR HKSHEDPRVVAAVRESVNYLVSQQNMLLIPTSFSPLK
SEQ ID No. 51SEQ ID no. 51
>gi|33188452|ref |NP_859427.1| peroxiredoxin 2 isoform b [Homo sapiens]> gi | 33188452 | ref | NP_859427.1 | peroxiredoxin 2 isoform b [Homo sapiens]
MASGNARIGKPAPDFKATAVVDGAFKEVKLSDYKGINTPRKEGGLGPLNIPLLADVTRRLSEDYGVLKTDMASGNARIGKPAPDFKATAVVDGAFKEVKLSDYKGINTPRKEGGLGPLNIPLLADVTRRLSEDYGVLKTD
EGIAYRGLFIIDGKGVLRQITVNDLPVGRSVDEALRLVQAFQYTDEHGEVCPAGWKPGSDTIKPNVDDSKEGIAYRGLFIIDGKGVLRQITVNDLPVGRSVDEALRLVQAFQYTDEHGEVCPAGWKPGSDTIKPNVDDSK
EYFSKHNEYFSKHN
SEQ ID No. 52SEQ ID no. 52
>gi I 438069 I emb| CAA80269.1 I thiol-specific antioxidant protein [Homo sapiens]> gi I 438069 I emb | CAA80269.1 I thiol-specific antioxidant protein [Homo sapiens]
MASGNARIGKPAPDFKATAVVDGAFKEVKLSDYKGKYVVLFFYPLDFTFVCPTEIIAFTTVKRTSAKLGC EVLGVSVDSQFTHLAWINTPRKEGGLGPLNIPLLADVTRRLSEDYGVLKNDEGIAYRGLFIIDGKGVLRQ ITVNDLPVGRSVDEALRLVQAFQYTDEHGEVCPAAWKPGRDTIKPNVDDSKEYFSKHNMASGNARIGKPAPDFKATAVVDGAFKEVKLSDYKGKYVVLFFYPLDFTFVCPTEIIAFTTVKRTSAKLGC EVLGVSVDSQFTHLAWINTPRKEGGLGPLNIPLLADVTRRLSEDYGVLKNDEGIAYRGLFIIDGKGVLRQ ITVNDLPVGRSVDEALRLVQAFQYTDEHGEVCPAAWKPGRDTIKPNVDDSKEYFSKHN
SEQ ID No. 53SEQ ID no. 53
>gi I 440308 I gb I AAA50465.1 I enhancer protein> gi I 440308 I gb I AAA50465.1 I enhancer protein
MASGNARIGKPAPDFKATAVVDGAFKEVKLSDYKGKYVVLFFYPLDFTFVCPTEIIAFSNRAEDFRKLGC EVLGVSVDSQFNHLAWINTPRKEGGLGPLNIPLLGDVTRRLSEDYGVLKTDEGIAYRGLFIIDGKGVLRQ ITVNDLPVGRSVDEALRLVQAFQYTDEHGEVCPAGWKPGSDTIKPNVDDSKEYFSKHNMASGNARIGKPAPDFKATAVVDGAFKEVKLSDYKGKYVVLFFYPLDFTFVCPTEIIAFSNRAEDFRKLGC EVLGVSVDSQFNHLAWINTPRKEGGLGPLNIPLLGDVTRRLSEDYGVLKTDEGIAYRGLFIIDGKGVLRQ ITVNDLPVGRSVDEALRLVQAFQYTDEHGEVCPAGWKPGSDTIKPNVDDSKEYFSKHN
SEQ ID No. 54SEQ ID no. 54
>gi I 16198390 I gb| AAH15848.1 I Chromosome 17 open reading frame 25 [Homo sapiens]> gi I 16198390 I gb | AAH15848.1 I Chromosomes 17 open reading frame 25 [Homo sapiens]
MAARRALHFVFKVGNRFQTARFYRDVLGMKVLRHEEFEEGCKAACNGPYDGKWSKTMVGFGPEDDHFVAEMAARRALHFVFKVGNRFQTARFYRDVLGMKVLRHEEFEEGCKAACNGPYDGKWSKTMVGFGPEDDHFVAE
LTYNYGVGDYKLGNDFMGITLASSQAVSNARKLEWPLTEVAEGVFETEAPGGYKFYLQNRSLPQSDPVLKLTYNYGVGDYKLGNDFMGITLASSQAVSNARKLEWPLTEVAEGVFETEAPGGYKFYLQNRSLPQSDPVLK
VTLAVSDLQKSLNYWCNLLGMKIYEKDEEKQRALLGYADNQCKLELQGVKGGVDHAAAFGRIAFSCPQKEVTLAVSDLQKSLNYWCNLLGMKIYEKDEEKQRALLGYADNQCKLELQGVKGGVDHAAAFGRIAFSCPQKE
LPDLEDLMKRENQKILTPLVSLDTPGKATVQVVILADPDGHEICFVGDEAFRELSKIDPEGSKLLDDAMALPDLEDLMKRENQKILTPLVSLDTPGKATVQVVILADPDGHEICFVGDEAFRELSKIDPEGSKLLDDAMA
ADKSDEWFAKHNKPKASGADKSDEWFAKHNKPKASG
SEQ ID No. 55SEQ ID no. 55
>gi| 34850074 | ref | NP_057164.2 | hypothetical protein LOC51031 [Homo sapiens]> Gi | 34850074 | ref | NP_057164.2 | hypothetical protein LOC51031 [Homo sapiens]
MAARRALHFVFKVGNRFQTARFYRDVLGMKVLRHEEFEEGCKAACNGPYDGKWSKTMVGFGPEDDHFVAEMAARRALHFVFKVGNRFQTARFYRDVLGMKVLRHEEFEEGCKAACNGPYDGKWSKTMVGFGPEDDHFVAE
LTYNYGVGDYKLGNDFMGITLASSQAVSNARKLEWPLTEVAEGVFETEAPGGYKFYLQNRSLPQSDPVLKLTYNYGVGDYKLGNDFMGITLASSQAVSNARKLEWPLTEVAEGVFETEAPGGYKFYLQNRSLPQSDPVLK
VTLAVSDLQKSLNYWCNLLGMKIYEKDEEKQRALLGYADNQCKLELQGVKGGVDHAAAFGRIAFSCPQKEVTLAVSDLQKSLNYWCNLLGMKIYEKDEEKQRALLGYADNQCKLELQGVKGGVDHAAAFGRIAFSCPQKE
LPDLEDLMKRENQKILTPLVSLDTPGKATVQVVILADPDGHEICFVGDEAFRELSKMDPEGSKLLDDAMSLPDLEDLMKRENQKILTPLVSLDTPGKATVQVVILADPDGHEICFVGDEAFRELSKMDPEGSKLLDDAMS
ADKSDEWFAKHNKPKASGADKSDEWFAKHNKPKASG
SEQ ID No. 56SEQ ID no. 56
>gi|4929769|gb|AAD34145.1|AF151908_l CGI-150 protein [Homo sapiens]> gi | 4929769 | gb | AAD34145.1 | AF151908_l CGI-150 protein [Homo sapiens]
MRLTPFSLSTGNSFRYSRRLKKNIFGTAPALRVSEMSLRPSSRIFPCFSRNGLDFTIVITLAQPPVPGIS FIVAKPRLFPGAGSAGCGLLERLFLSLLLGTGLRWCLRGCFPGARFCSTTSPEGHTTFTGLRRSARTQRL AQGPKPGPPAATVARQTSRVSPAPPCSLRPGLRHESAPSGIGDVTARGALRGLGCTVRVTAACGGNHGCS QMLHFVFKVGNRFQTARFYRDVLGMKVLRHEEFEEGCKAACNGPYDGKWSKTMVGFGPEDDHFVAELTYN YGVGDYKLGNDFMGITLASSQAVSNARKLEWPLTEVAEGVFETEAPGGYKFYLQNRSLPQSDPVLKVTLA VSDLQKSLNYWCNLLGMKIYEKDEEKQRALLGYADNQCKLELQGVKGGVDHAAAFGRIAFSCPQKELPDL EDLMKRENQKILTPLVSLDTPGKATVQVVILADPDGHEICFVGDEAFRELSKMDPEGSKLLDDAMAADKS DEWFAKHNKPKASGMRLTPFSLSTGNSFRYSRRLKKNIFGTAPALRVSEMSLRPSSRIFPCFSRNGLDFTIVITLAQPPVPGIS FIVAKPRLFPGAGSAGCGLLERLFLSLLLGTGLRWCLRGCFPGARFCSTTSPEGHTTFTGLRRSARTQRL AQGPKPGPPAATVARQTSRVSPAPPCSLRPGLRHESAPSGIGDVTARGALRGLGCTVRVTAACGGNHGCS QMLHFVFKVGNRFQTARFYRDVLGMKVLRHEEFEEGCKAACNGPYDGKWSKTMVGFGPEDDHFVAELTYN YGVGDYKLGNDFMGITLASSQAVSNARKLEWPLTEVAEGVFETEAPGGYKFYLQNRSLPQSDPVLKVTLA VSDLQKSLNYWCNLLGMKIYEKDEEKQRALLGYADNQCKLELQGVKGGVDHAAAFGRIAFSCPQKELPDL EDLMKRENQKILTPLVSLDTPGKATVQVVILADPDGHEICFVGDEAFRELSKMDPEGSKLLDDAMAADKS DEWFAKHNKPKASG
SEQ ID No. 57 >gi|19684181|gb|AAH26033.1 | Gelsolin (amyloidosis, Finnish type) [Homo sapiens]SEQ ID no. 57 > gi | 19684181 | gb | AAH26033.1 | Gelsolin (amyloidosis, Finnish type) [Homo sapiens]
MAPHRPAPALLCALSLALCALSLPVRAATASRGASQAGAPQGRVPEARPNSMVVEHPEFLKAGKEPGLQIMAPHRPAPALLCALSLALCALSLPVRAATASRGASQAGAPQGRVPEARPNSMVVEHPEFLKAGKEPGLQI
WRVEKFDLVPVPTNLYGDFFTGDAYVILKTVQLRNGNLQYDLHYWLGNECSQDESGAAAIFTVQLDDYLNWRVEKFDLVPVPTNLYGDFFTGDAYVILKTVQLRNGNLQYDLHYWLGNECSQDESGAAAIFTVQLDDYLN
GRAVQHREVQGFESATFLGYFKSGLKYKKGGVASGFKHVVPNEVVVQRLFQVKGRRVVRATEVPVSWESFGRAVQHREVQGFESATFLGYFKSGLKYKKGGVASGFKHVVPNEVVVQRLFQVKGRRVVRATEVPVSWESF
NNGDCFILDLGNNIHQWCGSNSNRYERLKATQVSKGIRDNERSGRARVHVSEEGTEPEAMLQVLGPKPALNNGDCFILDLGNNIHQWCGSNSNRYERLKATQVSKGIRDNERSGRARVHVSEEGTEPEAMLQVLGPKPAL
PAGTEDTAKEDAANRKLAKLYKVSNGAGTMSVSLVADENPFAQGALKSEDCFILDHGKDGKIFVWKGKQAPAGTEDTAKEDAANRKLAKLYKVSNGAGTMSVSLVADENPFAQGALKSEDCFILDHGKDGKIFVWKGKQA
NTEERKAALKTASDFITKMDYPKQTQVSVLPEGGETPLFKQFFKNWRDPDQTDGLGLSYLSSHIANVERVNTEERKAALKTASDFITKMDYPKQTQVSVLPEGGETPLFKQFFKNWRDPDQTDGLGLSYLSSHIANVERV
PFDAATLHTSTAMAAQHGMDDDGTGQKQIWRIEGSNKVPVDPATYGQFYGGDSYIILYNYRHGGRQGQIIPFDAATLHTSTAMAAQHGMDDDGTGQKQIWRIEGSNKVPVDPATYGQFYGGDSYIILYNYRHGGRQGQII
YNWQGAQSTQDEVAASAILTAQLDEELGGTPVQSRVVQGKEPAHLMSLFGGKPMIIYKGGTSREGGQTAPYNWQGAQSTQDEVAASAILTAQLDEELGGTPVQSRVVQGKEPAHLMSLFGGKPMIIYKGGTSREGGQTAP
ASTRLFQVRANSAGATRAVEVLPKAGALNSNDAFVLKTPSAAYLWVGTGASEAEKTGAQELLRVLRAQPVASTRLFQVRANSAGATRAVEVLPKAGALNSNDAFVLKTPSAAYLWVGTGASEAEKTGAQELLRVLRAQPV
QVAEGSEPDGFWEALGGKAAYRTSPRLKDKKMDAHPPRLFACSNKIGRFVIEEVPGELMQEDLATDDVMLQVAEGSEPDGFWEALGGKAAYRTSPRLKDKKMDAHPPRLFACSNKIGRFVIEEVPGELMQEDLATDDVML
LDTWDQVFVWVGKDSQEEEKTEALTSAKRYIETDPANRDRRTPITVVKQGFEPPSFVGWFLGWDDDYWSVLDTWDQVFVWVGKDSQEEEKTEALTSAKRYIETDPANRDRRTPITVVKQGFEPPSFVGWFLGWDDDYWSV
DPLDRAMAELAADPLDRAMAELAA
SEQ ID No. 58 Gelsolin precursorSEQ ID no. 58 gelsolin precursor
MAPHRPAPAL LCALSLALCA LSLPVRAATA SRGASQAGAP QGRVPEARPN SMVVEHPEFL KAGKEPGLQI WRVEKFDLVP VPTNLYGDFF TGDAYVILKT VQLRNGNLQY DLHYWLGNEC SQDESGAAAI FTVQLDDYLN GRAVQHREVQ GFESATFLGY FKSGLKYKKG GVASGFKHVV PNEVVVQRLF QVKGRRVVRA TEVPVSWESF NNGDCFILDL GNNIHQWCGS NSNRYERLKAMAPHRPAPAL LCALSLALCA LSLPVRAATA SRGASQAGAP QGRVPEARPN SMVVEHPEFL KAGKEPGLQI WRVEKFDLVP VPTNLYGDFF TGDAYVILKT VQLRNGNLQY DLHYWLGNEC SQDESGAAAI FTVQLDDYLN GRAVQHREVQ GFESATFLGY FKSGLKYKKG GVASGFKHVV PNEVVVQRLF QVKGRRVVRA TEVPVSWESF NNGDCFILDL GNNIHQWCGS NSNRYERLKA
TQVSKGIRDN ERSGRARVHV SEEGTEPEAM LQVLGPKPAL PAGTEDTAKE DAANRKLAKLTQVSKGIRDN ERSGRARVHV SEEGTEPEAM LQVLGPKPAL PAGTEDTAKE DAANRKLAKL
YKVSNGAGTM SVSLVADENP FAQGALKSED CFILDHGKDG KIFVWKGKQA NTEERKAALK TASDFITKMD YPKQTQVSVL PEGGETPLFK QFFKNWRDPD QTDGLGLSYL SSHIANVERV PFDAATLHTS TAMAAQHGMD DDGTGQKQIW RIEGSNKVPV DPATYGQFYG GDSYIILYNY RHGGRQGQII YNWQGAQSTQ DEVAASAILT AQLDEELGGT PVQSRVVQGK EPAHLMSLFG GKPMIIYKGG TSREGGQTAP ASTRLFQVRA NSAGATRAVE VLPKAGALNS NDAFVLKTPSYKVSNGAGTM SVSLVADENP FAQGALKSED CFILDHGKDG KIFVWKGKQA NTEERKAALK TASDFITKMD YPKQTQVSVL PEGGETPLFK QFFKNWRDPD QTDGLGLSYL SSHIANVERV PFDAATLHTS TAMAAQHGMD DDGTGQKQIW RIEGSNKVPV DPATYGQFYG GDSYIILYNY RHGGRQGQII YNWQGAQSTQ DEVAASAILT AQLDEELGGT PVQSRVVQGK EPAHLMSLFG GKPMIIYKGG TSREGGQTAP ASTRLFQVRA NSAGATRAVE VLPKAGALNS NDAFVLKTPS
AAYLWVGTGA SEAEKTGAQE LLRVLRAQPV QVAEGSEPDG FWEALGGKAA YRTSPRLKDKAAYLWVGTGA SEAEKTGAQE LLRVLRAQPV QVAEGSEPDG FWEALGGKAA YRTSPRLKDK
KMDAHPPRLF ACSNKIGRFV IEEVPGELMQ EDLATDDVML LDTWDQVFVW VGKDSQEEEK TEALTSAKRY IETDPANRDR RTPITVVKQG FEPPSFVGWF LGWDDDYWSV DPLDRAMAELKMDAHPPRLF ACSNKIGRFV IEEVPGELMQ EDLATDDVML LDTWDQVFVW VGKDSQEEEK TEALTSAKRY IETDPANRDR RTPITVVKQG FEPPSFVGWF LGWDDDYWSV DPLDRAMAEL
AAAA
SEQ ID No. 59SEQ ID no. 59
>gi I 3766197 |gb| AAC64396.1 I ATP-specific succinyl-CoA synthetase beta subunit [Homo sapiens]> gi I 3766197 | gb | AAC64396.1 I ATP-specific succinyl-CoA synthetase beta subunit [Homo sapiens]
FNNHGLQVQQQQQRNLSLHEYMSMELLQEAGVSVPKGYVAKSPDEAYAIAKKLGSKDVVIKAQVLAGGRGFNNHGLQVQQQQQRNLSLHEYMSMELLQEAGVSVPKGYVAKSPDEAYAIAKKLGSKDVVIKAQVLAGGRG
KGTFESGLKGGVKIVFSPEEAKAVSSQMIGKKLFTKQTGEKGRICNQVLVCERKYPRREYYFAITMERSFKGTFESGLKGGVKIVFSPEEAKAVSSQMIGKKLFTKQTGEKGRICNQVLVCERKYPRREYYFAITMERSF
QGPVLIGSSHGGVNIEDVAAETPEAIIKEPIDIEEGIKKEQALQLAQKMGFPPNIVESAAENMVKLYSLFQGPVLIGSSHGGVNIEDVAAETPEAIIKEPIDIEEGIKKEQALQLAQKMGFPPNIVESAAENMVKLYSLF
LKYDATMIEINPMVEDSDGAVLCMDAKINFDSNSAYRQKKIFDLQDWTQEDERDKDAAKANLNYIGLDGNLKYDATMIEINPMVEDSDGAVLCMDAKINFDSNSAYRQKKIFDLQDWTQEDERDKDAAKANLNYIGLDGN
IGCLVNGAGLAMATMDIIKLHGGTPANFLDVGGGATVHQVTEAFKLITSDKKVLAILVNIFGGIMRCDVIIGCLVNGAGLAMATMDIIKLHGGTPANFLDVGGGATVHQVTEAFKLITSDKKVLAILVNIFGGIMRCDVI
AQGIVMAVKDLEIKIPVVVRLQGTRVDDAKALIADSGLKILACDDLDEAARMVVKLSEIVTLAKQAHVDVAQGIVMAVKDLEIKIPVVVRLQGTRVDDAKALIADSGLKILACDDLDEAARMVVKLSEIVTLAKQAHVDV
KFQLPIKFQLPI
SEQ ID No. 60SEQ ID no. 60
>gi I 56204104 I emb I CAI22099.il TAR DNA binding protein [Homo sapiens]> gi I 56204104 I emb I CAI22099.il TAR DNA binding protein [Homo sapiens]
MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGN LVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLK TGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREF FSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGVHL ISNVYGRSTSLKVVLMSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGN LVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLK TGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREF FSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGVHL ISNVYGRSTSLKVVL
SEQ ID No. 61SEQ ID no. 61
2, 4-dienoyl-CoA reductase, mitochondrial precursor2, 4-dienoyl-CoA reductase, mitochondrial precursor
MKLPARVFFT LGSRLPCGLA PRRFFSYGTK ILYQNTEALQ SKFFSPLQKA MLPPNSFQGK VAFITGGGTG LGKGMTTLLS SLGAQCVIAS RKMDVLKATA EQISSQTGNK VHAIQCDVRD PDMVQNTVSE LIKVAGHPNI VINNAAGNFI SPTERLSPNA WKTITDIVLN GTAFVTLEIG KQLIKAQKGA AFLSITTIYA ETGSGFVVPS ASAKAGVEAM SKSLAAEWGK YGMRFNVIQP GPIKTKGAFS RLDPTGTFEK EMIGRIPCGR LGTVEELANL AAFLCSDYAS WINGAVIKFD GGEEVLISGE FNDLRKVTKE QWDTIEELIR KTKGSMKLPARVFFT LGSRLPCGLA PRRFFSYGTK ILYQNTEALQ SKFFSPLQKA MLPPNSFQGK VAFITGGGTG LGKGMTTLLS SLGAQCVIAS RKMDVLKATA EQISSQTGNK VHAIQCDVRD PDMVQNTVSE LIKVAGHPNI VINNAAGNFI SPTERLSPNA WKTITDIVLN GTAFVTLEIG KQLIKAQKGA AFLSITTIYA ETGSGFVVPS ASAKAGVEAM SKSLAAEWGK YGMRFNVIQP GPIKTKGAFS RLDPTGTFEK EMIGRIPCGR LGTVEELANL AAFLCSDYAS WINGAVIKFD GGEEVLISGE FNDLRKVTKE QWDTIEELIR KTKGS
SEQ ID No. 62SEQ ID no. 62
>gi|49168580|emb|CAG38785.1 I MDH2 [Homo sapiens]> gi | 49168580 | emb | CAG38785.1 I MDH2 [Homo sapiens]
MLSALVRPVSAALRRSFSTSAQNNAKVAVLGASGGIGQPLSLLLKNSPLVSRLTLYDIAHTPGVAADLSHMLSALVRPVSAALRRSFSTSAQNNAKVAVLGASGGIGQPLSLLLKNSPLVSRLTLYDIAHTPGVAADLSH
IETKAAVKGYLGPEQLPDCLKGCDVVVIPAGVPRKPGMTRDDLFNTNATIVATLTAACAQHCPEAMICVIIETKAAVKGYLGPEQLPDCLKGCDVVVIPAGVPRKPGMTRDDLFNTNATIVATLTAACAQHCPEAMICVI
ANPVNSTIPITAEVFKKHGVYNPNKIFGVTTLDIVRANTFVAELKGLDPARVNVPVIGGHAGKTIIPLISANPVNSTIPITAEVFKKHGVYNPNKIFGVTTLDIVRANTFVAELKGLDPARVNVPVIGGHAGKTIIPLIS
QCTPKVDFPQDQLTALTGRIQEAGTEVVKAKAGAGSATLSMAYAGARFVFSLVDAMNGKEGVVECSFVKSQCTPKVDFPQDQLTALTGRIQEAGTEVVKAKAGAGSATLSMAYAGARFVFSLVDAMNGKEGVVECSFVKS
QETECTYFSTPLLLGKKGIEKNLGIGKVSSFEEKMISDAIPELKASIKKGEDFVKTLK SEQ ID No. 63QETECTYFSTPLLLGKKGIEKNLGIGKVSSFEEKMISDAIPELKASIKKGEDFVKTLK SEQ ID no. 63
Heat-shock protein beta-1Heat-shock protein beta-1
MTERRVPFSL LRGPSWDPFR DWYPHSRLFD QAFGLPRLPE EWSQWLGGSS WPGYVRPLPP ΆAIESPAVAA PAYSRALSRQ LSSGVSEIRH TADRWRVSLD VNHFAPDELT VKTKDGVVEI TGKHEERQDE HGYISRCFTR KYTLPPGVDP TQVSSSLSPE GTLTVEAPMP KLATQSNEIT IPVTFESRAQ LGGPEAAKSD ETAAKMTERRVPFSL LRGPSWDPFR DWYPHSRLFD QAFGLPRLPE EWSQWLGGSS WPGYVRPLPP ΆAIESPAVAA PAYSRALSRQ LSSGVSEIRH TADRWRVSLD VNHFAPDELT VKTKDGVVEI TGKHEERQDE HGYISRCFTR KYTLPPGVDP TQVSSSLSPE GTLTVEAPMP KLATQSNEIT IPVTFESRAQ LGGPEAAKSD ETAAK
SEQ ID No. 64SEQ ID no. 64
>gi I 21735621 I ref |NP_005909.2 I mitochondrial malate dehydrogenase precursorI ref | NP_005909.2 I mitochondrial malate dehydrogenase precursor
[Homo sapiens][Homo sapiens]
MLSALARPASAALRRSFSTSAQNNAKVAVLGASGGIGQPLSLLLKNSPLVSRLTLYDIAHTPGVAADLSH IETKAAVKGYLGPEQLPDCLKGCDVVVIPAGVPRKPGMTRDDLFNTNATIVATLTAACAQHCPEAMICVI ANPVNSTIPITAEVFKKHGVYNPNKIFGVTTLDIVRANTFVAELKGLDPARVNVPVIGGHAGKTIIPLIS QCTPKVDFPQDQLTALTGRIQEAGTEVVKAKAGAGSATLSMAYAGARFVFSLVDAMNGKEGVVECSFVKS QETECTYFSTPLLLGKKGIEKNLGIGKVSSFEEKMISDAIPELKASIKKGEDFVKTLKMLSALARPASAALRRSFSTSAQNNAKVAVLGASGGIGQPLSLLLKNSPLVSRLTLYDIAHTPGVAADLSH IETKAAVKGYLGPEQLPDCLKGCDVVVIPAGVPRKPGMTRDDLFNTNATIVATLTAACAQHCPEAMICVI ANPVNSTIPITAEVFKKHGVYNPNKIFGVTTLDIVRANTFVAELKGLDPARVNVPVIGGHAGKTIIPLIS QCTPKVDFPQDQLTALTGRIQEAGTEVVKAKAGAGSATLSMAYAGARFVFSLVDAMNGKEGVVECSFVKS QETECTYFSTPLLLGKKGIEKNLGIGKVSSFEEKMISDAIPELKASIKKGEDFVKTLK
SEQ ID No. 65SEQ ID no. 65
>gi|27753613|gb|AAO22156.1|AF202897_l prostate and colon associated protein> gi | 27753613 | gb | AAO22156.1 | AF202897_l prostate and colon associated protein
[Homo sapiens][Homo sapiens]
MDCREMDLYEDYQSPFDFDAGVNKSYLYLSPSGNSSPPGSPTLQKFGLLRTDPVPEEGEDVAATISATET LSEEEQEELRRELAKVEEEIQTLSQVLAAKEKHLAEIKRKLGINSLQELKQNIAKGWQDVTATSAYKKTS ETLSQAGQKASAAFSSVGSVITKKLEDVKNSPTFKSFEEKVENLKSKVGGTKPAGGDFGEVLNSAANASA TTTEPLPEKTQESLMDCREMDLYEDYQSPFDFDAGVNKSYLYLSPSGNSSPPGSPTLQKFGLLRTDPVPEEGEDVAATISATET LSEEEQEELRRELAKVEEEIQTLSQVLAAKEKHLAEIKRKLGINSLQELKQNIAKGWQDVTATSAYKKTS ETLSQAGQKASAAFSSVGSVITKKLEDVKNSPTFKSFEEKVENLKSKVGGTKPAGGDFGEVLNSAANASA TTTEPLPEKTQESL
SEQ ID No. 66SEQ ID no. 66
>gi I 3757661 | emb | CAA76365.1 | secretagogin [Homo sapiens]> gi I 3757661 | emb | CAA76365.1 | secretagogin [Homo sapiens]
MDSSREPTLGRLDAAGFWQVWRRFDADEKGYIEEKELDAFFLHMLMKLGTDDTVMKANLHKVKQQFMTTQMDSSREPTLGRLDAAGFWQVWRRFDADEKGYIEEKELDAFFLHMLMKLGTDDTVMKANLHKVKQQFMTTQ
DASKDGRIRMKELAGMFLSEDENFLLLFRRENPLDSSVEFMQIWRKYDADSSGFISAAELRNFLRDLFLHDASKDGRIRMKELAGMFLSEDENFLLLFRRENPLDSSVEFMQIWRKYDADSSGFISAAELRNFLRDLFLH
HKKAISEAKLEEYTGTMMKIFDRNKDGRLDLNDLARILALQENFLLQFKMDACSTEERKRDFEKIFAYYDHKKAISEAKLEEYTGTMMKIFDRNKDGRLDLNDLARILALQENFLLQFKMDACSTEERKRDFEKIFAYYD
VSKTGALEGPEVDGFVKDMMELVQPSISGVDLDKFREILLRHCDVNKDGKIQKSELALCLGLKINPVSKTGALEGPEVDGFVKDMMELVQPSISGVDLDKFREILLRHCDVNKDGKIQKSELALCLGLKINP
SEQ ID No. 67SEQ ID no. 67
>gi|49457021|emb|CAG46831.1| TPD52 [Homo sapiens]> Gi | 49457021 | emb | CAG46831.1 | TPD52 [Homo sapiens]
MDRGEQGLLRTDPVPEEGEDVAATISATETLSEEEQEELRRELAKVEEEIQTLSQVLAAKEKHLAEIKRKMDRGEQGLLRTDPVPEEGEDVAATISATETLSEEEQEELRRELAKVEEEIQTLSQVLAAKEKHLAEIKRK
LGINSLQELKQNIAKGWQDVTATSAYKKTSETLSQAGQKASAAFSSVGSVITKKPEDVKNSPTFKSFEEKLGINSLQELKQNIAKGWQDVTATSAYKKTSETLSQAGQKASAAFSSVGSVITKKPEDVKNSPTFKSFEEK
VENLKSKVGGTKPAGGDFGEVLNSAANASATTTEPLPEKTQESLVENLKSKVGGTKPAGGDFGEVLNSAANASATTTEPLPEKTQESL
SEQ ID No. 68SEQ ID no. 68
>gi|54695758 | gb | AAV38251.1 | tumor protein D52 [Homo sapiens]> gi | 54695758 | gb | AAV38251.1 | tumor protein D52 [Homo sapiens]
MDRGEQGLLRTDPVPEEGEDVAATISATETLSEEEQEELRRELAKVEEEIQTLSQVLAAKEKHLAEIKRK LGINSLQELKQNIAKGWQDVTATSAYKKTSETLSQAGQKASAAFSSVGSVITKKLEDVKNSPTFKSFEEK VENLKSKVGGTKPAGGDFGEVLNSAANASATTTEPLPEKTQESLMDRGEQGLLRTDPVPEEGEDVAATISATETLSEEEQEELRRELAKVEEEIQTLSQVLAAKEKHLAEIKRK LGINSLQELKQNIAKGWQDVTATSAYKKTSETLSQAGQKASAAFSSVGSVITKKLEDVKNSPTFKSFEEK VENLKSKVGGTKPAGGDFGEVLNSAANASATTPLPLSQESL
SEQ ID No. 69SEQ ID no. 69
>gi I 62898994 I dbj IBAD97351.il N8 protein long isoform (Fragment) variant> gi I 62898994 I dbj IBAD97351.il N8 protein long isoform (fragment) variant
[Homo sapiens][Homo sapiens]
RESPAEARRSSARRGGRSEPGRAAGGGAAEDTRRRAGDMDRGEQGLLRTDPVPEEGEDVAATISATETLSRESPAEARRSSARRGGRSEPGRAAGGGAAEDTRRRAGDMDRGEQGLLRTDPVPEEGEDVAATISATETLS
EKEQEELRRELAKVEEEIQTLSQVLAAKEKHLAEIKRKLGINSLQELKQNIAKGWQDVTATSAYKKTSETEKEQEELRRELAKVEEEIQTLSQVLAAKEKHLAEIKRKLGINSLQELKQNIAKGWQDVTATSAYKKTSET
LSQAGQKASAAFSSVGSVITKKLEDVKNSPTFKSFEEKVENLKSKVGGTKPAGGDFGEVLNSAANASATTLSQAGQKASAAFSSVGSVITKKLEDVKNSPTFKSFEEKVENLKSKVGGTKPAGGDFGEVLNSAANASATT
TEPLPEKTQESLTEPLPEKTQESL
SEQ ID No. 70SEQ ID no. 70
>gi I 70608174 I ref |NP_001020424.1 I tumor protein D52 isoform 2 [Homo sapiens]> gi I 70608174 I ref | NP_001020424.1 I tumor protein D52 isoform 2 [Homo sapiens]
MDRGEQGLLRTDPVPEEGEDVAATISATETLSEEEQEELRRELAKVEEEIQTLSQVLAAKEKHLAEIKRKMDRGEQGLLRTDPVPEEGEDVAATISATETLSEEEQEELRRELAKVEEEIQTLSQVLAAKEKHLAEIKRK
LGINSLQELKQNIAKGWQDVTATSAYKKTSETLSQAGQKASAAFSSVGSVITKKLEDVKLQAFSHSFSIRLGINSLQELKQNIAKGWQDVTATSAYKKTSETLSQAGQKASAAFSSVGSVITKKLEDVKLQAFSHSFSIR
SIQHSISMPAMRNSPTFKSFEEKVENLKSKVGGTKPAGGDFGEVLNSAANASATTTEPLPEKTQESLSIQHSISMPAMRNSPTFKSFEEKVENLKSKVGGTKPAGGDFGEVLNSAANASATTTEPLPEKTQESL
SEQ ID No. 71SEQ ID no. 71
>gi I 4507645 I ref I NP_000356.1 I triosephosphate isomerase 1 [Homo sapiens]> I 4507645 I ref I NP_000356.1 I triosephosphate isomerase 1 [Homo sapiens]
MAPSRKFFVGGNWKMNGRKQSLGELIGTLNAAKVPADTEVVCAPPTAYIDFARQKLDPKIAVAAQNCYKV TNGAFTGEISPGMIKDCGATWVVLGHSERRHVFGESDELIGQKVAHALAEGLGVIACIGEKLDEREAGIT EKVVFEQTKVIADNVKDWSKVVLAYEPVWAIGTGKTATPQQAQEVHEKLRGWLKSNVSDAVAQSTRIIYG GSVTGATCKELASQPDVDGFLVGGASLKPEFVDIINAKQ MAPSRKFFVGGNWKMNGRKQSLGELIGTLNAAKVPADTEVVCAPPTAYIDFARQKLDPKIAVAAQNCYKV TNGAFTGEISPGMIKDCGATWVVLGHSERRHVFGESDELIGQKVAHALAEGLGVIACIGEKLDEREAGIT EKVVFEQTKVIADNVKDWSKVVLAYEPVWAIGTGKTATPQQAQEVHEKLRGWLKSNVSDAVAQSTRIIYG GSVTGATCKELASQPDVDGFLVGGASLKPEFVDIINAKQ

Claims

Patentansprüche claims
1. Verfahren zur Diagnose von Pankreaskrebs oder einer Vorläufer- und/oder Begleiterkrankungen, wobei eine Bestimmung mindestens eines Biomarkers ausgewählt aus der Gruppe a.) Keratin 8 protein (SEQ ID No. 1), Vimentin (SEQ ID No. 2), Mitochondrial malate dehydrogenase (SEQ ID No. 3), Beta tropomyosin (SEQ ID No. 4), ACTGl protein (SEQ ID No. 5), Thioredoxin delta 3 (SEQ ID No. 6), B Chain B Triosephosphate Isomerase (SEQ ID No. I)1 Annexin A2 (SEQ ID No. 8), TPM4-ALK fusion oncoprotein type 2 (SEQ ID No. 9), Peptidylprolyl isomerase A (SEQ ID No. 10), Smooth muscle mysoin light chain (SEQ ID No. 11), Desmin (SEQ ID No. 12), Major vault protein 1 (SEQ ID No. 13),A method for diagnosing pancreatic cancer or a precursor and / or comorbidities, wherein a determination of at least one biomarker selected from the group a.) Keratin 8 protein (SEQ ID No. 1), Vimentin (SEQ ID No. 2), mitochondrial malate dehydrogenase (SEQ ID No. 3), beta tropomyosin (SEQ ID No. 4), ACTGl protein (SEQ ID No. 5), thioredoxin delta 3 (SEQ ID No. 6), B chain B triosephosphate isomerase (SEQ ID No I) 1 annexin A2 (SEQ ID No. 8), TPM4-ALK fusion oncoprotein type 2 (SEQ ID No. 9), peptidylprolyl isomerase A (SEQ ID No. 10), smooth muscle mysoin light chain (SEQ ID No. 9). 11), desmin (SEQ ID No. 12), major vault protein 1 (SEQ ID No. 13),
Heterogeneous nuclear ribonucleoprotein Al (SEQ ID No. 14), SlOOAlO (SEQ ID No. 15), EFla-like protein (SEQ ID No. 16), Regulatory myosin light chain long version (SEQ ID No. 17), Tropomyosin 1 alpha chain isoform 3 (SEQ ID No. 18), Tropomyosin 2 (beta) isoform 2 (SEQ ID No. 19), Myosin regulatory light chain MRCL3 (SEQ ID No. 20), Alpha-2-globin (SEQ ID No. 21), Tropomyosin 4 (SEQ ID No. 22), Transgelin (SEQ ID No. 23), Keratin 7 (SEQ ID No. 24), ACTB protein (SEQ ID No. 25), M2-type pyruvate kinase (SEQ ID No. 26), Actin related protein 2/3 complex subunit 5 (SEQ ID No. 27), Anterior gradient 2 homolog (AGR 2) (SEQ ID No.28), Stratifin (14-3-3 sigma) (SEQ ID No. 29), Coactosin-like 1 (SEQ ID No. 30), Chaperonin heat shock 6OkD protein 1 (SEQ ID No. 31), Transgelin 2 (SEQ ID No. 32), Aldehyde dehydrogenase 1 (SEQ ID No. 33), Sarcomeric tropomyosin kappa (SEQ ID No. 34), Annexin A3 (SEQ ID No. 35), Delta-globin (SEQ ID No. 36), Serum albumin (SEQ ID No. 37), Protein PP4-X (Annexin A4) (SEQ ID No. 38), Crystallin (SEQ ID No. 39), Myosin regulatory light chain MRCL3 (SEQ ID No. 40), oder Gruppe b.) aldehyde dehydrogenase 1 (SEQ ID No. 41), Aldehyde dehydrogenase IAl (SEQ ID No. 42), T-complex protein 1 subunit beta (SEQ ID No. 43), Apolipoprotein A4 (SEQ ID No. 44), Malate dehydrogenase mitochondrial precursor (SEQ ID No. 45), Voltage-dependent anion selective Channel protein 1 (SEQ ID No. 46), glyceraldehydes-3-phosphate dehydrogenase (SEQ ID No. 47), uracil DNA glycosylase (SEQ ID No. 48), aging- associated-associated 9 protein (SEQ ID No. 49), Nipsnap homolog 3A (SEQ ID No. 50), peroxiredoxin 2 isoform b (SEQ ID No. 51), thiol-specific antioxidant protein (SEQ ID No. 52), enhancer protein (SEQ ID No. 53), Chromosome 17 open reading frame 25 (SEQ ID No. 54), hypothetical protein LOC51031 (SEQ ID No. 55), CGI-150 protein (SEQ ID No. 56), Gelsolin isoform a (SEQ ID No. 57), Gelsolin ' precursor (SEQ ID No. 58), ATP-specific succinyl-CoA synthetase beta subunit (SEQ ID No. 59), TAR DNA binding protein (SEQ ID No. 60), 2, 4-dienoyl-CoA reductase mitochondrial precursor (SEQ ID No. 61), MDH2 (SEQ ID No. 62), heat shock protein beta-1 (SEQ ID No. 63), mitochondrial malate dehydrogenase precursor MDH-2 (SEQ ID No. 64), prostate and colon associated protein (SEQ ID No. 65), secretagogin (SEQ ID No. 66), TPD 52 (SEQ ID No. 67), tumor protein D52 (SEQ ID No. 68), N8 protein long isoform (Fragment) variant (SEQ ID No. 69), tumor protein D52 isoform 2 (SEQ ID No. 70), triosephosphate isomerase 1 (SEQ ID No. 71) oder Fragmenten und Teilpeptiden davon an einem zu' untersuchenden Patienten erfolgt.Heterogeneous nuclear ribonucleoprotein Al (SEQ ID No. 14), SlOOAlO (SEQ ID No. 15), EFla-like protein (SEQ ID No. 16), regulatory myosin light chain long version (SEQ ID No. 17), tropomyosin 1α chain isoform 3 (SEQ ID No. 18), tropomyosin 2 (beta) isoform 2 (SEQ ID No. 19), myosin regulatory light chain MRCL3 (SEQ ID No. 20), alpha-2-globin (SEQ ID No. 21 ), Tropomyosin 4 (SEQ ID No. 22), transgelin (SEQ ID No. 23), keratin 7 (SEQ ID No. 24), ACTB protein (SEQ ID No. 25), M2-type pyruvate kinase (SEQ ID No 26), actin related protein 2/3 complex subunit 5 (SEQ ID No. 27), anterior gradient 2 homologue (AGR 2) (SEQ ID No.28), stratifin (14-3-3 sigma) (SEQ ID No 29), coactosin-like 1 (SEQ ID No. 30), chaperonin heat shock 6OkD protein 1 (SEQ ID No. 31), transgelin 2 (SEQ ID No. 32), aldehyde dehydrogenase 1 (SEQ ID No. 33), sarcomeric tropomyosin kappa (SEQ ID No. 34), annexin A3 (SEQ ID No. 35), delta-globin (SEQ ID No. 36), serum albumin (SEQ ID No. 37), protein PP4-X (annexin A4) (SEQ ID No. 38), crystallin (SEQ ID No. 39) , Myosin regulatory light chain MRCL3 (SEQ ID No. 40), or group b.) Aldehyde dehydrogenase 1 (SEQ ID No. 41), aldehyde dehydrogenase IAI (SEQ ID No. 42), T-complex protein 1 subunit beta (SEQ ID No. 43), apolipoprotein A4 (SEQ ID No. 44), malate dehydrogenase mitochondrial precursor (SEQ ID No. 45), voltage-dependent anion selective channel protein 1 (SEQ ID No. 46), glyceraldehyde-3-phosphate dehydrogenase (SEQ ID No. 47), uracil DNA glycosylase (SEQ ID No. 48), aging-associated-9 protein (SEQ ID No. 49), Nipsnap homolog 3A (SEQ ID No. 50), peroxiredoxin 2 isoform b ( SEQ ID No. 51), thiol-specific antioxidant protein (SEQ ID No. 52), enhancer protein (SEQ ID No. 53), chromosomes 17 open reading frame 25 (SEQ ID No. 54), hypothetical protein LOC51031 (SEQ ID No. 55), CGI-150 protein (SEQ ID No. 56), gelsolin isoform a (SEQ ID No. 57) , gelsolin 'precursor (SEQ ID No. 58), ATP-specific succinyl-CoA synthetase beta subunit (SEQ ID No. 59), TAR DNA binding protein (SEQ ID No. 60), 2, 4-dienoyl-CoA reductase mitochondrial precursor (SEQ ID No. 61), MDH2 (SEQ ID No. 62), heat shock protein beta-1 (SEQ ID No. 63), mitochondrial malate dehydrogenase precursor MDH-2 (SEQ ID No. 64), prostate and colon associated protein (SEQ ID No. 65), secretagogin (SEQ ID No. 66), TPD 52 (SEQ ID No. 67), tumor protein D52 (SEQ ID No. 68), N8 protein long isoform (fragment) variant (SEQ ID No. 69), tumor protein D52 isoform 2 (SEQ ID No. 70), triosephosphate isomerase 1 (SEQ ID No. 71) or fragments and partial peptides thereof is carried out at a under investigation for 'patients.
2. Verfahren zur Diagnose von Pankreaskrebs oder einer2. Method for the diagnosis of pancreatic cancer or a
Vorläufer- und/oder Begleiterkrankungen nach Anspruch 1, dadurch gekennzeichnet, dass die Diagnose eine in-vitro Diagnose ist.Precursor and / or comorbidities according to claim 1, characterized in that the diagnosis is an in vitro diagnosis.
3. Verfahren zur Diagnose von Pankreaskrebs oder einer Vorläufer- und/oder Begleiterkrankungen nach Anspruch 1 oder 2, wobei die Vorläufer- und/oder Begleiterkrankungen PDAC (Pancreatic ductal adenocarcinoma) , PanIN (Pankreatische intraepitheliale Neoplasien) , Pankreasläsionen, CP (Chronische Pankreatitis) , einschließlich endokriner Tumoren der Pankreas ist.3. A method of diagnosing pancreatic cancer or precursor and / or comorbidities according to claim 1 or 2, wherein the precursor and / or comorbidities PDAC (pancreatic ductal adenocarcinoma), PanIN (pancreatic intraepithelial neoplasia), pancreatic lesions, CP (chronic pancreatitis) including endocrine tumors of the pancreas.
4. Verfahren zur Diagnose von Pankreaskrebs oder einer Vorläufer- und/oder Begleiterkrankungen nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass eine Kombination der Biomarker nach Anspruch 1 zumindest Stratifin (14-3-3 sigma) (SEQ ID No. 29) und / oder Vimentin (SEQ ID No. 2) und / oder Major vault protein 1 (SEQ ID No. 13) und / oder Anterior gradient 2 homolog (AGR 2) (SEQ ID No.28), und / oder SlOOAlO (SEQ ID No. 15) und / oder EFla-like protein (SEQ ID No. 16) und / oder Annexin A2 (SEQ ID No. 8) und / oder Annexin A4 (SEQ ID No. 38) enthält/enthalten.4. Method for the diagnosis of pancreatic cancer or precursor and / or comorbidities according to claim 1 or 2, characterized in that a combination of the biomarkers according to claim 1 at least stratifin (14-3-3 sigma) (SEQ ID No. 29) and or vimentin (SEQ ID No. 2) and / or major vault protein 1 (SEQ ID No. 13) and / or anterior gradient 2 homolog (AGR 2) (SEQ ID No. 28), and / or SlOOAlO (SEQ ID No. 15) and / or EFla-like protein (SEQ ID No. 16) and / or Annexin A2 (SEQ ID No. 8) and / or Annexin A4 (SEQ ID No. 38) /contain.
5. Verfahren zur Diagnose von Pankreaskrebs nach Anspruch 1 bis 4 zur Durchführung von klinischen Entscheidungen, insbesondere weiterführende Behandlung und Therapie mittels Arzneimitteln.5. A method for the diagnosis of pancreatic cancer according to claim 1 to 4 for the implementation of clinical decisions, in particular further treatment and therapy by means of drugs.
6. Verfahren zur Diagnose nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, dass die Diagnose zur Prognose, zur differentialdiagnostischen Früherkennung und Erkennung, zur Beurteilung des Schweregrades und zur therapiebegleitenden Verlaufsbeurteilung erfolgt.6. A method of diagnosis according to any one of claims 1 to 5, characterized in that the diagnosis for prognosis, for the differential diagnostic early detection and detection, for the assessment of the severity and the therapy-accompanying course assessment takes place.
7. Verfahren nach einem der vorherigen Ansprüche, dadurch gekennzeichnet, dass parallele oder simultane Bestimmungen der Marker durchgeführt werden.7. The method according to any one of the preceding claims, characterized in that parallel or simultaneous determinations of the markers are carried out.
8. Verfahren nach einem der vorherigen Ansprüche, dadurch gekennzeichnet, dass eine 2D-Elektrophorese erfolgt, wobei in der ersten Dimension eine isoelektrische Fokussierung, in der zweiten Dimension eine Gelelektrophorese durchgeführt wird.8. The method according to any one of the preceding claims, characterized in that a 2D electrophoresis takes place, wherein in the first dimension an isoelectric focusing, in the second dimension, a gel electrophoresis is performed.
9. Verfahren nach einem der vorherigen Ansprüche, dadurch gekennzeichnet, dass die Bestimmungen an mindestens einer Patientenprobe durchgeführt werden. 9. The method according to any one of the preceding claims, characterized in that the determinations are carried out on at least one patient sample.
10. Verfahren nach einem der vorherigen Ansprüche, dadurch gekennzeichnet, dass die Bestimmungen mittels einem Schnelltest, insbesondere in Einzel- oder Multiparameterbestimmungen durchgeführt werden.10. The method according to any one of the preceding claims, characterized in that the determinations are carried out by means of a rapid test, in particular in single or multiparameter determinations.
11. Kit zur Diagnose nach einem der Ansprüche 1-10 enthaltend Nachweisreagenzien und weitere Hilfsmittel.11. kit for diagnosis according to any one of claims 1-10 containing detection reagents and other auxiliaries.
12. Diagnostische Vorrichtung zur Durchführung eines Verfahrens nach einem der Ansprüche 1 bis 10, insbesondere ein Proteinbiochip, Array oder Assay. 12. A diagnostic device for performing a method according to any one of claims 1 to 10, in particular a protein biochip, array or assay.
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