MX2008006724A - 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

POLIPEPTIDE MARKER FOR THE DIAGNOSIS AND EVALUATION OF VASCULAR DISEASES DESCRIPTION OF THE INVENTION The present invention relates to the use of the presence or absence of one or more peptide markers in a sample from a subject for the diagnosis and evaluation of severity of Vascular diseases (VD) and to a method for the diagnosis and evaluation of such vascular disease, wherein the presence or absence of the marker or peptide markers is indicative of the severity of a VD. Vascular diseases are diseases that affect the vessels of an organism and consequently organs such as the heart, brain, kidney, etc. They include, for example, arteriosclerosis, disturbed circulation, hypertension and cardiac dysrhythmia. Blood vessels Atherosclerosis refers to the hardening of arteries by vascular deposits. Deposits of cholesterol crystals lead to the formation of inflammatory foci (atheromas) in which blood components, lipids, metabolic slags and lime salts tend to sediment. The so-called plaques are formed, which are two-dimensional sclerosis, where the vascular wall becomes stiffer and narrower. Artery Ref .: 193243 it loses its elasticity and has difficulty in carrying out its task, that is, the transport of blood from the heart to the individual regions of the body. Secondary diseases include, for example, angina pectoris, myocardial infarction, circulatory collapse, attack. The disturbed circulation mostly affects the lower portion of the body, from the vein aorta ventral to the arteries of the foot, and leads to the reduction of blood flow and supply of oxygen to muscle tissue, which gradually becomes necrotic. In the last stage, the ulcers form and occlude the vessels to the extent that amputation becomes unavoidable. Hypertension does not have a definite cause; In this way, the intake of medications or the excessive secretion of adrenal hormones can cause the blood pressure to increase. There are also high blood pressures in permanent tension, which results in angiospasms. Hypertension damages the vascular walls, so there is a risk of tearing or obstruction. If the regularity of the heartbeat is disturbed, the condition is referred to as cardiac dysrhythmia. The heartbeat can be either very fast (tachycardia), very slow (bradycardia) or irregular (arrhythmia). Vascular diseases can be avoided by prevention, since they are also caused by an unhealthy and unnatural behavior of lifetime. For a radical reversal of lifestyle, arteriosclerosis at an early stage can be stopped, for example, by reducing blood pressure and blood lipid levels. The progress of vascular diseases may additionally be slowed by drug therapies (eg, acetylsalicylic acid, beta-receptor blockers, ACE inhibitors, etc.). However, it should be noted that damaged vessels are irreparable, and the process at an advanced stage is irreversible. Therefore, the early detection of vascular diseases is particularly important. Heart: In a coronary heart disease, the diagnosis of RV is made first indirectly by the evaluation of risk factors and by non-invasive examinations, such as blood pressure measurement, resting and exercise electrocardiograms, and blood graphs to determine the state lipid (LDL cholesterol, HDL cholesterol, triglycerides), fasting blood glucose level and, if necessary, HbAlc. If such examinations produce the presence of high-risk characteristics, that is, severe vascular events (death, myocardial infarction) are expected in the near future, a more accurate diagnosis is made by means of invasive diagnoses, for example, in the form of a catheter or angiography examination coronary In this way, the heart and coronary vessels and other vessels are examined by means of a catheter or with an X-ray method, an X-ray contrast agent is used for a better visualization of the heart and vessels in the image of X-rays. Indications for coronary angiography include a low or medium preliminary test while non-invasive diagnoses fail to provide reliable results, patients in whom non-invasive testing is not possible due to disabilities or diseases, and patients for whom exclusion With certainty of a suspected coronary disease is essential for reasons related to work (for example, pilots, firemen). However, coronary angiography can be performed only if several complications, such as hyperthyroidism or allergy to contrast medium, are excluded, in addition to the preliminary examinations mentioned above. In addition, since the contrast medium is secreted through the kidney, sufficient renal function must be ensured, or for subjects dependent on dialysis, dialysis should always be performed subsequent to the examination. In this way, it becomes clear that there is a need for a non-invasive possibility of an early and reliable diagnosis of vascular diseases.

Kidney: Vascular diseases of the kidney include: * Stenosis of the renal artery * Renal artery thrombosis * Renal artery embolism * Thrombosis of the renal vein A stenosis of the renal artery is a constriction of double side of the renal artery or its branches main. It may be the cause of high blood pressure, which is then referred to as renovascular hypertension. Its cause is arteriosclerosis (predominantly in old age) in approximately 70% of cases, and fibromuscular dysplasia (a connective tissue abnormality) in approximately 20% of cases. Rarely, aneurysms of the aorta or renal artery, vasculitis, mechanical compression from tumors or cysts, embolisms or thrombosis are causally involved. The constriction of the renal artery leads to reduced blood flow through the affected kidney. In order to compensate for the presumed (local) reduction in blood pressure, the kidney increases the production of renin, which leads to an increase in blood volume and an increase in blood pressure of the whole organism through the angiotensin mechanism -aldosterone and this form to arterial hypertension. Therefore, stenosis of the renal artery is discovered more when hypertension develops, but only and approximately 1-2% of all hypertensions are caused by the same. In terms of therapy, there are different possibilities: • PTA (percutaneous transluminal catheter angioplasty): dilation of the constriction by means of an inserted balloon catheter (balloon dilation); «Stress: insertion of a wire mesh (stent) that is to say to keep the vessel open; • Surgical removal of stenosis A common cause of renal artery thrombosis is embolism from the heart, for example, during atrial fibrillation, which is accompanied by symptoms such as flank pain, proteinuria, very high LDH. Flank pain is also observed in renal vein thrombosis, but additionally proteinuria and, in some cases, hematuria or a nephrotic syndrome are observed. Brain: Constrained vessels in the brain region result in a reduced oxygen supply, and when the artery is occluded (for example, by acute coagulation due to changes from arterial sclerosis), an attack occurs with loss of perception, paralysis, disturbed speech, etc. In the cerebral arteries, such as in the large arteries, arterial sclerosis may in rare cases lead to aneurysms of the vascular walls, and along with risk factors such as hypertension, the vascular wall can tear and result in internal bleeding that endangers life. Surprisingly, it has now been found that particular peptide markers in a urine sample from a subject can be used for the diagnosis of DV and thus decide whether or not drug therapy is necessary. In this way, the present invention relates to the use of the presence or absence of at least one peptide marker, ideally several polypeptide markers, in a urine sample from a subject for the diagnosis of vascular diseases, wherein the polypeptide marker or markers are selected from the polypeptide markers No. 1 to No. 526, which are characterized by the molecular masses and migration times as set forth in Table 1.

Table 1: Polypeptide markers for the diagnosis of vascular diseases and their molecular masses and migration times (EC time in minutes): 15 ? fifteen fifteen fifteen fifteen V) Preferably, labels 1-104 and / or 107-413 are employed. With the present invention, it is also possible to determine the severity of the RV. This information footprint helps to decide which therapeutic measures are employed. The migration time by capillary electrophoresis (CE) is determined, for example, as indicated in the Example under article 2. In this way, a glass capillary 90 cm in length and with an internal diameter (ID by its acronym in English) of 75 μm and an external diameter (OD for its acronym in English) of 360 μm is operated at a voltage of 30 kV. It is used as the solvent for the sample, 30% methanol, 0.5% formic acid. It is known that EC migration times may vary. However, the order in which the polypeptide labels are eluted is typically the same for any CE system used. In order to balance the differences in migration time, the system can be normalized using standards for which migration times are known. These standards can be, for example, the polypeptides established in the examples (see the Example, article 3). The characterization of the polypeptide markers shown in Tables 1 to 3 is determined by means of capillary electrophoresis-mass spectrometry (CE-MS), a method which has been described in detail, for example, by Neuhoff et al. (Rapid Communications in mass spectrometry, 2004, Vol. 20, pp. 149-156). The variation of the molecular masses between the individual measurements or between the different mass spectrometers is relatively small, typically within a range of + 0.1%, preferably within a range of + 0.05%, more preferably within a range of + 0.03%, more preferably within a range of + 0.03%, even more preferably within a 0.01% range. The polypeptide markers according to the invention are proteins or peptides or products of degradation of proteins or peptides. They can be chemically modified, for example, by posttranslational modifications, such as glycosylation, phosphorylation, alkylation or disulphide bridges, or by other reactions, for example, within the scope of degradation. In addition, polypeptide markers can also be chemically altered, for example, oxidized, within the purification range of the samples. Proceeding from the parameters that determine the polypeptide markers (molecular weight and migration time), it is possible to identify the sequence of the corresponding polypeptides by methods known in the prior art. The polypeptides according to the invention (see Tables 1 to 4) are used to diagnose the severity of the RV. "Diagnosis" means the process of knowledge gain by assigning symptoms or phenomena to an illness or injury. In the present case, the severity of the RV is concluded from the presence or absence of particular polypeptide markers. In this way, the polypeptide markers according to the invention are determined in a sample from a subject, where their presence or absence allows to conclude the severity of the RV. The presence or absence of a polypeptide tag can be average by any method known in the prior art. The methods which may be known are exemplified later. A polypeptide tag is considered present if its measured value is at least as high as its threshold value. If the measured value is lower, then the polypeptide marker is considered absent. The threshold value can be determined either by the sensitivity of the measurement method (detection limit) or empirically. In the context of the present invention, considers that the threshold value is preferably exceeded if the measured value of the sample for a certain molecular mass is at least twice as high as that of the control sample (for example, only buffer or solvent). The polypeptide tag or markers are / are used in such a way that their presence or absence is measured, where the presence or absence is indicative of the severity of the RV (frequency marker). Thus, there are polypeptide markers which are typically present in subjects with RV, but occur less frequently or are absent in subjects without RV, for example, 1-24 (Table 2). In addition, there are polypeptide markers which are present in patients with RV, such as polypeptide markers No. 25 to 106, but less frequently or not present in patients without RV. Table 2: Polypeptide markers (frequency markers) for the diagnosis of vascular diseases, their molecular masses and migration times, and their presence and absence in patients who suffer from VD (VD) and control groups (Control) as a factor (1 = 100%, 0 = 0%, sample processing and measurement as described in the Example).

In addition or alternatively to the frequency markers (determination of presence or absence), the amplitude markers as set out in Table 3 can also be used for the diagnosis of RV (No. 107- 526). The amplitude markers are used in such a way that the presence or absence is not critical, but the height of the signal (the amplitude) decides whether the signal is present in both groups. In Tables 3 and 4, the mean amplitudes of the corresponding signals (characterized by mass and migration time) averaged over all the measured samples are established. Two standardization methods are possible to achieve comparability between the differently concentrated samples or different measurement methods. In the first procedure, all the signals of Peptides from a sample are normalized to a total amplitude of 1 million counts. Therefore, the respective mean amplitudes of the individual markers are stated as parts per million (ppm). The amplitude markers obtained by this method are shown in Table 3 (No. 107-413). In addition, it is possible to define additional amplitude markers by an alternative normalization method. In this case, all peptide signals from a sample are scaled with a common normalization factor. In this way, a linear regression is formed between the peptide amplitudes of the individual samples and the reference values of all known polypeptides. The slope of the regression line corresponds just to the relative concentration and is used as a normalization factor for this sample. The biomarkers obtained by this standardization methods are shown in Table 4 (No. 414-526). All groups employed consist of at least 20 individual patients or control samples in order to obtain a reliable mean amplitude. The decision for a diagnosis (VD or not) is made as a function of how high the amplitude of the respective polypeptide markers is in the patient sample compared to the average amplitudes in the control groups or the VD group. If the amplitude corresponds to the mean amplitudes of the group VD, is considered "the exi tence of a vascular disease, and if it corresponds rather to the average amplitudes of the control group, the non-existence of RV is considered." The distance between the measured value and the average amplitude can be considered A probability of the samples belonging to a certain group, an explanation of example should be given by means of marker No. 137 (Table 3) The mean amplitude of the marker is significantly increased in VD (12044 ppm against 5726 ppm in the control group.) Now, if the value for this marker in a patient sample is from 0 to 5726 ppm or exceeds this range by a maximum of 20%, that is, from 0 to 6871 ppm, then this sample belongs to the control group. If the value is 12044 ppm or up to 20% later, or higher, it is to say, between 9635 and very high values, this is to be considered an indication of a vascular disease. Alternatively, the distance between the measured value and the average amplitude can be considered a probability of belonging to a certain group. A frequency marker is a variant of an amplitude marker in which the amplitude is low in some samples. It is possible to convert such frequency markers to amplitude markers by including the corresponding samples in which the marker is not found in the calculation of the amplitude with a very small amplitude, in order to limit the detection.

UJ Table 4: Amplitude markers with normalization according to procedure 2 The subject from which the sample is derived in which the presence or absence of one or more polypeptide markers is determined can be any subject which is capable of suffering a DV. Preferably, the subject is a mammal, and more preferably, a human. In a preferred embodiment the invention, not only a polypeptide tag, but a combination of polypeptide tags are used to determine the severity of the RV, where the severity of the RV can be concluded from its presence or absence. By comparing a plurality of polypeptide markers, a bias in the total result can be reduced or avoided from a few individual deviations from the probability of typical presence in the sick or control individual. The sample in which the presence or absence of the peptide marker or markers according to the invention is measured can be any mixture which is obtained from the body of the subject. The sample is a sample which has a suitable polypeptide composition to provide information about the state of the subject (VD or not). For example, it may be a sample of blood, urine, synovial fluid, a tissue fluid, a body secretion, sweat, cerebrospinal fluid, lymph, intestinal, gastric or pancreatic juice, bile, lacrimal fluid, a tissue sample, sperm, vaginal fluid or stool.

Preferably, it is a liquid sample. In a preferred embodiment, the sample is a urine sample or blood sample, wherein a blood sample can be serum (blood) or plasma sample (blood). In a preferred embodiment, the sample is a urine sample or blood sample, wherein the blood sample can be a serum (blood) or plasma (blood) sample. Urine samples can be taken as preferred in the prior art. Preferably, a medium-current urine sample is used as the urine sample in the context of the present invention. For example, the urine sample may also be taken by means of a urinating apparatus as described in patent application WO 01/74275. The blood samples can be taken by methods known in the prior art, for example, from the vein, artery or capillary. Usually, a blood sample is obtained by drawing venous blood by means of a syringe, for example, from an arm of the subject. The term "blood sample" includes samples obtained from blood by further purification and separation methods, such as blood plasma or blood serum. The presence or absence of a marker The polypeptide in the sample can be determined by any method known in the prior art that is suitable for measuring polypeptide markers. Such methods are known to the skilled person. In principle, the presence or absence of a polypeptide tag can be determined by direct methods, such as mass spectrometry, or indirect methods, for example, by means of ligands. If required or desired, the sample from the subject, eg, the urine or blood sample, can be pretreated by any suitable means and, for example, purified or separated before the presence or absence of the marker or markers is measured. of polypeptide. The treatment may comprise, for example, purification, separation, dilution or concentration. The methods may be, for example, centrifugation, filtration, ultrafiltration, dialysis, precipitation or chromatographic methods, such as separation of affinity or separation by means of ion exchange chromatography, electrophoretic separation, i.e. separation by different migration behaviors. electrically charged particles in solution before application of an electric field. Particular examples thereof are gel electrophoresis, two polyacrylamide gel electrophoresis (2D-PAGE), capillary electrophoresis, metal affinity chromatography, metal affinity chromatography immobilized (IMAC), lecithin-based affinity chromatography, liquid chromatography, high performance liquid chromatography (HPLC), normal and inverse phase HPLC, cation exchange chromatography and selective bond to surfaces. All these methods are well known to the skilled person, and the skilled person will be able to select the method as a function of the sample employed and the method for determining the presence or absence of the polypeptide marker or markers. In one embodiment of the invention, the sample, before being separated by capillary electrophoresis, is separated, purified by ultracentrifugation and / or divided by ultrafiltration into fractions which contain polypeptide markers of a particular molecular size. Preferably, a mass spectrometric method is used to determine the presence or absence of a polypeptide tag, wherein a purification or separation of the sample can be performed upstream of such a method. As compared to methods currently employed, mass spectrometric analysis has the advantage that the concentration of many (> 100) polypeptides of a sample can be determined by a simple analysis. Any type of mass spectrometer can be used. By means of mass spectrometry, it is possible measuring 10 fmoles of a polypeptide tag, i.e. 0.1 ng of a 10 kD protein, as a routine matter with a measurement accuracy of about + 0.01% in a complex mixture. In mass spectrometers, an ion forming unit is coupled with a suitable analytical device. For example, electrospray ionisation (ESI) interfaces are mostly used to measure ions in liquid samples, while MALDI (matrix-assisted laser deaeration / ionization) is used to measure ions from a sample crystallized in a matrix. To analyze the formed ions, quadrupoles, ion traps or flight time analyzers (TOF for its acronym in English) can be used, for example. In electrospray ionization (ESI), the molecules present in solution are atomized, inter alia, under the influence of high voltage (for example, 1-8 kV), which forms first charged droplets that become smaller starting of the evaporation of the solvent. Finally, the so-called Coulomb explosions result in the formation of free ions, which can then be analyzed and detected. In the analysis of ions by means of TOF, a particular acceleration voltage is applied which confers an equal amount of kinetic energy to the ions. After from. this, the time it takes to travel to the respective ions a particular drift distance through the flight tube is measured very accurately. Since with equal amounts of kinetic energy, the velocity of the ions depends on their masses, the latter can thus be determined. The TOF analyzers have a very high scanning speed and therefore achieve a good resolution. Preferred methods for determining the presence and absence of polypeptide markers include gas phase ion spectrometry, such as deaeration / laser ionization mass spectrometry, MALDI-TOS MS, SELDI-TOF MS (deaeration / laser ionization) superficially increased), LC MS (liquid chromatography / mass spectrometry), 2D-PAGE / MS and capillary electrophoresis-mass spectrometry (CE-MS). All the mentioned methods are known to the skilled person. A particularly preferred method is CE-MS, in which capillary electrophoresis is coupled with mass spectrometry. This method has been described in some detail, for example, in German Patent Application DE 10021737, in Kaiser et al., (J. Chromatogr A, 2003, Vol. 1013: 157-171 and Electrophoresis, 2004, 25: 2044 -2055) and in Wittke et al. (J. Chromatogr. A, 2003, 1013: 173-181). The CE-MS technology allows the presence of several hundred polypeptide markers in a sample to be determined simultaneously within a short time and in a small volume with high sensitivity. After a sample has been measured, a standard of the measured polypeptide markers is prepared, and this pattern can be compared with reference standards of sick or healthy subjects. In most cases, it is sufficient to use a limited number of polypeptide markers for the diagnosis of UAS. A CE-MS method which includes CE coupled online to an ESI-TOF MS is also preferred. For CE-MS, the use of volatile solvents is preferred, and it works best under essentially salt-free conditions. Examples of such solvents include acetonitrile, isopropanol, methanol and the like. The solvents can be diluted with water or a weak acid (for example, 0.1% to 1% formic acid) in order to protonate the analyte, preferably the polypeptides. By means of capillary electrophoresis, it is possible to separate molecules by their charge and size. The neutral particles will migrate at the speed of the electro-osmotic flow before application of a current, while the cations are accelerated towards the cathode, and the anions are delayed. The advantage of the capillaries in the electrophoresis resides in the favorable proportion of surface to volume, which allows a good dissipation of the Joule heat generated during the current flow. This in turn allows high voltages (usually up to 30 kV) are applied and in this way a high separation behavior and short analysis times. In capillary electrophoresis, silica glass capillaries having internal diameters of typically 50 to 75 μm are usually employed. The lengths used are, for example, 30-100 cm. In addition, the separation capillaries are usually made of silica glass coated with plastic. Capillaries can be either untreated, that is, exposing their hydrophilic groups on the inner surface, or coated on the inner surface. A hydrophobic coating can be used to improve resolution. In addition to the voltage, a pressure may be applied, which is typically within the range of 0 to 1 psi. The pressure can also be applied only during separation or altered in between. In a preferred method for measuring polypeptide markers, the markers in the sample are separated by capillary electrophoresis, then directly ionized and transferred in line to a coupled mass spectrometer for detection. In the method according to the invention, it is advantageous to use several polypeptide markers to diagnose the RV. In particular, at least three polypeptide markers can be used, for example, markers 1, 2 and 3; 1, 2 and 4; etc. The use of at least 4, 5, or 6 markers is preferred. The use of at least 11 markers, for example, markers 1 to 11, is even more preferred. The use of all the 526 labels set forth in Tables 1 to 4 is more preferred. In order to determine the probability of the existence of a severe RV when using several markers, statistical methods known to the skilled person may be used. For example, the Random Forest method described by Weissinger et al. (Kidney Int., 2004, 65: 2426-2434) can be used by using a computer program such as S-Plus, or the support vector machines as described in the same publication. Example 1. Sample Preparation To detect the polypeptide markers to diagnose RV, urine is used, urine is collected from healthy donors (control group) as well as from patients suffering from severe RV. For the subsequent CE-MS measurement, the proteins which are also contained in the urine of patients in a high concentration, such as albumin and immunoglobulins, have to be separated by ultrafiltration. In this way, 700 μl of urine is collected and mixed with 700 μm of filter buffer (2M urea), 10 mM ammonia, 0.02% SDS). This sample of 1.4 ml volume is ultrafiltered (20 kDa, Sartorius, Gottingen, Germany). Ultrafiltration is performed at 3000 rpm in a centrifugation until 1.1 ml of the ultrafiltrate is obtained. The 1.1 ml of filtrate obtained is then applied to a PD 10 column (Amersham Bioscience, Uppsala, Sweden) and eluted with 2.5 ml of 0.01% NH4OH, and lyophilized. For the measurement of CE-MS, the polypeptides are then resuspended with 20 μl of water (grade CLAR, Merck). 2. Measurement of CE-MS The measurements of CE-MS are made with a capillary electrophoresis system from Beckman Coulter (P / ACE MDQ System; Beckman Coulter Inc., Fullerton, CA, USA) and an ESI-TOF mass spectrometer from Bruker (micro-TOF MS, Bruker Daltonik, Bremen, Germany). CE capillaries are supplied by Beckman Coulter and have an ID / OD of 50/360 μm and a length of 90 cm. The mobile phase for EC separation consists of 20% acetonitrile and 0.25% formic acid in water. For the "pod flow" in the MS, 30% isopropanol with 0.5% formic acid is used, here at a flow rate of 2 μl / minutes. The coupling of CE and MS is carried out by the dispersing equipment of CE-ESI-MS (Agilent Technologies, Waldbronn, Germany). To inject the sample, a pressure of 1 to a maximum of 6 psi is applied, and the duration of the injection is 99 seconds. With these parameters, approximately 150 of the sample are injected into the capillary, which corresponds to approximately 10% of the capillary volume. A stacking technique is used to concentrate the sample in the capillary. In this way, before the sample is injected, a solution of 1 M NH3 is injected for 7 seconds (in 1 psi), and after the sample is injected, a solution of 2 M formic acid is injected for 5 seconds. When the separation voltage (30 kV) is applied, the analytes are automatically concentrated between these solutions. Subsequent CE separation is performed with a pressure method: 40 minutes at 0 psi, then 0.1 psi for 2 minutes, 0.2 psi for 2 minutes, 0.3 psi for 2 minutes, 0.4 psi for 2 minutes, and finally 0.5 psi for 32 minutes. The total duration of a separation run is thus 80 minutes. In order to obtain a good signal strength as possible on the MS side, the nebulizer gas is returned to its lowest possible value. The applied voltage for the spray needle to generate the electrospray is 3700-4100 V. The remaining fixations in the mass spectrometer are optimized in the mass spectrometer for peptide detection according to the manufacturer's instructions. The spectra are recorded in a mass range of m / z 400 to m / z 3000 and accumulate every 3 seconds. 3. Standards for EC measurement To check and standardize CE measurement, the following proteins or polypeptides are used which are characterized by the established EC migration times: The proteins / polypeptides are used in a concentration of 10 pmoles / μl each in water. "REV", "ELM", "KINCON", and "GIVLY" are synthetic peptides. The molecular masses of the peptides and the m / z ratios of the individual charge states visible in MS are established in the following Table: At the outset, it is known to the skilled person that slight variations of migration times may occur in separations by capillary electrophoresis. However, under the conditions described, the migration order will not change. For the skilled person who knows the established masses and EC times, it is possible without difficulty to assign their own measurements to the polypeptide markers according to the invention. For example, one can proceed as follows: First, one of the polypeptides found in the measurement is selected (peptide 1) and the aim is to find one or more identical masses within a set period of time of the CE (for example, + minutes). If only an identical mass is found within this range, the assignment is completed. If several coupling masses are found, a decision will still be made around the assignment. In this way, another peptide (peptide 2) is selected from the measurement and an identification of a suitable polypeptide marker is sought, again taking into account a corresponding time frame. Again, if several markers with a corresponding mass can be found, the most likely assignment is that there is a substantially linear relationship between the change for peptide 1 and that for peptide 2. Depending on the complexity of the problem of assignment, it is suggested by itself to the skilled person to optionally use additional proteins from their sample for assignment, for example, ten proteins. Typically, migration times are either extended or shortened by particular absolute values, or compressions or expansions of course all occur. However, the co-emigration peptides will also co-migrate under such conditions. In addition, the skilled person can make use of the migration patterns described by Zuerbig et al. in Electrophoresis 27 (2006), p. 2111-2126. If the measurement in the form of m / z is plotted against migration time by means of a simple diagram (for example with MS Excel), the described line patterns also become visible. Now, a simple allocation of the individual polypeptides by counting the lines is possible. Other assignment procedures are also possible. Basically, the skilled person can also use the peptides mentioned above as internal standards for assigning CE measurements. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (13)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A process for diagnosing vascular diseases VD, characterized in that it comprises the step of determining the presence or absence of at least one polypeptide marker in a shows, where the polypeptide marker is selected from the markers 1 to 526, which are characterized by the following molecular masses and migration times: ? n
2. 2. The process according to claim 1, characterized in that an evaluation of the determined presence or absence of markers 1 to 106 is carried out by means of the following reference values:
3. 3. The process according to claim 1, characterized in that an evaluation of the amplitude of markers 107 to 413 is carried out by means of the following reference values: or ^ 1 0 5 And for the markers 414 to 526, it is carried out by means of the following reference values: ^ 1 ^ 1
4. 4. The process according to claim 1, characterized in that at least two or at least three or at least five or six or at least ten or all polypeptide markers are used according to claim 1.
5. 5. The process according to any of claims 1 to 4, characterized in that the sample from a subject is a urine sample or blood sample (serum or plasma sample). The process according to any of claims 1 to 5, characterized in that capillary electrophoresis, CLAR, gas phase ion spectrometry and / or mass spectrometry are used to detect the presence or absence of the polypeptide marker or markers . 7. The process according to any of claims 1 to 6, characterized in that capillary electrophoresis is performed before the molecular mass of the polypeptide markers is measured. 8. The process according to any of claims 1 to 7, characterized in that mass spectrometry is used to detect the presence or absence of the polypeptide marker or markers. 9. The use of at least one polypeptide marker selected from markers No. 1-526, where it is determined by the molecular mass values and migration times according to claim 1, to diagnose vascular diseases. 10. A method for diagnosing vascular diseases VD, characterized in that it comprises: a) separating a sample into at least three, preferably 10, subsamples; b) analyzing at least two subsamples to determine the presence or absence or amplitude of at least one polypeptide tag in the sample, wherein the polypeptide tag is selected from tags 1 to 526, which are determined by the masses Molecular and migration times (CE times) according to claim 1. 11. The method according to claim 10, characterized in that at least 10 sub-samples are measured. The method according to at least one of claims 1 to 11, characterized in that the CE time is based on a 90 cm long glass capillary which has an internal diameter (ID) of 50 μm in a voltage applied 25 kV, where 20% acetonitrile, 0.25 M formic acid in water is used as the mobile solvent. 13. A combination of markers, characterized in that it comprises at least 10 markers selected from markers 1 to 526, which is characterized by the molecular masses and migration times (CE times) according to claim 1.