WO2013150132A2

WO2013150132A2 - Polypeptide markers for diagnosis and assessment of strokes

Info

Publication number: WO2013150132A2
Application number: PCT/EP2013/057182
Authority: WO
Inventors: Harald Mischak
Original assignee: Mosaiques Diagnostics And Therapeutics Ag
Priority date: 2012-04-05
Filing date: 2013-04-05
Publication date: 2013-10-10
Also published as: WO2013150132A3

Abstract

A method for diagnosis and assessment of ischemic stroke and transient ischemic attacks comprising the step of determining a presence or absence or amplitude of at least three polypeptide markers in a urine sample, wherein said polypeptide markers are selected from the markers that are characterised in table 1 by values for the molecular masses and the migration time.

Description

Polypeptide marker for diagnosis and

Assessment of strokes

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 of an individual for the diagnosis and assessment of ischemic stroke and transistoric, ischemic attacks as well as a method for the diagnosis and assessment of ischemic stroke and transistoric ischemic attacks or absence of the peptide marker (s) is indicative of ischemic stroke and of transient ischemic attacks.

A transient ischemic attack (TIA) is a circulatory disorder of the brain that causes neurological deficits that completely regress within 24 hours.

If there is no complete regression of the symptoms, it is by definition an ischemic stroke (IS).

Typical symptoms include hemiplegia of the arm and / or leg, speech problems and - possibly half-sided - visual disturbances. Especially in the case of TIA, some of the symptoms have already subsided during hospital admission. The diagnosis is essentially based on imaging techniques.

However, even if a specialist evaluates, it is estimated that about one-fifth of patients with ischemic stroke and up to 50% of patients with TIA will be diagnosed with another condition. In particular, delineation to a cerebral hemorrhage is often difficult.

In one study, the sensitivity of magnetic resonance imaging with diffusion weighting was 83%, while computed tomography without contrast medium had only a sensitivity of 16%. Thus, the false-negative rate of magnetic resonance imaging is still 17%. The ability or ability to easily and reliably detect and assess a stroke, especially a minor stroke or a TIA would have many advantages.

The object of the present invention was to provide a method with which this diagnosis can take place, in particular also the severity of a stroke can be assessed.

The object is achieved by a method for diagnosing a stroke, comprising the step of determining the presence or absence or amplitude of at least three polypeptide markers in a urine sample, the polypeptide markers being selected from the markers shown in Table 1 by values for the molecular masses, the migration time and / or optionally their peptide sequence are characterized.

The evaluation of the measured polypeptides can be based on the presence or absence and / or amplitude of the markers taking into account the following reference or regulation values. The specified logarithm of the amplitude is a measure of a higher or lower occurrence of a marker in the respective groups.

The control group is a group of patients who did not have a stroke.

The sequences are shown in Table 3.

In one embodiment of the invention only markers are used whose sequence is given in Table 3.

Specificity is defined as the number of actual negative samples divided by the sum of the number of actual negatives and the number of false positives. A specificity of 100% means that a test identifies all healthy persons as healthy, i. no healthy person is identified as ill. This does not say anything about how well the test detects sick patients.

Sensitivity is defined as the number of actual positive samples divided by the sum of the number of actual positives and the number of false negatives. A sensitivity of 100% means that the test detects all patients. He does not say how well the test detects healthy people.

By the markers according to the invention it is possible to detect IS or TIA with a specificity of at least 60, preferably at least 70, more preferably 80, even more preferably at least 90 and most preferably at least 95%.

By the markers according to the invention it is possible to detect IS or TIA with a sensitivity of at least 60, preferably at least 70, more preferably 80, even more preferably at least 90 and most preferably at least 95%.

Migration time (CE time) is determined by capillary electrophoresis (CE) - e.g. executed in point 2 - determined. In this example, a 90 cm long glass capillary with an inner diameter (ID) of 50 μm and an outer diameter (OD) of 360 μm is operated at an applied voltage of 30 kV. The eluent used is, for example, 30% methanol, 0.5% formic acid in water or 20% acetonitrile, 0.25% formic acid. All percentages in% by volume.

It is known that the CE migration time can vary. However, the order in which the polypeptide labels elute is typically the same for any CE system used under the conditions indicated. To the- To compensate for any remaining differences in migration time, the system can be normalized using standards for which migration times are well known. These standards can be, for example, the polypeptides given in the examples (see Example 3), or certain known peptides from urine, as described, for example, in Jantos-Siwy et al. (Quantitative Urinary Proteome Analysis for Biomarker Evaluation in Chronic Kidney Disease, J. Proteome Res., 8: 268-281 (2009)).

The characterization of the polypeptides shown in Table 1 was determined by capillary electrophoresis mass spectrometry (CE-MS), a method which is described e.g. in detail from e.g. Neuhoff et al. Rapid Communications in mass spectrometry, 2004, Vol. 20, pages 149-156), Mischak, H. et al. (Cap- philary electrophoresis mass spectrometry as a powerful tool in biomarker dis- covery and clinical di- agnosis: An update of recent developments: Mass Spectrom Rev. 28, 703-724 (2009)), Mischak, H. et al , (Comprehensive human urine standards for comparability and standardization in clinical proteome analysis, Proteomics Clin Appl., 4, 464-478 (2010)), Good, D.M. et al. (Natu- rally occurring human urinary peptides for use in the diagnosis of chronic kidney disease, Mol. Cell Proteomics 9, 2424-2437 (2010)). The variation of molecular masses between individual measurements or between different mass spectrometers is relatively small with exact calibration, typically in the range of ± 0.05%, more preferably ± 0.03%, even more preferably ± 0.01% or 0.005%.

The polypeptide markers according to the invention are proteins or peptides or degradation products of proteins or peptides. They may be chemically modified, e.g. by post-translational modifications such as glycation, phosphorylation, alkylation or disulfide bridging, or by other reactions, e.g. in the context of mining, to be changed. Based on the parameters that determine the polypeptide markers (molecular mass and migration time), it is possible to identify the sequence of the corresponding polypeptides by methods known in the art.

The polypeptides of the invention are used to diagnose IS or TIA. Diagnosis is the process of the recognition Winning by assigning symptoms or phenomena to a disease or injury. The presence or absence of a polypeptide marker can be measured by any method known in the art. Methods that can be used are exemplified below.

A polypeptide marker is present when its reading is at least as high as the threshold. If its reading is below that, the polypeptide marker is absent. The threshold value can either be determined by the sensitivity of the measurement method (detection limit) or defined based on experience.

In the context of the present invention, the threshold is preferably exceeded when the sample reading for a given molecular mass is at least twice that of a blank (e.g., only buffer or solvent). The polypeptide marker (s) is / are used to measure its presence or absence, the presence or absence being indicative of IS or TIA. Thus, there are polypeptide markers that are typically present in individuals with IS or TIA, but are less common or absent in individuals without IS or TIA. Furthermore, there are polypeptide markers which are present in patients with IS or TIA, but are absent or only rarely present in patients without IS or TIA. The frequency with which a marker appears in the group with IS or TIA or in the control group is given in Table 2 as frequency.

In addition or alternatively to the frequencies (determination of the presence or absence), the amplitudes can also be used for diagnosis. The amplitudes are used in such a way that it is not the presence or absence that is decisive, but the height of the signal (the amplitude) when the signal is present in both groups. A nomination procedure makes sense in order to achieve comparability between differently concentrated samples or different measurement methods. For this purpose, collagen fragments are preferably used, as in Jantos Siwy et al. (Quantitative Urinary Proteome Analysis for Biomarker Evaluation in Chronic Kidney Disease, J. Proteome Res., 8: 268-281 (2009)). wrote. It is determined the linear correlation between the reference values for the amplitude of the given known peptides (so-called "housekeeping peptides") and the experimentally determined values. The increase in the regression line just corresponds to the relative concentration and is used as a normalization factor for the calibration of all peptide signals of this sample with a common normalization factor.

The decision to make a diagnosis depends on how high the amplitude of the respective polypeptide markers in the patient sample is compared to the mean amplitudes in the control group or the "sick" group. If the value is close to the mean amplitude of the "sick" group, it is to be assumed that the occurrence of a stroke, it corresponds more to the mean amplitudes of the control group, is not to assume a stroke. The distance to the mean amplitude can be interpreted as a probability of belonging to a group. Alternatively, the distance between the measured value and the mean amplitude may be considered as a probability of belonging to a group.

Preferably, both the frequency and the amplitude are used for the evaluation. In each case, it is possible to derive probabilities for the assignment to the groups "IS or TIA" or "healthy" from measurement data of an unknown sample, from which then a total probability results.

The p-value is a measure of the likelihood that the association of the markers with the two groups (IS or TIA and control) is based on a random distribution that is not related to IS or TIA. The smaller the Wilcox p-value, the more likely it is to correlate with IS or TIA. The individual from whom the sample is derived, in which the presence or absence or amplitude of one or more polypeptide markers is determined, may be any individual who may be suffering from IS or TIA. Preferably, the subject is a mammal, most preferably a human.

In a preferred embodiment of the invention not only three polypeptide markers but a larger combination of markers are used. By comparison of a plurality of polypeptide markers, the adulteration of the overall result can be reduced or avoided by individual individual deviations from the typical probability of presence in the individual.

The sample measuring the presence or absence of the polypeptide marker (s) of the invention may be any sample recovered from the subject's body. The sample is a sample having a polypeptide composition suitable for making statements about the condition of the individual. For example, it may be blood, urine, synovial fluid, tissue fluid, body secretions, sweat, cerebrospinal fluid, lymph, intestinal, gastric, pancreatic, bile, tear fluid, tissue sample, sperm, vaginal fluid or stool sample , Preferably, it is a liquid sample. In a preferred embodiment, the sample is a urine sample.

Urine samples may be known as known in the art. Preferably, in the context of the present invention, a mid-jet urine sample is used. The urine sample may e.g. by means of a catheter or also with the aid of a urination apparatus, as described in WO 01/74275.

The presence or absence or amplitude of a polypeptide marker in the sample can be determined by any method known in the art suitable for measuring polypeptide markers. Those skilled in such methods are known. In principle, the presence or absence of a polypeptide marker can be determined by direct methods such as e.g. Mass spectrometry, or indirect methods, such as by ligands or specific probes such as antibodies.

If necessary or desirable, the sample of the subject, eg, the urine sample, may be pretreated and, for example, purified or separated, prior to measuring the presence or absence of the polypeptide marker (s) by any suitable means. The treatment may include, for example, purification, separation, dilution or concentration. The methods may include, for example, centrifugation, filtration, ultrafiltration, dialysis, precipitation or chromatographic methods such as affinity separation or separation by ion exchange chromatography, or electrophoretic separation. Specific examples thereof are gel electrophoresis, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary electrophoresis, metal affinity chromatography, immobilized metal affinity chromatography (IMAC), affinity chromatography on the basis of lectins, liquid chromatography, high performance liquid chromatography (HPLC), normal and reverse phase HPLC , Cation exchange chromatography and selective bonding to surfaces. All of these methods are well known to those skilled in the art and one skilled in the art will be able to select the method depending on the sample used and the method for determining the presence or absence of the polypeptide marker (s).

Preferably, a mass spectrometric method is used to determine the presence or absence or amplitude of a polypeptide marker, which method may precede purification or separation of the sample. The mass spectrometric analysis has the advantage over current methods that the concentration of many (> 100) polypeptides of a sample can be determined by a single analysis. Any type of mass spectrometer can be used. With mass spectrometry, it is possible to routinely measure 10 fmoles of a polypeptide marker, that is, 0.1 ng of a 10 kDa protein with a measurement accuracy of approximately ± 0.01% from a complex mixture. In mass spectrometers, an ion-forming unit is coupled to a suitable analyzer. For example, electrospray ionization (ESI) interfaces are commonly used to measure ions from liquid samples, whereas the matrix assisted laser desorption / ionization (MALDI) technique is used to measure ions from sample crystallized with a matrix , For analysis of the resulting ions, e.g. Quadrupoles, ion traps or time-of-flight (TOF) analyzers are used.

In electrospray ionization (ESI), the molecules present in solution are sprayed, inter alia, under the influence of high voltage (eg 1-8 kV), resulting in charged droplets formed by evaporation of the solvent get smaller. Finally, so-called Coulomb explosions lead to the formation of free ions, which can then be analyzed and detected.

In the analysis of the ions by means of TOF, a specific acceleration voltage is applied, which gives the ions an equal kinetic energy. Then, the time required for the respective ions to travel a drift path through the flight tube is measured very accurately. Since with the same kinetic energy, the speed of the ions depends on your mass, this can thus be determined. TOF analyzers have a very high scanning speed and achieve a very high resolution.

Preferred methods for determining the presence or absence of polypeptide markers include gas phase ion spectrometry, such as laser desorption / ionization mass spectrometry, 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). All of the methods mentioned are known to the person skilled in the art.

A particularly preferred method is CE-MS, in which capillary electrophoresis is coupled with mass spectrometry. This method is described in detail e.g. in German patent application DE 10021737, in Kaiser et al. (J. Chromatogr. A, 2003, Vol. 1013: 157-171, and Electrophoresis, 2004, 25: 2044-2055), in Wittke et al. (J. Chromatogr. A, 2003, 1013: 173-181) and Ref. The CE-MS technique allows to determine the presence of several hundreds of polypeptide markers of a sample simultaneously in a short time, a small volume and high sensitivity. After a sample has been measured, a pattern of the measured polypeptide markers is prepared (see below). This can be compared with reference patterns of ill or healthy individuals. In most cases it is sufficient to use a limited number of polypeptide markers for the diagnosis of diseases. More preferred is a CE-MS method which includes CE coupled online to an ESI-TOF-MS.

For CE-MS, the use of volatile solvents is preferred, and it is best to work under substantially salt-free conditions. conditions. Examples of suitable solvents include acetonitrile, methanol and the like. The solvents may be diluted with water and an acid (eg 0.1% to 1% formic acid) added to protonate the analyte, preferably the polypeptides.

Capillary electrophoresis makes it possible to separate molecules according to their charge and size. Neutral particles migrate at the rate of electroosmotic flow upon application of a current, cations are accelerated to the cathode and anions are retarded. The advantage of capillaries in electrophoresis is the favorable ratio of surface area to volume, which enables a good removal of the Joule heat arising during the current flow. This in turn allows the application of high voltages (usually up to 30 kV) and thus a high separation efficiency and short analysis times.

In capillary electrophoresis, quartz glass capillaries with internal diameters of typically 50 to 75 μm are normally used. The used lengths are 30-100 cm. In addition, the capillaries usually consist of plastic-coated quartz glass. The capillaries may be both untreated, i. on the inside show their hydrophilic groups, as well as be coated on the inside. A hydrophobic coating can be used to improve the resolution. In addition to the voltage, a pressure which is typically in the range of 0-1 psi may also be applied. The pressure can also be created during the separation or changed during the process.

In a preferred method of measuring polypeptide markers, the markers of the sample are separated by capillary electrophoresis, then directly ionized and transferred online to a mass spectrometer coupled thereto for detection. In the method according to the invention, several polypeptide markers can advantageously be used for diagnostics. Preferred is the use of at least 5, 6, 8, or 10 markers. In one embodiment, 20 to 50 markers are used.

In order to determine the probability of the presence of a disease when using multiple markers, methods known to the person skilled in the art can be used be used. For example, the Weissinger et al. Kidney Int., 2004, 65: 2426-2434) using a computer program such as S-Plus or the support vector machines described in the same publication. Further information can be found, for example, Dakna et al. BMC Bioinformatics 11 (2010) 594.

These methods combine multiple biomarkers into a single variable associated with both the likelihood of disease and the severity of the disease. This variable can thus also be used to determine the severity or severity of the disease (eg shown in Schiffer et al., Prediction of muscle invasive bladder cancer using urinary proteomics., Clin. Cancer Res. 15, 4935-4943 (2009 for bladder carcinoma) as well as to reflect a possible response to therapy (as shown, for example, in Haubitz et al., Identification and Validation of Urinary Biomarkers for Differential Diagnosis and Evaluation of Therapeutic Intervention in ANCA Associated Vasculitis, Mol. Cell. Proteomics 8, 2296-2307 (2009) for ANCA-associated vasculitius, or Andersen et al., Urinary proteome analysis, assessing renoprotective treatment in type 2 diabetic patients with microalbuminuria, BMC Nephro, 11, 29 (2010) for diabetic nephropathy ).

The biomarkers identified according to the invention can also be used to determine the severity of the stroke. The biomarkers show correlation with the National Institute of Health Stroke Scale (NIHSS). The combination of the normalized amplitudes of the individual biomarkers can thus be used to determine the severity of the stroke, thus giving valuable information on the therapy.

Example 1:

1. Sample preparation:

Urine was used to detect polypeptide markers for diagnosis. Urine was collected from healthy donors (peer group) and from patients who had a stroke. For the subsequent CE-MS measurement, those also had to be in urine of patients in higher concentration occurring proteins such as albumin and immunoglobulins are separated by ultrafiltration. For this purpose, 700 .mu.l of urine were removed and treated with 700 .mu.l Filtrationspuffer (2M urea, lOmM ammonia, 0.02% SDS). These 1.4 ml sample volumes were ultrafiltered (20 kDa, Sartorius, Göttingen, DE). The UF was carried out at 3000 rpm in a centrifuge until 1.1 ml ultrafiltrate was obtained.

The resulting 1.1 ml filtrate was then applied to a PD 10 column (Amersham Bioscience, Uppsala, Sweden) and eluted with 2.5 ml of 0.01% NH ₄ OH and lyophillized. For CE-MS measurement, the polypeptides were then resuspended with 20 μΙ water (HPLC grade, Merck).

2. CE-MS measurement:

The CE-MS measurements were performed with a capillary electrophoresis system from Beckman Coulter (P / ACE MDQ System, Beckman Coulter Inc, Fullerton, USA) and a Bruker ESI-TOF mass spectrometer (micro-TOF MS, Bruker Daltonik, Bremen, D). The CE capillaries were purchased from New Objective, they had an ID / OD of 50/360 μιτι and a length of 90 cm. The mobile phase for the CE separation consisted of 20% acetonitrile and 0.25% formic acid in water. 30% isopropanol with 0.5% formic acid was used for the "sheath flow" at the MS, here with a flow rate of 20 μΙ / h. The coupling of CE and MS was realized by a CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, DE). To inject the sample, 1 to max. 6 psi pressure applied, the duration of the injection was 99 seconds. With these parameters about 300 nl of the sample were injected into the capillary, this corresponds to about 10% of the capillary volume. To concentrate the sample in the capillary, a "stacking" technique was used. An IM NH ₃ solution is injected for 7 seconds (at 1 psi) prior to sample injection. After applying the separation voltage (25 kV), the analytes are automatically concentrated between these solutions. The following CE separation was performed with a pressure method: 0 psi for 30 minutes, 0.1 psi for 1 min, 0.2 psi for 1 min, 0.3 psi for 1 min, 0.4 psi for 1 min, and 0.5 psi for 35 min. The total duration of a separation run was thus 70 minutes. In order to get the best possible signal intensity on the side of the MS, no "Nebulizer gas" was used. The voltage applied to the spray needle to generate the electrospray was 4000-4800 V. The remaining settings on the mass spectrometer were optimized as instructed by the manufacturer of peptide detection. The spectra were recorded over a mass range of m / z 400 to m / z 3000 and accumulated every 3 seconds.

3. Standards for CE measurement

To control and calibrate the CE measurement, the following proteins or polypeptides were used, which are characterized under the selected conditions by the CE migration times listed below:

The proteins / polypeptides are each used in a concentration of 10 pmol / μΙ in water. "REV", "ELM", "KINCON" and "GIVLY" represent synthetic peptides.

It is known in principle to those skilled in the art that small variations in the migration times can occur in the case of capillary electrophoretic separations. Under the conditions described, however, the migration order does not change. It is readily possible for a person skilled in the art with knowledge of the indicated masses and CE times to assign their own measurements to the polypeptide markers according to the invention. For this he can proceed as follows, for example: first, he selects one of the polypeptides found in his measurement (peptide 1) and attempts to find one or more matching masses within a time window of the indicated CE time (for example + 5 min). If he only finds a matching mass within this interval, the assignment is completed. Does he find several suitable masses, must still be made a decision on the assignment. For this purpose, a further peptide (peptide 2) is selected from the measurement and attempts to identify a suitable polypeptide marker, again taking into account a corresponding time window. If, in turn, several markers can be found with a corresponding mass, the most probable assignment is that in which there is a substantially linear relationship between the shift for the peptide 1 and for the peptide 2. Depending on the complexity of the assignment problem, it is obvious to a person skilled in the art to optionally use further proteins from his sample for the assignment, for example ten proteins. Typically, migration times are either lengthened or shortened by certain absolute values, or upsets or knocks occur throughout the course. Co-migrating peptides also co-migrate under such conditions.

In addition, the person skilled in the art can use the methods described by Zuerbig et al. in Electrophoresis 27 (2006), pages 2111-2125. If he uses a simple diagram to plot his measurements in terms of m / z versus migration time, the line patterns described will also be visible. By counting the lines, a simple assignment of the individual polypeptides is now possible. Other procedures for assignment are possible. In principle, one skilled in the art could also use the above peptides as an internal standard to assign its CE measurements

4. Sequencing of polypeptides

The sequencing of the polypeptides determined by this method is known in principle to the person skilled in the art. The following method can be used.

The urine samples are analyzed according to Carty DM et al. (Urinary proteomics for predication of preeclampsia, Hypertension 2011; 57: 561-9) with a Dionex Ultimate 3000 RSLS nanoflous system (Dionex, Camberley, UK) (LC / MS). Samples (5 μΙ) were spun at a flow rate of 5 μΙ / min in 0.1% formic acid and 2% acetonitrile onto a Dionex C18 nano trap. Column (0.1 x 20 mm, 5 μm) abandoned. The sample was eluted at a flow rate of 0.3 μΙ / min onto an Acclaim-PepMap C18 nano-column (75μm x 15cm, 2μm, 100%), the trap column and the nanofluid column were opened

Kept at 35 ° C. The samples were eluted for 100 minutes with a gradient of solvent A, 0.1% formic acid, solvent B, acetonitrile starting with 5% B and increasing to 50% B. The column was washed with 90% B before being equilibrated before the next sample. The eluent from the column was ionized into an Orbitrap Velos FTMS using a Proxeon nanospray ESI source (Thermo Fisher, Hemel Hempstead, UK) operating in the positive ion mode. The ionization voltage was 2.5 kV and the capillary temperature was 200 ° C. The mass spectrometer was operated in the MS / MS mode with a scan range from m / z 380 to 2000 amu. The 10 largest multiply charged ions were selected from each scan for MS / MS analysis; the fragmentation method was HCD with 35% collision energy. The ions were selected by a data-dependent method with a repetition number of 1 and an exclusion time of 15 s for MS2. The ion dissolution was 60000 in MSI and 7500 for HCD-MS2. The files were used for a search against the human non-redundant IPI database using the Open Mass Spectrometry Search Algorithm (OMSSA, see pubchem.ncbi.nlm.nih.gov/omssa) and SEQUEST (using the Thermo Proteome Discoverer), without any enzyme specificity. No fixed modification and oxidation of methionine and proline were chosen as variable modifications. The permissible mass error window for MS or MS / MS was 10 ppm or 0.05 Da. In the case of SEQUEST, the peptide data were extracted using high peptide-confidence and top-one peptide rank filters. 1% FDR was used as the threshold for identifying identified peptides. The correlation between peptide loading at the working pH of 2 and CE migration time was exploited to minimize the occurrence of false positives (Zürbig, P. et al., Bio- marker discovery by CE-MS enables sequence analysis via MS / MS with platform-independent separation Electrophoresis 27, 2111-2125 (2006)). The calculated on the basis of the number of basic amino acids CE migration time was compared to the experimental migration time. Only those peptides that were found with both search algorithms (OMSSA and SEQUEST) and that had a mass deviation of less than ± 10 ppm, fragment ions with a mass deviation of less than ± 0.05 Da and a deviation of the CE migration time of less than ± 2 min were accepted.

Example 2

Sample 14289 and Sample 14238 were classified with a classifier based on the biomarkers of the invention in a blinded assay. When all 229 biomarkers were used, the scoring for subject was 14289-0788, so no stroke, the scoring for subject 14238 was 1,058, so positive for stroke.

Example 3

Using only 3 biomarkers, the following results were obtained for 2 subjects:

The evaluation is as follows:

Subject 14238:

Marker 123902 has an amplitude of 2.35 in the control group and an amplitude of 1.86 in the stroke group. The subject found an amplitude of 2.62, i. still above the amplitude for the control group. This results in a negative score for the patient.

Marker 70413 has an amplitude of 2.16 in the control group and a score of 3.02 in the stroke group. The found amplitude is 2.85; this is closer to the group for stroke. He gets a positive score.

Marker 4609 has a score of 0.06 in the healthy group and a score of 1.27 in the stroke group. The amplitude of 2.31 is still above the amplitude of the sick group. This gives a strong positive score. This results in a total score for subject 14238 of +0.910.

Test person 14289:

For subject 14289, there is an amplitude for marker 123902 of 2.57. This is thus above the control group "healthy" and thus receives a negative score.

Marker 70413 has an amplitude of 2.48. The score is slightly closer to the healthy than to the sick group, so there is a small negative score.

Marker 4906 was not found in the sample. The marker is rare in the control (3%) and also with small amplitudes. His absence is a strong negative score.

Overall, this resulted in a score of -1.020.

Example 4

After unblinding, subject 14289 was found to be the urine sample of a patient who did not have a stroke, while subject 14238 was from a stroke patient, so in both cases the diagnosis was correct.

Claims

claims

A method for diagnosing and assessing ischemic stroke and transient ischemic attacks comprising the step of determining the presence or absence or amplitude of at least three polypeptide markers in a urine sample, wherein the polypeptide markers are selected from the markers given in Table 1 by values for the molecular masses and the migration time are characterized.

2. Method according to claim 1, characterized in that an evaluation of the determined presence or absence or amplitude of the markers is carried out on the basis of the regulation listed in Table 2.

3. The method according to at least one of claims 1 to 2, wherein at least five, at least six, at least eight, at least ten, at least 20 or at least 50 polypeptide markers are used, as defined in claim 1.

The method of any one of claims 1 to 3, wherein the sample of an individual is a midbeam urine sample.

5. The method according to any one of claims 1 to 4, wherein capillary electrophoresis, HPLC, gas phase ion spectrometry and / or mass spectrometry is used to determine the presence or absence or amplitude of the polypeptide marker.

6. The method according to any one of claims 1 to 5, wherein before a measurement of the molecular mass of the polypeptide marker, a separation, preferably carried out by capillary electrophoresis.

7. A method according to any one of claims 1 to 6, wherein mass spectrometry is used to detect the presence or absence of the polypeptide marker (s).

8. Use of at least three polypeptide markers selected from the markers according to Table 1, which is characterized by the values for the molecular masses and the migration time, for the diagnosis of ischemic stroke and transistoric ischemic attacks.

9. A method for diagnosing ischemic stroke and transistoric ischemic attacks comprising the steps a) the separation of a sample into at least five, preferably 10 subsamples,

b) analysis of at least five subsamples to determine the presence or absence or amplitude of at least one polypeptide marker in the subsample, the polypeptide marker being selected from the markers of Table 1 characterized by molecular masses and migration time (CE time) ,

10. The method of claim 9, wherein at least 10 subsamples are measured.

11. The method according to at least one of claims 1 to 10, characterized in that the CE time is based on a 90 cm long glass capillary having an inner diameter (ID) of 50 microns at an applied voltage of 25 kV, being used as eluent 20th % Acetonitrile, 0.25% formic acid in water is used.

12. The method according to at least one of claims 1 to 7 or 9 to 11, wherein the sensitivity is at least 60% and the specificity at least 40%.

13. The method according to at least one of claims 1 to 12, wherein the assessment of a transistoric ischemic attack takes place.

14. The method according to at least one of claims 1 to 13, wherein the assessment of an ischemic stroke occurs.

15. A method for evaluating the development of a transistoric ischemic attack comprising performing the method according to one of claims 1 to 13 at two different times.

16. The method of claim 15 for evaluating a therapeutic intervention.