CN101317095A - Polypeptide marker for the diagnosis and evaluation of vascular diseases - Google Patents

Abstract

The invention relates to a method for the diagnosis of vascular diseases (VE). In said method, the presence or absence of at least one polypeptide marker in a sample is determined, said polypeptide marker being selected from markers 1 - 526, which are characterised by values for the molecular masses and the migration time (CE-time).

Description

Be used to diagnose polypeptide marker with evaluation of vascular diseases

The present invention relates to whether exist one or more peptide marks to be used for diagnosis and evaluation of vascular diseases (vascular disease from experimenter's sample, the purposes of the order of severity VD), and relate to the method for diagnosing and estimating these angiosises, wherein whether exist described peptide mark to indicate the order of severity of VD.

Angiosis is invasion and attack organism blood vessel and therefore attacks the disease of organ such as heart, brain, kidney etc.They comprise for example artery sclerosis, dyshaemia, hypertension and arrhythmia cordis.Blood vessel:

Artery sclerosis refers to cause artery sclerosis by the blood vessel sediment.The sediment of cholesterol crystal causes the formation of inflammatory lesions (atheroma), and blood constituent, lipid, metabolism residue (metabolicslags) and lime salt (lime salts) are easy to be deposited in the described inflammatory lesions.Form so-called spot (two dimension sclerosis), so vascular wall becomes harder and comparatively narrow.Artery loses its elasticity and is difficult to carry out its task (being about to blood is transported to health from heart each zone).Secondary disease comprises for example angina pectoris, miocardial infarction, circulatory collapse, apoplexy.Dyshaemia mainly influences lower body part, to sufficient artery, causes reducing the supply to musculature of blood flow and oxygen from abdominal aorta, and it is downright bad that described musculature gradually becomes.Late, ulcer forms and occluding vascular to the amputation inevitable degree that becomes.Hypertension has the uncertain cause of disease, and therefore, ingestion of drugs or excessive secretion adrenal hormone can cause blood pressure to increase.Hypertension also can appear in the continuation anxiety (permanent sress), and it causes vasopasm.Therefore hypertension infringement vascular wall has the risk of breaking or blocking.If the regular multilated of heartbeat, this illness is called as arrhythmia cordis.Heartbeat may be too fast (tachycardia (tachycardia)), too slow (bradycardia (bradycardia)) or irregular (arrhythmia cordis).Angiosis can be avoided by prevention, because it is also caused by unhealthy or abnormal life behavior.By fundamental shifts life style, artery sclerosis in early days the stage be prevented from, for example by bringing high blood pressure down and blood lipid level.The process of angiosis can slack-off by drug therapy in addition (for example acetylsalicylic acid, beta receptor blocking agent, ACE inhibitor etc.).Yet, should be noted that the blood vessel that is damaged can not repair, and the process in late period is irreversible.Therefore, the early detection of angiosis is even more important.

Heart:

In coronary heart disease, realize the diagnosis of VD by the estimated risk factor and by the non-intrusion type inspection at first indirectly, described non-intrusion type inspection is such as blood pressure measurement, resting electrocardiogram and exercise electrocardiogram and to detect down the blood picture of lising: lipid state (LDL cholesterol, HDL cholesterol, triglyceride), fasting blood G/W gentle (if desired) HbA1c.If drawing, such inspection has the excessive risk feature, promptly be expected at serious vascular events (dead, miocardial infarction) takes place the foreseeable future, utilize non-invasive diagnosing to diagnose more accurately so, for example with the form of catheterization or coronary arteriography.Therefore, utilize conduit or x-ray method to check heart and coronary vasodilator and other blood vessel.X-ray contrast agent is used for manifesting better heart and blood vessel on the X-ray image.The suitable person of coronary arteriography comprise preliminary test possibility (preliminary test probability) for minuent or moderate but non-invasive diagnosing fails to provide reliable results, because defective or disease can not carry out the patient of non-intrusion type inspection and because the work related causes must be got rid of the patient (for example pilot, fireman) that may have coronary heart disease.Yet,,, can implement coronary arteriography as long as various complication are excluded such as hyperthyroidism or to the allergic reaction of contrast preparation except the above-mentioned trial inspection of mentioning.In addition, because contrast preparation by renal secretion, must guarantee enough renal functions,, must always after checking, then implement dialysis perhaps for the dependent patient of dialysis.Therefore, clearly need early stage and the non-intrusion type possibility reliable diagnostic angiosis.

Kidney:

The angiosis of kidney comprises:

RAS

Arteria renalis thrombosis

Thrombosis of renal artery

The renal vein thrombosis

RAS is that the one-sided or bilateral of the arteria renalis or its main branch is narrow.It may be the reason of Arterial Hypertention, thereby described Arterial Hypertention is called as renovascular hypertension.

In about 70% case, its reason is artery sclerosis (being topmost in advanced age), and in about 20% case, its reason is muscle fibre dysplasia (connective tissue is unusual).Singularly, its reason comprises mechanical pressure, embolism or the thrombosis of sustainer or arteriorenal aneurysm, vasculitis, tumour or tumour.

RAS causes reducing by the blood flow of influenced kidney.In order to remedy (the part of supposition! ) the blood pressure reduction, kidney increases the generation of feritin, and feritin causes whole organic blood volume to increase and elevation of blood pressure by angiotensins-aldosterone mechanism, and causes Arterial Hypertention.Therefore, when hypertension forms gradually, find RAS usually, but cause thus about only 1～2% in all hypertension.

For treatment, have different possibilities:

PTA (percutaneous puncture conduit revascularization (Percutaneous transluminal catheterangioplasty)): utilize and insert ball ductal ectasia narrow (ball expansion);

Support: insert silk screen (support) to keep vessel open;

Operation is eliminated narrow.

The thrombotic common cause of the arteria renalis is the thrombus that is derived from heart, and for example, during atrial fibrillation, described atrial fibrillation simultaneous phenomenon is flank pain, albuminuria, very high LDH for example.Also in the renal vein thrombosis, observe the flank pain, but except albuminuria, in some cases, also observe blood urine or nephrotic syndrome.

Brain:

Narrow blood vessel causes the oxygen supply to reduce in the brain zone, and when artery gets clogged (for example the acute clot that is caused by the variation of artery sclerosis blocks), apoplexy occurs and follow unconsciousness, paralysis, disfluency etc.In cerebral artery, such as in main artery, artery sclerosis can cause the vascular wall aneurysm under few situation, and with risks and assumptions hypertension for example, vascular wall may break and cause life-threatening internal haemorrhage.

Therefore surprisingly, have been found that now from the expressing polypeptide mark in experimenter's the urine samples and can be used for the diagnosis of VD and judge whether need drug therapy.

Therefore, whether the existence that the present invention relates to be used at least a peptide mark in experimenter's urine samples (being several polypeptide markers ideally) diagnoses the purposes of angiosis, wherein said one or more polypeptide markers are selected from polypeptide marker No.1 to No.526, and described polypeptide marker No.1 to No.526 is characterised in that to have molecular mass as described in Table 1 and transit time.

Table 1: the polypeptide marker that is used to diagnose angiosis with and molecular mass and transit time (CE time, in minute)

No.	Quality	The CE time	No.	Quality	The CE time
						451	2442.16	34.11	501	1173.58	37.51
452	1422.66	21.72	502	1100.55	36.99
						453	1623.8	24.15	503	1128.44	33.71
454	1624.61	37.73	504	3149.6	31.22
						455	3298.48	36.06	505	1068.56	21.69
456	1016.31	35.67	506	1349.48	36.47
						457	1580.94	24.31	507	1689.81	40.57
458	1157.58	37.41	508	2305.7	34.8
						459	1250.61	27.94	509	840.44	23.94
460	1378.67	28.85	510	911.3	34.39
						461	1392.68	21.75	511	1299.64	22.42
462	1409.64	22.06	512	911.47	25.92
						463	1425.65	22.34	513	1025.51	25.44
464	1451.71	29.19	514	3400.07	42.03
						465	1576.66	26.5	515	1901.89	43.92
466	1651.85	40.6	516	1110.42	34.37
						467	1876.94	22.29	517	1032.5	25.89
468	1911.12	24.98	518	1040.52	25.11
						469	2064.01	21.95	519	1265.64	27.14
470	2150.04	27.76	520	1171.55	29.24
						471	2751.59	29.16	521	1012.53	35.08
472	4289.94	28.69	522	1286.49	36.78
						473	4306.05	28.78	523	2932.36	34.11
474	4800.18	23.83	524	1215.49	27.61
						475	1111.32	35.47	525	1423.68	21.47
476	1161.49	36.79	526	1487.71	29.58
						477	3168.38	24.69
478	1229.57	36.29
						479	1579.78	29.83
480	1680.82	30.02
						481	1725.56	38.3
482	5228.15	27.04
						483	1769.78	28.25
484	1114.54	25.52
						485	1390.5	37.05
486	2046.99	32.56
						487	2899.33	49.62
488	1096.53	26.12
						489	1257.49	34.26
490	868.45	23.35
						491	1160.43	35.6
492	1539.8	40.36
						493	3318.91	36.01
494	1084.48	25.31
						495	1388.39	58.99
496	3129.86	35.93
						497	1255.56	36.33
498	1383.69	39.02
						499	1561.75	40.72
500	3108.55	31.25

Preferably, service marking thing 1～104 and/or 107～413.

Use the present invention, also can measure the order of severity of VD.This information helps decision to use what kind of treatment measure.

Utilize Capillary Electrophoresis (CE) to measure transit time, for example, described in the 2nd of embodiment.Therefore, working length is that 90cm, internal diameter (ID) are that 75 μ m and external diameter (OD) are the glass capillary of 360 μ m under the voltage of 30kV.Use contains the solvent of the water of 30% methyl alcohol, 0.5% formic acid as sample.

Known CE transit time can change.But, for used any CE system, the order of polypeptide marker wash-out is normally identical.For the difference of balance transit time, can use the described system of standard correction of known transit time.These standards can be the polypeptide described in the embodiment (seeing embodiment, the 3rd) for example.

Utilize capillary electrophoresis-mass spectrometry (CE-MS) to measure the feature of table 1 to polypeptide marker shown in 3, described capillary electrophoresis-mass spectrometry (CE-MS) method is described in detail in, (Rapid Communications in massspectrometry such as Neuhoff for example, 2004, Vol, 20pp.149-156).Between each is measured or the molecular mass between the different mass spectrometer change less relatively, typically in the scope of soil 0.1%, preferably in ± 0.05% scope, more preferably in ± 0.03% scope, even more preferably in ± 0.01% scope.

Polypeptide marker according to the present invention is the catabolite of protein or peptide or protein or peptide.They can for example by posttranslational modification, such as glycosylation, phosphorylation, alkylation or disulfide bond, or be passed through for example other reaction in the degraded scope by chemical modification.In addition, polypeptide marker also can be by chemical modification in the scope of sample purifying, and is for example oxidized.

Set about from the parameter (molecular mass and transit time) of measuring polypeptide marker, can determine the sequence of corresponding polypeptide by the prior art known method.

The order of severity that is used to diagnose VD according to polypeptide of the present invention (seeing Table 1 to 4)." diagnosis " refers to by symptom or phenomenon are obtained the process of knowledge owing to i or I.In the present circumstance, whether the order of severity of VD is released by the existence of special peptides mark.Therefore, measure according to polypeptide marker of the present invention in from experimenter's sample, wherein whether its existence can be used for inferring the order of severity of VD.Whether the existence of polypeptide marker can measure by any known method of prior art.Illustrate possible known method below.

If the measured value of polypeptide marker is equally high with its threshold value at least, think so to have this polypeptide marker.If measured value is lower, think so not have polypeptide marker.Can be by the sensitivity (detection limit) of measuring method or rule of thumb definite threshold value.

In the context of the present invention, if be the twice of blank sample (for example, only buffering agent or solvent) at least at the measured value of a certain molecular mass in the sample, preferably think to surpass threshold value.

Use this polypeptide marker in the mode that whether exists of measuring polypeptide marker, wherein whether have the order of severity (frequency mark thing (frequency marker)) that indicates VD.Therefore, some polypeptide marker is present among the experimenter who suffers from VD usually, but more rarely or not is present among the experimenter who does not suffer from VD, for example, and 1-24 (table 2).In addition, some polypeptide marker is present among the VD patient, such as polypeptide marker No.25～106, but is present among the patient who does not suffer from VD more singularly or not.

Table 2: be used for diagnosing polypeptide marker (frequency mark thing), its molecular mass and the transit time of angiosis and represent with factor form whether it is present in VD patient (VD) and control group (contrast) (1=100%, 0=0%; Sample preparation as be shown in the examples and measurement)

Substitute outside the frequency mark thing (measure and whether exist) or as it, amplitude mark as described in Table 3 also can be used for the diagnosis (Nos.107～526) of VD.In the mode of the amplitude of use mark, whether it exists not is most important, if but all there is signal in two groups, the height of signal (amplitude) plays a decisive role so.In table 3 and 4, the average amplitude of the corresponding signal (is feature with quality and transit time) that relative all measuring samples average has been described.Two kinds of method for normalizing can be realized between the different concentrating samples or the comparability between the different measuring method.In first method, all peptide signals of sample are normalized to total amplitude of 100 ten thousand countings.Therefore, the average amplitude separately of each mark is defined as umber/1,000,000 (ppm).The amplitude mark that obtains by this method is displayed on (Nos.107～413) in the table 3.

In addition, can define other amplitude mark by method for normalizing as an alternative: in this case, utilize shared normalized factor to weigh all peptide signals of a sample.Therefore, form linear regression between the reference point of the peptide amplitude of single sample and all known peptides.The slope of the tropic is just in time corresponding to relative concentration and be used as the normalized factor of this sample.The biomarker that obtains by this method for normalizing is displayed on (Nos.414～526) in the table 4.

All used group sample or the control sample by at least 20 different patients formed to obtain reliable average amplitude.Make diagnosis decision (whether being VD) with following function: compare with the average amplitude of control group or VD group, in patient's sample separately the amplitude of polypeptide marker how highly be.If amplitude more is equivalent to the average amplitude of VD group, think to have angiosis, and if amplitude more be equivalent to the average amplitude of control group, think not have VD.Distance between measured value and the average amplitude can be considered to the probability that sample belongs to a certain group.To utilize mark No.137 to provide example explanation (table 3).The average amplitude of mark described in the VD significantly increases (12044ppm is with respect to the 5726ppm in the control group).Now, if the value of this mark is 0 to 5726ppm or exceeds this scope 20% at most in patient's sample, promptly from 0 to 6871ppm, this sample belongs to control group so.If value is 12044ppm or exceeds and be lower than its value of 20%, or higher, promptly 9635 and very high value between, then be considered to the indication of angiosis.

As an alternative, the distance between measured value and the average amplitude can be considered to the probability that sample belongs to a certain group.

In some samples, the frequency mark thing is the variant of the amplitude mark of low amplitude.Be included in by respective sample that will wherein not find mark in the amplitude calculating of very little amplitude (detection limit rank), such frequency mark thing may be changed into the amplitude mark.

Table 3: according to method 1 normalized amplitude mark

Table 4: according to method 2 normalized amplitude marks

No.	The average amplitude control group	Average amplitude CVD group	No.	The average amplitude control group	Average amplitude CVD group
						414	3214	2678	471	359	423
415	514	250	472	3360	3317
						416	1359	615	473	4000	2575
417	581	174	474	1388	2785
						418	630	499	475	335	3122
419	212	141	476	302	237
						420	681	381	477	351	847
421	445	227	478	186	145
						422	1178	103	479	3094	2397
423	540	348	480	4737	2446
						424	188	206	481	1468	2644
425	1540	687	482	566	974
						426	569	914	483	535	429
427	301	106	484	2818	4530
						428	976	511	485	17423	37226
429	972	515	486	3087	1793
						430	1320	729	487	25	319
431	278	210	488	3397	6633
						432	1582	1196	489	2904	6138
433	589	267	490	239	198
						434	384	502	491	1794	3083
435	1006	399	492	2558	1701
						436	1064	800	493	426	419
437	270	216	494	1326	2891
						438	3453	2235	495	181	768
439	837	790	496	212	207
						440	1353	684	497	741	600
441	710	733	498	135	197
						442	809	627	499	4632	4647
443	8328	3904	500	331	461
						444	596	661	501	302	414
445	380	593	502	206	306
						446	2389	1375	503	1521	3346
447	297	285	504	349	561
						448	4154	2314	505	211	315
449	532	953	506	208	247
						450	1145	733	507	1270	1039
451	744	845	508	305	334
						452	2878	2433	509	213	266
453	8725	4307	510	2436	3827
						454	1109	1620	511	460	294
455	750	409	512	389	974
						456	687	971	513	197	273
457	2155	1229	514	152	448
						458	1622	1964	515	743	575
459	50522	45582	516	428	713
						460	2259	2915	517	185	298
461	4142	4664	518	219	296
						462	6265	8932	519	4218	7618
463	1969	2931	520	75	146
						464	36818	19376	521	96	214
465	1352	1591	522	56	92
						466	5789	1562	523	208	316
467	2562	1358	524	349	729
						468	91735	90455	525	543	1220
469	7723	4053	526	388	684
						470	2496	1342

The source experimenter who has measured the sample whether one or more polypeptide markers exist can be any experimenter that possible suffer from VD.Preferably, described experimenter is a mammal, and most preferably, is the people.

In a preferred embodiment of the invention, not only a kind of polypeptide marker, but the combination of polypeptide marker is used to measure the order of severity of VD, wherein whether the order of severity of VD can be inferred from the existence of described polypeptide marker.By many peptide species marks, the deviation in the total result that can reduce or avoid to cause owing to some individual deviations that have possibility in patient or the contrast individuality usually.

It is described that to measure the sample whether peptide mark according to the present invention exist therein can be any sample that obtains from experimenter's health.Described sample is the sample that contains the peptide composition of the information that is suitable for providing relevant experimenter's state (whether being VD).For example, it can be blood, urine, synovia, tissue fluid, body exudates (body secretion), sweat, cerebrospinal fluid, lymph, intestinal juice, gastric juice or pancreatic juice, bile, tear, tissue sample, seminal fluid, vaginal secretion or fecal specimens.Preferably, it is a fluid sample.

In a preferred embodiment, described sample is urine samples or blood sample, and wherein blood sample can be serum or plasma sample.

Can preferably obtain urine samples according to prior art.Preferably, the midstream urine sample is used for the said urine samples of the context of the invention.For example, also can utilize the equipment of urinating described in the WO 01/74275 to collect urine samples.

Can utilize method commonly known in the art to obtain blood sample, for example, from vein, artery or kapillary.Usually, utilize syringe for example to obtain blood sample from experimenter's arm venous blood samples.Term " blood sample " comprises the sample that obtains from blood with separation method by being further purified, such as blood plasma or serum.

The existence that can utilize polypeptide in any known method working sample that is suitable for measuring polypeptide marker in the prior art whether.Such method is that the technician is known.In principle, the existence that can for example measure polypeptide marker by part such as mass spectrum or indirect method by direct method whether.

If necessary or needs are arranged, can measure polypeptide marker whether exists before by any suitable method pre-service and for example purifying or separation are for example urinated or blood sample from experimenter's sample.Described processing can comprise for example purifying, separation, dilution or concentrate.Described method can be for example centrifugal, filtration, ultrafiltration, dialysis, precipitation or chromatography, such as affine separation or utilize the separation, electrophoretic separation (promptly applying electric field utilizes in the solution the different migratory behaviours of electrically charged particle to separate) of ion-exchange chromatography to separate.Its instantiation is gel electrophoresis, two-dimentional polyacrylamide gel electrophoresis (2D-PAGE), Capillary Electrophoresis, metal affinity chromatography, immobilization metal affinity chromatography (IMAC), the affinity chromatography based on lectin, liquid chromatography, high performance liquid chromatography (HPLC), positive and reversed-phase HPLC, cation-exchange chromatography and selective surface's combination.All these methods are that the technician knows, and the technician will select to be used to measure the method whether polypeptide marker exists as the method and the selection of specimen in use function.

In one embodiment of the invention, utilizing before Capillary Electrophoresis separates, utilize ultracentrifugation separation, purification of samples and/or utilize ultrafiltration that sample is divided into fraction, described fraction contains the polypeptide marker of specific molecular size.

Preferably, the existence that mass spectroscopy is used to measure polypeptide marker whether wherein can be from reverse purifying or the separation of carrying out sample of such method.Compare with at present used method, mass spectrophotometry has can be by the advantage of the concentration of many (＞100) polypeptide in the single assay determination sample.Can use the mass spectrum of any kind.Utilize mass spectrum, may detect the polypeptide marker (being the 10kD protein of 0.1ng) of 10fmol in the potpourri of complexity, according to routine, measuring accuracy is about ± 0.01%.In mass spectrometer, ion forms unit and suitable analytical equipment coupling connection.For example electro-spray ionization (ESI) interface is mainly used in the ion of measuring in the fluid sample, and MALDI (substance assistant laser desorpted attached/ionization) is used for measuring the ion from matrix crystallization sample.In order to analyze the ion of formation, can use for example quadrupole rod, ion trap or flight time (TOF) analytical instrument.

In electro-spray ionization (ESI), the molecule that exists in solution is atomized, and in addition, under the influence of high pressure (for example 1 to 8kV), at first forms charged droplet, and described droplet becomes littler and evaporates from solvent.At last, so-called COULOMB EXPLOSION causes forming dissociated ion, can analyze and detect described dissociated ion then.

Utilizing TOF to carry out applying specific accelerating potential in the ion analysis, this voltage is given the kinetic energy of ion equivalent.Very accurately measure each ion and move certain drift apart from required time by tof tube thereafter.Because the kinetic energy with equivalent, the speed of ion depends on their quality, therefore can detect their quality (latter).The TOF analyser has very high sweep velocity and reaches good resolution thus.

Be used to detect the method for optimizing whether polypeptide marker exist and comprise the gaseous ion chromatogram, such as laser desorption/ionization mass spectrum, MALDI-TOF MS, SELDI-TOF MS (surface-enhanced laser desorption/ionization), LC MS (liquid chromatography/mass spectrometry), 2D-PAGE/MS and capillary electrophoresis-mass spectrometry (CE-MS).Above-mentioned all methods are that the technician is known.

Particularly preferred method is CE-MS, wherein Capillary Electrophoresis and mass spectrum coupling connection.This method quite at length has been described in for example (J.Chromatogr A such as German patent application DE 10021737, Kaiser, 2003, Vol.1013:157-171, and Electrophoresis, 2004,25:2044-2055) and Wittke etc. (J.Chromatogr A, 2003,1013:173-181) in.The CE-MS technology allow at short notice with small size in the existence of hundreds of polypeptide markers in the working sample simultaneously in high sensitivity.After measuring a kind of sample, prepare the pattern (pattern) of measured polypeptide marker, and this pattern can be compared with patient or health volunteer's reference pattern.As a rule, the diagnosis of using the polypeptide marker of Finite Number to be used for UAS is enough.The CE-MS method that comprises with the CE of the online coupling connection of ESI-TOF MS is further preferred.

For CE-MS, it is preferred using volatile solvent, and is preferably under the salt-free basically condition and carries out work.The example of these solvents comprises acetonitrile, isopropyl alcohol, methyl alcohol etc.Described solvent available water or weak acid (for example 0.1% to 1% formic acid) dilution is with protonated with analyte (preferred polypeptide).

Utilize Capillary Electrophoresis, can be by the electric charge and the described molecule of size separation of molecule.After applying electric current, neutral particle will move with electroosmotic flow speed, and kation quickens towards anode, and negative ion then is delayed.Advantage capillaceous is to keep favourable surface in the electrophoresis: volume ratio, the Joule heat that it can dissipate and produce well during electric current.Thereby allow to apply high voltage (usually up to 30kV) and therefore allow high score from behavior and short analysis time.

In Capillary Electrophoresis, use internal diameter to be typically the silex glass kapillary of 50～75 μ m usually.Used length is for example 30～100cm.In addition, separation capillary is made by the silex glass of applied plastic usually.Kapillary can be untreated (hydrophilic radical that is them is exposed on the inside surface) or coating is arranged on inside surface.

Hydrophobic coating can be used for improving resolution.Except voltage, also can exert pressure, institute's plus-pressure is usually in 0 to 1psi scope.Also can only exert pressure simultaneously in separation or during changing.

A method that preferably is used for measuring polypeptide marker, utilize the mark of capillary electrophoresis separation sample, be used for detecting in direct ionization and the online mass spectrometer of transferring to coupling connection then.

In the method according to the invention, it is favourable using several polypeptide markers to be used to diagnose VD.Particularly, can use three peptide species marks at least, for example mark 1,2 and 3; 1,2 and 4; Or the like.

Use at least 4,5 or 6 kind of mark are preferred.

Use at least 11 kinds of marks, for example mark 1 to 11, or even preferred.

Use table 1 all cited 526 kinds of marks in 4 are most preferred.

For there is the probability of serious VD in mensuration when using several mark, but the known statistical method of operation technique personnel.For example, by utilizing computer program such as S-Plus, perhaps Weissinger etc. (Kidney Int., 2004,65:2426-2434) described support vector machine can be used the Random Forests method described in the above-mentioned same document.

Embodiment:

1. specimen preparation

For detecting polypeptide marker, use urine with diagnosis VD.Collect urine from healthy donors (control group) and the patient that suffers from serious VD.

Detect for subsequently CE-MS, must fall with high concentration to be contained in protein (such as albumin and immunoglobulin (Ig)) in patient's urine equally by ultra-filtration and separation.Therefore, collect 700 μ l urine and mix mutually with the filtration damping fluid (2M urea, 10mM ammonia, 0.02%SDS) of 700 μ m.Sample (20kDa, the Sartorius of this 1.4ml volume of ultrafiltration

Germany).In hydro-extractor, carry out described ultrafiltration under the 3000rpm up to obtaining the 1.1ml ultrafiltrate.

1.1ml ultrafiltrate with gained is applied to PD 10 posts (Amersham Bioscience, Uppsala, Sweden) and uses 2.5ml 0.01%NH then ₄OH wash-out, and freeze-drying.For CE-MS detects, use 20 μ l water (HPLC level, Merck) resuspended described polypeptide then.

2.CE-MS detect

Utilize capillary electrophoresis system (the P/ACE MDQ System of Beckman Coulter; Beckman Coulter Inc., Fullerton, CA, USA) the ESI-TOF mass spectrometer (micro-TOF MS, Bruker Daltonik, Bremen, Germany) with Bruker carries out the CE-MS detection.

The CE kapillary is provided by Beckman Coulter and ID/OD is that 50/360 μ m, length are 90cm.The moving phase that CE separates is made up of the water that contains 20% acetonitrile and 0.25% formic acid.For " the sheath stream " on the MS, use 30% isopropyl alcohol that contains 0.5% formic acid, flow velocity herein is 2 μ l/ minutes.The coupling UNICOM of CE and MS crosses CE-ESI-MS Sprayer cover box (AgilentTechnologies, Waldbronn, Germany) and realizes.

For injected sample, use 1 to maximum 6psi pressure, and the continuous injection time is 99 seconds.Utilize these parameters, about 150nl sample is expelled in the kapillary, it is equivalent to about 10% of kapillary volume.In kapillary with the technology of piling up (stacking technique) concentrating sample.Therefore, before the injected sample, injection 1M NH ₃Solution 7 seconds (under the 1psi), and after injected sample, injection 2M formic acid solution 5 seconds.When applying separation voltage (30kV), analyte is concentrated between these solution automatically.

The CE that utilizes pressure application to carry out subsequently separates: 0psi 40 minutes, 0.1psi is 2 minutes then, 0.2psi 2 minutes, 0.3psi 2 minutes, 0.4psi 2 minutes, last 0.5psi 32 minutes.Therefore the total duration of lock out operation is 80 minutes.

In order to obtain signal intensity as well as possible in MS one side, atomization gas is set to minimum possible values.Being applied to the voltage that nozzle needle is used to produce electron spray is 3700～4100V.Instructions according to manufacturer carries out optimization to carry out the peptide detection to all the other settings on the mass spectrometer.Record spectrogram and per 3 seconds add up in m/z400 to m/z3000 mass range.

3. be used for the standard that CE detects

Detect for check and standardization CE, use characteristic is the following proteins or the polypeptide of described CE transit time:

Concentration with 10pmol/ μ l in water is used every kind of protein/polypeptide." REV ", " ELM ", " KINCON " and " GIVLY " are synthetic peptides.

The molecular mass of peptide and in MS the m/z ratio of visible single charge state be described in the following table:

H is single	1.0079	1.0079	1.0079	1.0079	1.0079	1.0079	1.0079
								m/z	Aprotinin	Ribonuclease	Lysozyme	REV	KINCON	ELM	GIVLY
	The simple substance amount	The simple substance amount	The simple substance amount	The simple substance amount	The simple substance amount	The simple substance amount	The simple substance amount
								0	6513.0900	13681.3200	14303.8800	1732.9600	2333.1900	2832.4100	2048.0300
1	6514.0979	13682.3279	14304.8879	1733.9679	2334.1979	2833.4179	2049.0379
								2	3257.5529	6841.6679	7152.9479	867.4879	1167.6029	1417.2129	1025.0229
3	2172.0379	4561.4479	4768.9679	578.6612	778.7379	945.1446	683.6846
								4	1629.2804	3421.3379	3576.9779	434.2479	584.3054	709.1104	513.0154
5	1303.6259	2737.2719	2861.7839	347.5999	467.6459	567.4899	410.6139
								6	1086.5229	2281.2279	2384.9879	289.8346	389.8729	473.0762	342.3462
7	931.4494	1955.4822	2044.4193	248.5736	354.3208	405.6379	293.5836
								8	815.1442	1711.1729	1788.9929	217.6279	292.6567	355.0592	257.0117
9	724.6846	1521.1546	1590.3279	193.5590	260.2512	315.7201	228.5668
								10	652.3169	1369.1399	1431.3959	174.3039	234.3269	284.2489	205.8109
11	593.1070	1244.7643	1301.3606	158.5497	213.1161	258.4997	187.1924
								12	543.7654	1141.1179	1192.9979	145.4212	195.4404	237.0421	171.6771
13	502.0148	1053.4171	1101.3063	134.3125	180.4841	218.8856	158.5486

In principle, the slight variation that transit time may take place in the separation that utilizes Capillary Electrophoresis is that the technician is known.Yet under the described conditions, the migration order will can not change.For knowing described quality and the technician of CE time, can like a dream themselves mensuration be distributed to according to polypeptide marker of the present invention.For example, he can carry out according to following: at first, he selects a kind of polypeptide of finding (peptide 1) and attempts in the time slot of described CE time (for example ± 5 minute) and find the quality that one or more are identical in his measurement.If only find a kind of identical quality every interior during this time, then assigned.If find the quality of several couplings, the decision of relevant this distribution still needs to carry out.Therefore, from detect, select another peptide (peptide 2), and make great efforts to confirm suitable peptide mark, consider corresponding time slot once more.

In addition, have corresponding quality if can find several marks, most probable distribution is the relation that has substantial linear between the migration of the migration of wherein peptide 1 and peptide 2.

According to the complicacy of assignment problem, proposed techniques personnel self randomly use other protein from its sample to be used for distributing, for example ten kinds of protein.Usually, transit time prolongs or shortens specific absolute value, the compression or the extension of whole process perhaps occur.Yet, under such condition, move peptide altogether and also will move altogether.

In addition, the technician can utilize Zuerbig etc. at Electrophoresis 27 (2006), the migration model described in the pp.2111-2125.(for example utilizing MSExcel) maps to its measurement with respect to the form of transit time with m/z if he utilizes simple table, and it is obvious that described ray mode also becomes.Now, can distribute single polypeptide simply by the line counting is feasible.

Also can use other distribution method.Basically, the technician also can use above-mentioned peptide to be used to distribute his CE to measure as interior mark.

Claims (13)

1. One kind the diagnosis angiosis (VD) method, it comprises the step that whether has at least a polypeptide marker in the working sample, wherein said polypeptide marker is selected from mark 1～526, it is characterized in that having following molecular mass and transit time:

   No. Quality The CE time No. Quality The CE time No. Quality The CE time No. Quality The CE time No. Quality The CE time 1 1166.61 23.88 51 11058.18 21.88 101 3984.81 21.29 151 2082.01 33.67 201 1013.41 25.33 2 2431.50 24.10 52 1352.61 29.86 102 4830.78 26.61 152 1768.90 20.77 202 1016.49 25.88 3 1922.93 31.99 53 2802.85 36.35 103 3031.39 35.93 153 3442.09 33.32 203 1493.74 22.06 4 2509.16 25.76 54 4890.88 26.46 104 3788.76 25.21 154 876.42 35.07 204 2104.04 32.97 5 3194.22 30.34 55 5212.06 26.98 105 2567.2 34.76 155 2352.14 26.74 205 3718.81 32.39 6 1705.90 40.47 56 945.45 25.80 106 1447.8 19.49 156 937.50 34.12 206 4251.98 28.66 7 1962.95 31.77 57 1065.55 25.50 107 2241.51 24.11 157 1445.72 28.36 207 4538.67 26.20 8 3822.12 24.72 58 1137.58 26.41 108 2461.11 30.84 158 1893.10 28.85 208 206793 20.68 9 2212.32 24.94 59 1542.77 23.91 109 1965.96 23.62 159 2839.43 24.14 209 2292.11 27.26 10 3015.78 35.86 60 1693.83 23.47 110 2189.08 27.17 160 1600.76 29.61 210 3702.39 32.39 11 1784.95 20.94 61 3361.42 24.26 111 1127.58 20.82 161 1565.75 26.35 211 965.46 27.84 12 1902.92 31.87 62 3617.74 26.97 112 1400.71 20.35 162 1527.76 29.47 212 2186.07 25.89 13 2329.15 27.17 63 3737.69 37.15 113 1512.75 39.51 163 1812.90 39.98 213 2564.29 35.08 14 2154.05 21.78 64 980.54 22.44 114 1860.53 34.24 164 3137.52 30.29 214 2841.13 24.50 15 2166.03 27.89 65 1221.63 26.82 115 1442.69 27.72 165 1364.67 28.65 215 9666.78 20.85 16 2258.27 21.99 66 2952.27 25.14 116 2590.78 27.96 166 2298.07 33.82 216 1099.53 28.33 17 2573.84 20.49 67 3696.88 26.94 117 1556.72 27.90 167 3017.74 49.66 217 2471.25 34.69 18 1270.75 37.92 68 5574.45 23.24 118 2309.15 21.95 168 1235.61 26.67 218 3734.85 32.41 19 1611.84 40.12 69 1182.59 28.34 119 2389.33 22.34 169 1741.61 30.21 219 3927.86 33.50 20 1791.87 41.04 70 1963.96 31.76 120 1478.68 39.28 170 1818.90 30.93 220 3166.32 22.10 21 2030.00 25.23 71 882.54 23.81 121 1795.90 24.66 171 1892.95 22.22 221 2339.08 33.95 22 1290.40 30.87 72 4002.72 20.69 122 2211.03 35.06 172 3280.69 22.69 222 2563.76 22.05 23 1441.74 39.13 73 4059.96 20.44 123 1223.63 19.52 173 2658.34 19.50 223 3219.35 35.00 24 2924.25 24.05 74 1186.59 22.31 124 1829.04 21.22 174 1407.71 27.46 224 3359.66 31.84 25 816.41 21.10 75 1825.87 31.80 125 1878.66 30.19 175 1622.79 26.82 225 4097.98 24.59 26 963.52 21.71 76 3401.66 23.42 126 2009.96 32.27 176 1664.78 29.64 226 4654.14 25.81 27 1503.74 29.63 77 1496.75 30.36 127 2110.00 24.10 177 1321.65 28.39 227 1584.77 29.72 28 2849.59 23.02 78 1832.92 31.91 128 1552.79 29.75 178 1350.68 27.13 228 2148.10 25.54 29 3133.20 31.20 79 2281.35 36.34 129 1577.75 40.03 179 1549.76 39.52 229 2639.45 21.33 30 1283.62 27.30 80 2344.34 33.66 130 1936.94 34.71 180 2233.10 22.47 230 3013.27 22.27 31 1495.75 23.31 81 3944.82 24.59 131 2368.13 26.75 181 2679.27 23.48 231 3205.39 19.71 32 1513.70 29.29 82 3002.23 23.80 132 3633.05 33.25 182 1835.79 20.02 232 3831.86 28.39 33 1612.83 23.36 83 3416.77 36.76 133 1510.72 28.30 183 3421.66 25.96 233 1050.52 27.03 34 2313.19 33.80 84 3501.86 31.79 134 1668.87 40.49 184 1708.85 30.44 234 2157.06 22.19 35 2436.23 22.87 85 6783.03 26.61 135 2227.05 33.43 185 1993.96 32.16 235 2407.16 27.65 36 2557.42 28.22 86 14111.27 21.97 136 1495.75 39.41 186 2695.31 23.46 236 2837.93 23.99 37 2626.85 28.00 87 2616.02 28.35 137 1631.77 45.38 187 1204.65 21.93 237 3058.02 30.20 38 2933.46 27.68 88 2810.45 36.73 138 3158.60 29.62 188 1467.86 24.38 238 1658.67 21.53 39 2994.09 29.50 89 2940.95 29.07 139 1522.78 22.76 189 1767.07 24.10 239 3311.32 24.46 40 4101.34 28.51 90 2946.45 34.96 140 1727.87 39.61 190 8176.30 19.57 240 3556.63 23.64 41 935.49 23.69 91 1494.72 30.40 141 1883.94 40.14 191 1143.56 36.97 241 1085.50 21.70 42 1521.75 30.42 92 1080.53 27.86 142 1460.71 19.83 192 1834.90 31.05 242 1199.63 21.91 43 1669.79 21.48 93 2349.14 27.36 143 1805.88 29.92 193 2025.95 32.21 243 1247.58 22.00 44 2758.37 28.94 94 3303.00 23.07 144 1898.93 40.30 194 3489.70 31.45 244 1608.76 22.36 45 3546.94 26.22 95 4081.56 24.51 145 2237.06 27.12 195 1268.62 27.29 245 2501.20 34.30 46 3609.63 20.22 96 4670.27 25.84 146 3178.33 30.26 196 1659.82 29.35 246 3021.52 23.52 47 3697.49 23.71 97 4671.99 23.33 147 1844.56 34.28 197 2405.59 22.16 247 4153.75 33.41 48 4278.73 23.34 98 8933.94 22.57 148 1378.57 37.16 198 2483.21 27.54 248 5000.17 24.43 49 4421.04 20.73 99 1523.90 29.72 149 1764.86 29.88 199 2599.21 28.20 249 8917.48 22.53 50 4805.67 26.49 100 3956.82 25.20 150 1791.88 30.77 200 4170.01 33.51 250 1750.86 23.80

   No. Quality The CE time No. Quality The CE time No. Quality The CE time No. Quality The CE time 251 2235.13 34.10 301 1649.79 19.59 351 2073.17 27.43 401 1749.88 30.54 252 2644.25 21.13 302 4025.68 20.73 352 7958.65 34.32 402 1956.97 21.44 253 3943.96 33.53 303 980.33 35.59 353 1837.88 30.54 403 2189.12 26.54 254 11967.96 20.50 304 1096.41 35.95 354 2939.03 33.75 404 2257.63 36.10 255 2553.23 34.14 305 1698.65 37.60 355 2977.31 19.59 405 2917.54 28.99 256 1209.58 26.31 306 2361.21 20.77 356 3596.46 21.54 406 3633.69 26.99 257 1899.94 21.41 307 3148.50 24.22 357 3851.68 24.97 407 6055.77 21.03 258 1680.00 23.77 308 3157.23 34.74 358 1135.52 27.83 408 6186.02 24.99 259 2195.06 20.15 309 1304.59 27.95 359 3378.05 38.81 409 1858.92 24.17 260 3064.41 20.55 310 3575.78 32.27 360 3590.72 28.99 410 2274.11 33.47 261 3554.07 31.11 311 1510.75 20.12 361 3959.80 19.95 411 4522.51 26.20 262 3686.03 22.16 312 2485.20 34.25 352 1258.41 36.10 412 6237.35 31.39 263 3802.12 33.10 313 3076.33 19.64 363 1513.50 36.82 413 9883.82 20.84 264 4048.05 25.42 314 3343.39 31.80 364 1716.38 20.59 414 3385.6 25.47 265 2380.16 36.48 315 1405.71 20.16 365 2022.97 33.38 415 3745.6 26.65 266 1352.83 24.38 316 2587.16 21.07 366 2914.54 24.29 416 1408.7 39.13 267 1638.80 20.26 317 5213.25 22.47 367 5527.56 27.58 417 2551.3 34.75 268 2864.18 20.19 318 2320.16 20.73 368 931.51 20.00 418 3265.3 36.02 269 3754.66 37.16 319 4491.89 26.23 369 973.26 35.59 419 2739.3 28.4 270 4185.91 33.47 320 10199.91 21.11 370 1385.67 27.92 420 2065 24.48 271 858.42 23.26 321 854.38 34.92 371 2272.31 23.80 421 2264.1 22.67 272 1159.64 26.05 322 1084.56 36.85 372 4024.87 33.20 422 1058.5 24.94 273 1407.71 37.25 323 1814.78 37.29 373 2216.11 33.79 423 4467.9 29.05 274 1439.72 29.62 324 2078.05 22.47 374 2756.23 35.16 424 2887.4 35.66 275 1720.76 19.72 325 2175.08 33.26 375 2777.71 21.55 425 1635.8 40.33 276 1846.93 32.04 326 2411.78 26.97 376 3521.02 30.73 426 2525.2 27.72 277 3177.14 22.48 327 3738.59 24.76 377 3750.72 32.45 427 1526.8 23.63 278 4113.80 24.58 328 3935.57 34.15 378 4229.09 29.08 428 1664.8 29.87 279 2744.07 35.03 329 4863.21 26.66 379 4846.50 26.40 429 2583.3 28.31 280 2767.26 21.52 330 860.39 26.25 380 1046.55 25.35 430 2663.3 23.44 281 1310.64 27.11 331 1567.78 20.23 381 1608.80 30.94 431 1878.9 42.18 282 1613.88 23.95 332 2308.11 27.32 382 1878.78 31.58 432 1462.7 39.31 283 1703.90 33.64 333 2923.77 36.44 383 2589.16 22.45 433 1834.9 24 284 2761.40 21.46 334 3295.55 25.40 384 4369.06 20.25 434 1893.1 24.64 285 3242.42 22.78 335 3870.85 33.39 385 12717.08 26.92 435 1934 21.63 286 3338.17 23.36 336 1099.56 21.63 386 1210.43 36.48 436 1367.7 38.87 287 3371.74 22.96 337 1359.70 22.92 387 3092.54 36.22 437 1009.5 27.33 288 3593.53 20.25 338 2059.02 23.12 388 3246.61 25.65 438 3405.1 25.92 289 3677.52 24.49 339 2077.03 21.78 389 4012.41 20.81 439 2314.1 33.67 290 1624.80 30.81 340 3349.34 35.81 390 11016.34 21.31 440 3996.8 20.93 291 2210.92 37.55 341 8853.85 21.08 391 1284.61 29.17 441 2823.6 29.07 292 3290.37 24.12 342 1734.80 20.24 392 1460.83 22.53 442 1179.6 27.15 293 4413.76 29.03 343 1847.95 43.93 393 1807.88 23.98 443 1435.7 28.86 294 1482.73 22.47 344 2045.95 34.04 394 2596.33 34.86 444 2430.7 35.39 295 1813.78 31.87 345 2289.47 33.56 395 2686.97 29.06 445 1134.6 23.68 296 1934.87 20.04 346 2421.15 34.74 396 3871.59 27.51 446 2014 25.18 297 2249.89 34.14 347 2480.67 23.00 397 4069.63 25.30 447 2577.3 24.55 298 3280.59 25.76 348 2576.25 34.17 398 4288.98 25.94 448 1194.6 26.73 299 1096.56 21.46 349 3353.93 23.53 399 4426.21 20.09 449 1588.8 30.2 300 1125.58 21.76 350 1063.52 26.24 400 1071.55 21.41 450 2056 25.44

   No. Quality The CE time No. Quality The CE time 451 2442.16 34.11 501 1173.58 37.51 452 1422.66 21.72 502 1100.55 36.99 453 1623.8 24.15 503 1128.44 33.71 454 1624.61 37.73 504 3149.6 31.22 455 3298.48 36.06 505 1068.56 21.69 456 1016.31 35.67 506 1349.48 36.47 457 1580.94 24.31 507 1889.81 40.57 458 1157.58 37.41 508 2305.7 34.8 459 1250.61 27.94 509 840.44 23.94 460 1378.67 28.85 510 911.3 34.39 461 1392.68 21.75 511 1299.64 22.42 462 1409.64 22.06 512 911.47 25.92 463 1425.65 22.34 513 1025.51 25.44 464 1451.71 29.19 514 3400.07 42.03 465 1576.66 26.5 515 1901.89 43.92 466 1651.85 40.6 516 1110.42 34.37 467 1876.94 22.29 517 1032.5 25.89 468 1911.12 24.98 518 1040.52 25.11 469 2064.01 21.95 519 1265.64 27.14 470 2150.04 27.76 520 1171.55 29.24 471 2751.59 29.16 521 1012.53 35.08 472 4289.94 28.69 522 1286.49 36.78 473 4306.05 28.78 523 2932.36 34.11 474 4800.18 23.83 524 1215.49 27.61 475 1111.32 35.47 525 1423.68 21.47 476 1181.49 36.79 526 1487.71 29.58 477 31688.38 24.69 478 1229.57 36.29 479 1579.78 29.83 480 1680.82 30.02 481 1725.66 38.3 482 5228.15 27.04 483 1769.78 28.25 484 1114.54 25.52 485 1390.5 37.05 486 2046.99 32.56 487 2899.33 49.62 488 1096.53 26.12 489 1257.49 34.26 490 868.45 23.35 491 1160.43 35.6 492 1539.8 40.36 493 3318.91 36.01 494 1084.48 25.31 495 1388.39 58.99 496 3129.86 35.93 497 1255.56 36.33 498 1383.69 39.02 499 1561.75 40.72 500 3108.55 31.25

   。
2. 2. according to the method for claim 1, it is characterized in that utilizing following reference point to estimate to whether there being determining of mark 1～106:

   No. Contrast appears CVD appears No. Contrast appears CVD appears 1 0.01 0.58 54 0.52 0.08 2 0.18 0.75 55 0.59 0.17 3 0.14 0.67 56 0.65 0.21 4 0.25 0.75 57 0.48 0.04 5 0.08 0.56 58 0.57 0.13 6 0.05 0.54 59 0.56 0.13 7 0.36 0.83 60 0.44 0.00 8 0.31 0.79 61 0.57 0.13 9 0.26 0.71 62 0.44 0.00 10 0.14 0.58 63 0.73 0.29 11 0.06 0.50 64 0.53 0.08 12 0.15 0.58 65 0.50 0.04 13 0.31 0.75 66 0.49 0.04 14 0.32 0.75 67 0.70 0.25 15 0.12 0.54 68 0.91 0.46 16 0.03 046 69 0.71 0.25 17 0.25 0.67 70 0.63 0.17 18 0.18 0.58 71 0.47 0.00 19 0.47 0.88 72 0.81 0.33 20 0.18 0.58 73 0.59 0.13 21 0.09 0.50 74 0.90 0.42 22 0.27 0.67 75 0.77 0.29 23 0.18 0.58 76 0.94 0.46 24 0.10 0.50 77 0.69 0.21 25 0.44 0.04 78 0.49 0.00 26 0.44 0.04 79 0.66 0.17 27 0.44 0.04 80 0.53 0.04 28 0.48 0.08 81 0.53 0.04 29 0.74 0.33 82 0.67 0.17 30 0.62 0.21 83 0.50 0.00 31 0.45 0.04 84 0.50 0.00 32 0.41 0.00 85 0.62 0.13 33 0.62 0.21 66 0.88 0.38 34 0.74 0.33 87 0.63 0.13 35 0.41 0.00 88 0.55 0.04 36 0.78 0.38 89 0.52 0.00 37 0.45 0.04 90 0.61 0.08 38 0.70 0.29 91 0.91 0.38 39 0.78 0.38 92 0.75 0.21 40 0.41 0.00 93 0.67 0.13 41 0.67 0.25 94 0.73 0.17 42 0.59 0.17 95 0.60 0.04 43 0.67 0.25 96 0.69 0.13 44 0.54 0.13 97 0.85 0.29 45 0.63 0.21 98 0.60 0.04 46 0.42 0.00 99 0.70 0.13 47 0.80 0.38 100 0.71 0.13 48 0.54 0.13 101 0.58 0.00 49 0.42 0.00 102 0.59 0.00 50 0.58 0.17 103 0.80 0.17 51 0.42 0.00 104 0.77 0.08 52 0.72 0.29 105 0.98 0.49 53 0.52 0.08 106 0.90 0.49

   。
3. 3. according to the method for claim 1, it is characterized in that utilizing following reference point to estimate the amplitude of mark 107～413:

   No. The average amplitude control group Average amplitude CVD group No. The average amplitude control group Average amplitude CVD group No. The average amplitude control group Average amplitude CVD group 107 94 253 157 100 202 207 220 44 108 116 233 158 189 513 208 1195 546 109 50 123 159 1054 486 209 3909 1825 110 766 1878 160 161 412 210 406 167 111 45 175 161 123 456 211 149 58 112 89 419 162 229 517 212 1098 2400 113 69 146 163 273 554 213 769 164 114 174 418 164 196 517 214 527 1675 115 78 689 165 197 97 215 1173 416 116 47 99 166 176 506 216 711 324 117 99 188 167 1480 686 217 470 181 118 120 357 168 3107 880 218 723 333 119 317 2460 169 80 216 219 213 102 120 121 463 170 203 1328 220 345 169 121 172 380 171 344 848 221 677 334 122 796 1674 172 797 206 222 2489 744 123 167 888 173 1146 2842 223 132 63 124 1703 636 174 224 568 224 1451 717 125 768 3651 175 138 450 225 1324 299 126 340 1283 176 258 525 226 1689 741 127 193 583 177 15571 7296 227 88 238 128 335 320 178 367 745 228 191 701 129 243 566 179 185 575 229 361 118 130 95 214 180 250 130 230 5095 1789 131 161 768 181 1492 3433 231 601 241 132 118 299 182 784 351 232 1200 403 133 116 267 183 1158 2826 233 736 245 134 840 1950 184 919 371 234 1171 297 135 102 288 185 156 466 235 678 263 136 127 283 186 1694 4348 236 1597 482 137 5726 12044 187 201 66 237 115 353 138 263 728 188 1787 737 238 392 146 139 506 154 189 2810 6060 239 120 265 140 113 289 190 1703 766 240 1127 465 141 150 301 191 461 1095 241 623 240 142 136 290 192 707 6865 242 250 115 143 51 189 193 493 1490 243 633 306 144 168 343 194 346 799 244 224 81 145 196 769 195 3338 9120 245 120 60 146 119 250 196 240 654 246 1275 438 147 161 358 197 20 203 247 283 89 148 227 58 198 490 236 248 1514 737 149 130 289 199 259 533 249 264 91 150 97 196 200 1142 421 250 900 278 151 192 504 201 1241 2506 251 776 338 152 301 128 202 1511 749 252 411 97 153 442 108 203 294 107 253 227 103 154 154 1119 204 1090 2230 254 186 53 155 197 725 205 1151 456 255 890 332 156 82 201 206 983 475 256 469 1170

   No. The average amplitude control group Average amplitude CVD group No. The average amplitude control group Average amplitude CVD group No. The average amplitude control group Average amplitude CVD group No. The average amplitude control group Average amplitude CVD group 257 152 70 307 97 39 357 789 179 407 275 91 258 21200 7156 308 48 104 358 263 104 408 1590 291 259 282 115 309 176 375 359 184 84 409 1343 334 260 474 152 310 87 185 360 206 52 410 180 61 261 117 274 311 187 91 361 755 287 411 149 13 262 1359 517 312 143 43 362 246 26 412 298 135 263 421 195 313 381 143 363 316 106 413 469 42 264 183 83 314 402 194 364 1329 142 265 151 64 315 237 113 365 122 23 266 206 99 316 519 207 366 105 46 267 588 256 317 115 56 367 311 127 268 304 119 318 197 61 368 131 56 269 147 61 319 254 1335 369 206 38 270 172 65 320 283 140 370 104 41 271 338 157 321 88 201 371 126 43 272 292 138 322 119 56 372 345 110 273 227 110 323 129 46 373 416 151 274 142 387 324 125 50 374 209 86 275 166 79 325 3240 7677 375 268 54 276 179 385 326 114 51 376 549 188 277 200 75 327 236 89 377 115 36 278 212 81 328 163 79 378 353 110 279 169 68 329 702 204 379 379 135 280 359 157 330 481 159 380 503 52 261 141 284 331 407 175 381 753 360 282 244 104 332 228 79 382 335 2617 283 882 331 333 200 98 383 97 31 264 903 324 334 356 80 384 1280 178 285 231 98 335 152 72 385 438 64 286 1420 457 336 178 64 386 374 92 287 2096 591 337 281 50 387 329 109 288 676 261 338 293 104 388 283 124 289 470 234 339 796 299 389 273 36 290 169 49 340 174 83 390 3045 864 291 134 517 301 1025 194 391 51 25 292 624 309 342 209 95 392 711 69 293 279 111 343 407 145 393 187 92 294 444 130 344 144 462 394 74 29 295 752 1640 345 182 74 395 197 61 296 543 191 346 95 42 396 320 100 297 164 66 347 92 16 397 712 156 298 785 274 348 150 36 398 187 48 299 185 79 349 256 96 399 337 7 300 234 99 350 130 51 400 133 59 301 179 46 351 96 46 401 297 110 302 360 141 352 330 157 402 164 55 303 106 37 353 248 107 403 876 4574 304 146 47 354 205 69 404 820 309 305 730 323 355 310 36 405 845 288 306 323 40 356 411 139 406 475 119

   And utilize following reference point to estimate the amplitude of mark 414～526:

   No. The average amplitude control group Average amplitude CVD group No. The average amplitude control group Average amplitude CVD group 414 3214 2678 471 358 423 415 514 250 472 3360 3317 416 1359 615 473 4000 2575 417 581 174 474 1388 2785 418 630 499 475 335 3122 419 211 141 476 302 237 420 681 381 477 351 847 421 445 227 478 186 145 422 1178 103 479 3094 2397 423 540 348 480 4737 2446 424 188 206 481 1468 2644 425 1540 687 482 566 974 426 569 914 483 535 429 427 301 106 484 2818 4530 428 976 511 485 17423 37226 429 972 515 486 3087 1793 430 1320 729 487 25 319 431 178 210 488 3397 6633 432 1682 1196 489 2904 6138 433 589 287 490 239 198 434 384 502 491 1794 3083 435 1006 399 492 2558 1701 436 1054 800 493 4Z8 419 437 270 216 494 1326 2891 438 3453 2235 495 181 788 439 837 790 496 212 207 440 1353 684 497 741 600 441 710 733 498 135 197 442 809 627 499 4632 4647 443 8328 3904 500 331 461 444 596 661 501 302 414 445 380 593 502 206 306 446 2389 1375 503 1521 3346 447 297 285 504 349 561 448 4154 2314 505 211 315 449 532 953 506 208 247 450 1145 733 507 1270 1039 451 744 845 508 305 334 452 2878 2433 509 213 266 453 8725 4307 510 2436 3827 454 1109 1620 511 460 294 455 750 409 512 389 924 456 667 971 513 197 273 457 2155 1229 514 152 446 458 1522 1964 515 743 575 459 50512 45582 516 428 713 460 2259 2915 517 186 296 461 4142 4664 518 219 296 462 6265 8932 519 4218 7618 463 1969 2931 520 75 148 464 36818 19376 521 96 214 465 1352 1591 522 56 92 466 5789 1562 523 208 316 467 2562 1358 524 340 729 468 91735 90455 525 543 1220 469 7723 4053 526 388 684 470 2496 1342

   。
4. 4. according to the process of claim 1 wherein at least two kinds or at least three kinds or at least five kinds or six kinds or at least ten kinds or all polypeptide markers that use definition in the claim 1.
5. 5. according to each method in the claim 1 to 4, wherein said sample from the experimenter is urine samples or blood sample (serum or plasma sample).
6. 6. according to each method in the claim 1 to 5, wherein Capillary Electrophoresis, HPLC, gaseous ion chromatogram and/or mass spectrum are used for detecting whether have described polypeptide marker.
7. 7. according to each method in the claim 1 to 6, wherein before measuring described polypeptide marker molecular mass, carry out Capillary Electrophoresis.
8. 8. according to each method in the claim 1 to 7, wherein mass spectrum is used for detecting whether have described polypeptide marker.
9. 9. at least a polypeptide marker that is selected from mark No.1～526 is used to diagnose the purposes of angiosis, the feature of described mark to have to be molecular mass value and the transit time value according to claim 1.
10. One kind the diagnosis angiosis (VD) method, it may further comprise the steps:

    A) sample is divided at least three inferior samples, preferably at least ten inferior samples;

    B) analyze at least two inferior samples whether existing or its amplitude with at least a polypeptide marker in the working sample, wherein said polypeptide marker is selected from mark 1～526, and described mark is characterised in that according to the molecular mass of claim 1 and transit time (CE time).
11. 11., wherein measure at least ten inferior samples according to the method for claim 10.
12. 12. according at least one method in the claim 1 to 11, it is characterized in that the CE time is that 25kV, internal diameter (ID) are the glass capillary of 50 μ m, long 90cm based on applying voltage, the water that wherein contains 20% acetonitrile, 0.25M formic acid is as mobile solvent.
13. 13. a mark combination, it comprises at least ten kinds of marks that are selected from mark 1～526, and described mark is characterised in that molecular mass and the transit time (CE time) that has according to claim 1.