Polypeptide Markers for the Diagnosis and Evaluation of Vascular Diseases

摘要

A process for diagnosing vascular diseases (VD), comprising the step of determining the presence or absence of at least one polypeptide marker in a sample, wherein said polypeptide marker is selected from markers 1 to 526, which are characterized by values for the molecular masses and migration times (CE times).

主权项

1. A method of diagnosing vascular diseases (VD) in a subject patient, comprising:
obtaining a urine sample from said subject patient; purifying said urine sample to remove high concentration proteins; separating said purified urine sample into a plurality of polypeptides; identifying said plurality of purified polypeptides based on the separation characteristics of each of said plurality of purified polypeptides in the separating step; comparing said plurality of purified polypeptides to known polypeptide markers taken from control subjects with VD and without VD to obtain a subset of polypeptide markers from said plurality of purified polypeptides that substantially match said known polypeptide markers, wherein said known polypeptide markers are characterized by the following molecular masses and migration times (CE time):No. MassCE time11166.6123.8822431.5024.1031922.9331.9942509.1625.7653194.2230.3461705.8040.4771962.9531.7783822.1224.7292212.3224.94103015.7835.86111784.9520.94121902.9231.87132329.1527.17142154.0521.78152166.0327.89162258.2721.99172573.8420.49181270.7537.92191611.8440.12201791.8741.04212030.0025.23221290.4030.87231441.7439.13242924.2524.0525816.4121.1026963.5221.71271503.7429.63282849.5923.02293133.2031.20301283.6227.30311495.7523.31321513.7029.29331612.8323.36342319.1933.80352436.2322.87362557.4228.22372626.8528.00382933.4627.68392994.0929.50404101.3428.5141935.4923.69421521.7530.42431669.7921.48442758.3728.94453546.9426.22463609.6320.22473697.4923.71484278.7323.34494421.0420.73504805.6726.495111058.1821.88521352.6129.86532802.8536.35544890.8826.48555212.0626.9856945.4525.80571065.5525.50581137.5826.41591542.7723.91601693.8323.47613361.4224.26623617.7426.97633737.6937.1564980.5422.44651221.6326.82662952.2725.14673696.8826.94685574.4523.24691182.5928.34701963.9631.7671882.5423.81724002.7220.69734059.9620.44741186.5922.31751825.8731.80763401.6623.42771496.7530.36781832.9231.91792281.3536.34802344.3433.66813944.8224.59823002.2323.80833416.7736.76843501.8631.79856783.0326.618614111.2721.97872616.0228.35882810.4536.73892940.9529.07902946.4534.96911494.7230.40921080.5327.86932349.1427.36943303.0023.07954081.5624.51964670.2725.84974671.9923.33988933.9422.57991523.9029.721003956.8225.201013984.8121.291024830.7826.611033031.3935.931043788.7625.211052567.234.761061447.819.491072241.5124.111082461.1130.841091965.9623.621102189.0827.171111127.5820.821121400.7120.351131512.7539.511141860.5334.241151442.6927.721162590.7827.961171556.7227.901182309.1521.951192389.3322.341201478.6839.281211795.9024.661222211.0335.061231223.6319.521241829.0421.221251878.6630.191262009.9632.271272110.0024.101281552.7929.751291577.7540.031301936.9434.711312368.1326.751323633.0533.251331510.7228.301341668.8740.491352227.0533.431361495.7539.411371631.7745.381383158.6029.621391522.7822.761401727.8739.611411883.9440.141421460.7119.831431805.8829.921441898.9340.301452237.0627.121463178.3330.261471844.5634.281481378.5737.161491764.8629.881501791.8830.771512082.0133.671521768.9020.771533442.0933.32154876.4235.071552352.1426.74156937.5034.121571445.7228.361581893.1028.851592839.4324.141601600.7629.611611565.7526.351621627.7629.471631812.9039.981643137.5230.291651364.6728.651662298.0733.821673017.7449.661681235.6126.671691741.8130.211701818.9030.931711892.9522.221723280.6922.691732658.3419.501741407.7127.461751622.7926.821761684.7829.641771321.6528.391781350.6827.131791549.7639.521802233.1022.471812679.2723.481821835.7920.021833421.6625.961841708.8530.441851993.9632.161862695.3123.461871204.6521.931881467.8624.381891767.0724.101908176.3019.571911143.5636.971921834.9031.051932025.9532.211943489.7031.451951268.6227.291961659.8229.351972405.5922.161982483.2127.541992599.2128.202004170.0133.512011013.4125.332021016.4925.882031493.7422.062042104.0432.972053718.8132.392064251.9828.662074538.6726.202082067.9320.682092292.1127.262103702.3932.39211965.4627.842122186.0725.892132584.2935.082142841.1324.502159866.7820.852161099.5328.332172471.2534.692183734.8532.412193927.8633.502203166.3222.102212339.0833.952222563.7622.052233219.3535.002243359.6631.842254097.9824.592264654.1425.812271584.7729.722282148.1025.542292639.4521.332303013.2722.272313205.3919.712323831.8628.392331050.5227.032342157.0622.192352407.1627.652362837.9323.992373058.0230.202381658.6721.532393311.3224.462403556.6323.642411085.5021.702421199.6321.912431247.5822.002441608.7622.362452501.2034.302463021.5223.522474153.7533.412485000.1724.432498917.4822.532501750.8623.802512235.1334.102522644.2521.132533943.9633.5325411967.9620.502552553.2334.142561209.5826.312571899.9421.412581680.0023.772592195.0620.152603064.4120.552613554.0731.112623686.0322.162633802.1233.102644048.0525.422652380.1636.482661352.8324.382671638.8020.262682864.1820.192693754.6637.162704185.9133.47271858.4223.262721159.6426.052731407.7137.252741439.7229.622751720.7619.722761846.9332.042773177.1422.482784113.8024.582792744.0735.032802767.2621.522811310.6427.112821613.8823.952831703.9033.642842761.4021.462853242.4222.782863338.1723.362873371.7422.962883593.5320.252893677.5224.492901624.8030.812912210.9237.552923290.3724.122934413.7629.032941482.7322.472951813.7831.872961934.8720.042972249.8934.142983280.5925.762991098.5621.463001125.5821.763011649.7919.593024025.6820.73303980.3335.593041096.4135.953051698.6537.603062361.2120.773073148.5024.223083157.2334.743091304.5927.953103575.7832.273111510.7520.123122485.2034.253133076.3319.643143343.3931.803151405.7120.163162587.1621.073175213.2522.473182320.1620.733194491.8926.2332010199.9121.11321854.3834.923221084.5636.853231814.7837.293242078.0522.473252175.0833.263262411.7826.973273738.5924.763283935.5734.153294863.2126.66330860.3926.253311567.7820.233322308.1127.323332923.7736.443343295.5525.403353870.8533.393361099.5621.633371359.7022.923382059.0223.123392077.0321.783403349.3435.813418853.8521.083421734.8020.243431847.9543.933442045.9534.043452289.4733.563462421.1534.743472480.6723.003482576.2534.173493353.9323.533501083.5226.243512073.1727.433527958.6534.323531837.8830.543542939.0333.753552977.3119.593563596.4621.543573851.6824.973581135.5227.833593378.0538.813603590.7228.993613959.8019.953621258.4136.103631513.5036.823641716.3820.593652022.9733.383662914.5424.293675527.5627.58368931.5120.00369973.2635.593701385.6727.923712272.3123.803724024.8733.203732216.1133.793742756.2335.163752777.7121.553763521.0230.733773750.7232.453784229.0929.083794846.5026.403801046.5525.353811608.8030.943821878.7831.583832589.1622.453844369.0620.2538512717.0826.923861210.4336.483873092.5436.223883248.6125.653894012.4120.8139011016.3421.313911284.6129.173921460.8322.533931807.8823.983942596.3334.863952686.9729.063963871.5927.513974069.6325.303984288.9825.943994426.2120.094001071.5521.414011749.8830.544021956.9721.444032189.1226.544042257.6336.104052917.5428.994063633.6926.994076055.7721.034086186.0224.994091858.9224.174102274.1133.474114522.5126.204126237.3531.394139883.8220.844143385.625.474153745.626.654161408.739.134172551.334.754183265.336.024192739.328.4420206524.484212264.122.674221058.524.944234467.929.054242887.435.664251635.840.334262525.227.724271526.823.634281664.829.874292583.328.314302663.323.444311878.942.184321462.739.314331834.9244341893.124.64435193421.634361367.738.874371009.527.334383405.125.924392314.133.674403996.820.934412823.629.074421179.627.154431435.728.864442430.735.394451134.623.68446201425.184472577.324.554481194.626.734491588.830.2450205625.444512442.1634.114521422.6621.724531623.824.154541624.6137.734553298.4836.064561016.3135.674571580.9424.314581157.5837.414591250.6127.944601378.6728.854611392.6821.754621409.6422.064631425.6522.344641451.7129.194651576.6626.54661651.8540.64671876.9422.294681911.1224.984692064.0121.954702150.0427.764712751.5929.164724289.9428.694734306.0528.784744800.1823.834751111.3235.474761181.4936.794773168.3824.694781229.5736.294791579.7829.834801680.8230.024811725.6638.34825228.1527.044831769.7828.254841114.5425.524851390.537.054862046.9932.564872899.3349.624881096.5326.124891257.4934.26490868.4523.354911160.4335.64921539.840.364933318.9136.014941084.4825.314951388.3958.994963129.8635.934971255.5636.334981383.6939.024991561.7540.725003108.5531.255011173.5837.515021100.5536.995031128.4433.715043149.631.225051068.5621.695061349.4836.475071689.8140.575082305.734.8509840.4423.94510911.334.395111299.6422.42512911.4725.925131025.5125.445143400.0742.035151901.8943.925161110.4234.375171032.525.895181040.5225.115191265.6427.145201171.5529.245211012.5335.085221286.4936.785232932.3634.115241215.4927.615251423.6821.475261487.7129.58, wherein said CE times are based on capillary electrophoresis using a glass capillary of 90 cm in length with an inner diameter (ID) of 50 μm at an applied voltage of 30 kV,wherein 20% acetonitrile and 0.25% formic acid in water is used as the mobile phase for the capillary electrophoresis at a flow rate of 2 μl/min., andwherein said CE times are calibrated relative to the following values:Protein/PolypeptideMigration time (minutes)Aprotinin9.2Ribonuclease10.9Lysozyme8.9“REV” SEQ ID NO: 115.6“ELM” SEQ ID NO: 223.4“KINCON” SEQ ID NO: 320.0“GIVLY” SEQ ID NO: 436.8; determining if said subset of polypeptide markers comprises at least a first, second and third biomarker selected from:
a first biomarker that is at least one selected from the group consisting of: (1) markers 1 to 106 (frequency markers) having (a) a frequency of presence in patients with VD of at least 0.4 and (b) a frequency of presence in patients with VD at least three (3) times the frequency of presence in patients without VD; (2) markers 1 to 106 (frequency markers) having (a) a frequency of presence in patients without VD of at least 0.4 and (b) a frequency of presence in patients without VD at least three (3) times the frequency of presence in patients with VD; (3) markers 107 to 526 (amplitude markers) having a mean amplitude in patients with VD of at least 200 and a mean amplitude at least two (2) times greater than the mean amplitude of the same marker in patients without VD; and (4) markers 107 to 526 (amplitude markers) having a mean amplitude in patients without VD of at least 200 and a mean amplitude at least two (2) times greater than the mean amplitude of the same marker in patients with VD;a second biomarker which is different than said first biomarker and is at least one selected from the group consisting of: (1) markers 1 to 106 (frequency markers) having (a) a frequency of presence in patients with VD of at least 0.4 and (b) a frequency of presence in patients with VD at least three (3) times the frequency of presence in patients without VD; (2) markers 1 to 106 (frequency markers) having (a) a frequency of presence in patients without VD of at least 0.4 and (b) a frequency of presence in patients without VD at least three (3) times the frequency of presence in patients with VD; (3) markers 107 to 526 (amplitude markers) having a mean amplitude in patients with VD of at least 200 and a mean amplitude at least two (2) times greater than the mean amplitude of the same marker in patients without VD; and (4) markers 107 to 526 (amplitude markers) having a mean amplitude in patients without VD of at least 200 and a mean amplitude at least two (2) times greater than the mean amplitude of the same marker in patients with VD; anda third biomarker which is different than said first and second biomarkers and is at least one selected from the group consisting of markers 1 to 526 (frequency and amplitude markers); comparing the frequency and/or amplitude of presence of said at least first, second and third biomarkers in said sample of said subject patient to the frequency and/or amplitude of presence of the same at least first, second and third biomarkers from said known polypeptide markers from said control subjects; ranking said subject patient between said control subjects with VD and without VD based on the second comparing step; and diagnosing the probability of VD in said subject patient based on said ranking.