EP1955075A2 - Marqueur polypeptidique pour diagnostiquer et evaluer des maladies vasculaires - Google Patents

Marqueur polypeptidique pour diagnostiquer et evaluer des maladies vasculaires

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Publication number
EP1955075A2
EP1955075A2 EP06819861A EP06819861A EP1955075A2 EP 1955075 A2 EP1955075 A2 EP 1955075A2 EP 06819861 A EP06819861 A EP 06819861A EP 06819861 A EP06819861 A EP 06819861A EP 1955075 A2 EP1955075 A2 EP 1955075A2
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EP
European Patent Office
Prior art keywords
markers
sample
polypeptide
marker
absence
Prior art date
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Ceased
Application number
EP06819861A
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German (de)
English (en)
Inventor
Harald Mischak
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mosaiques Diagnostics and Therapeutics AG
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Mosaiques Diagnostics and Therapeutics AG
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Publication date
Application filed by Mosaiques Diagnostics and Therapeutics AG filed Critical Mosaiques Diagnostics and Therapeutics AG
Priority to EP06819861A priority Critical patent/EP1955075A2/fr
Publication of EP1955075A2 publication Critical patent/EP1955075A2/fr
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/493—Physical analysis of biological material of liquid biological material urine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • Polvpeptide marker for diagnosis and evaluation of vascular diseases
  • 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 evaluation of the severity of vascular diseases (VE) and a method for the diagnosis and evaluation of the vascular disease, wherein the presence or absence of the or the peptide marker (s) is indicative of the severity of a VE.
  • VE vascular diseases
  • Vascular disorders are diseases that affect the vessels of an organism and, subsequently, organs such as the heart, brain, kidney, etc. These are e.g. Atherosclerosis, circulatory disorders, hypertension, and arrhythmia.
  • Atherosclerosis refers to the hardening of arteries due to vascular deposits. Cholesterol crystal deposits lead to the formation of inflammatory foci (atheromas), in which blood constituents, fatty substances, metabolic waste products and calcium salts readily accumulate. It forms so-called plaques, area calcifications; which makes the vessel wall harder and tighter.
  • the artery loses its elasticity and is difficult to fulfill its task of transporting blood from the heart to the individual parts of the body. Consequences are, for example, angina pectoris, myocardial infarction, circulatory collapse, stroke.
  • Circulatory disorders usually affect the lower body area, from the abdominal aorta to the foot arteries, and lead to a reduction in blood flow and oxygenation in the muscle tissue, which gradually dies. In the last stage, ulcers form and close the vessels so that an amputation becomes inevitable. High blood pressure has no clear cause, so taking medication or excessive secretion of kidney hormones can make the blood pressure rocketing. High blood pressure levels are also associated with long-term stress, which leads to vascular spasms. Hypertension damages the vessel walls, causing there is a risk of tearing or locking. If the regularity of the heartbeat is disturbed, it is called cardiac arrhythmia. The heartbeat may be either too fast (tachycardia), too slow (bradycardia) or irregular (arrhythmia).
  • Vascular diseases can be prevented by prevention because they are also due to an unhealthy and unnatural lifestyle. By radically reversing the lifestyle, arteriosclerosis should be halted in the early stages, for example by lowering blood pressure and blood lipid levels. The progression of vascular diseases can also be slowed down by drug therapies (eg, acetylsalicylic acid, beta-adrenergic blockers, ACE inhibitors, etc.). However, it should be noted that damaged vessels are irreparable, the process at an advanced stage is irreversible. This makes early detection of vascular diseases particularly important.
  • drug therapies eg, acetylsalicylic acid, beta-adrenergic blockers, ACE inhibitors, etc.
  • VE in coronary heart disease is initially carried out indirectly via evaluation of risk factors and non-invasive examinations such as blood pressure, resting and exercise ECG, as well as blood tests to determine the lipid status (LDL cholesterol, HDL cholesterol, triglycerides), fasting blood sugar and if necessary HbAIc. If these examinations indicate the presence of high-risk features, ie serious vascular events (death, myocardial infarction) are expected within the next period, a more detailed diagnosis will be made using invasive diagnostics, eg catheterization or coronary angiography. For this, the heart and coronary vessels as well as other vessels are examined with the help of a catheter or X-ray procedure.
  • invasive diagnostics eg catheterization or coronary angiography
  • X-ray contrast agents are used. Indications for coronary angiography include a low or moderate pretest probability, with non-invasive diagnostics not providing reliable results, and patients undergoing noninvasive testing for disability or illness it is not possible and patients for whom a professional exclusion of coronary heart disease is indispensable if suspected (eg pilots, fire brigade).
  • coronary angiography can only be performed if, in addition to the previously mentioned preliminary examinations, various complications such as hyperthyroidism or a contrast agent allergy are excluded. Because the contrast agent is excreted via the kidney, there must also be adequate kidney function, or dialysis-dependent people must always undergo dialysis after the examination. This shows that there is a need for a non-invasive option for the early and reliable diagnosis of vascular disease.
  • Vascular diseases of the kidney are:
  • Renal artery stenosis is a one- or double-sided constriction of the renal artery or its major branches. It may be the cause of arterial hypertension, which is called renovascular hypertension.
  • the narrowing of the renal artery leads to a reduced perfusion of the affected kidney.
  • Blood pressure reduction increases the kidney produces renin, which via the angiotensin-aldosterone mechanism leads to an increase in blood volume and a blood pressure increase of the entire organism and thus to arterial hypertension.
  • the renal artery stenosis is therefore usually discovered in the diagnosis of hypertension, but it is only in about 1-2% of all hypertension the cause.
  • PTA percutaneous transluminal catheter angioplasty
  • Stent Insertion of a wire mesh (stent), which should hold the vessel open.
  • renal arterial thrombosis Common causes of renal arterial thrombosis are heart embolisms, e.g. in atrial fibrillation associated with symptoms such as flank pain, proteinuria, very high LDH. In renal vein thrombosis flank pain is also observed, but in addition a proteinuria and possibly a hematuria or a nephrotic syndrome.
  • Narrowed vessels in the brain area lead to a reduced oxygen supply, in occlusion of an artery (eg by an acute clot as a result of changes due to arteriosclerosis) it comes to stroke with loss of sensibility, paralysis, speech disorders, etc.
  • cerebral arteries as in the large arteries arise in the course arterial calcification in rare cases draining the vessel walls, in conjunction with risk factors such as high blood pressure threatens a rupture of the vessel wall and a life-threatening internal bleeding.
  • an object of the present invention is the use of the presence or absence of at least one, ideally more polypeptide marker (s) in a urine sample of an individual for the diagnosis of vascular diseases, said polypeptide marker (s) being selected from polypeptide labels Nos. 1 to No. 526, which are characterized by the molecular masses given in Table 1 and their migration times.
  • the markers 1-104 and / or 107-413 are preferably used.
  • the migration time is determined by means of capillary electrophoresis (CE), as described in Example 2, for example.
  • CE capillary electrophoresis
  • a 90 cm long glass capillary with an inner diameter (ID) of 50 ⁇ m and an outer diameter (OD) of 360 ⁇ m is operated at an applied voltage of 30 kV.
  • the eluent used is, for example, 30% methanol, 0.5% formic acid in water.
  • CE migration time can vary. Nevertheless, the order in which the polypeptide labels elute is typically the same for each CE system used under the conditions indicated. To even out any differences in migration time, the system can be normalized using standards for which migration times are known. These standards may e.g. be the polypeptides given in the examples (see example point 3).
  • the characterization of the polypeptides shown in Tables 1 to 4 was determined by capillary electrophoresis mass spectrometry (CE-MS), a method which is described e.g. in detail by Neuhoff et al. (Rapid Communications in mass spectrometry, 2004, Vol. 20, pages 149-156).
  • CE-MS capillary electrophoresis mass spectrometry
  • the variation of molecular masses between individual measurements or between different mass spectrometers is relatively small with exact calibration, typically in the range of ⁇ 0.1%, preferably in the range of ⁇ 0.05%, more preferably ⁇ 0.03%, even more preferably ⁇ 0.01%.
  • polypeptide markers according to the invention are proteins or peptides or degradation products of proteins or peptides. They may be chemically modified, for example by post-translational modifications such as glycolization, phosphorylation, alkylation or disulfide bridging, or altered by other reactions, eg in the context of degradation. In addition, the polypeptide markers can also be chemically modified, eg oxidized, during the purification of the samples. 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.
  • polypeptides of the invention are used to diagnose the severity of VE. Diagnosis is the process of gaining knowledge by assigning symptoms or phenomena to a disease or injury. In the present case, the presence or absence of certain polypeptide markers is indicative of the severity of the VE. For this purpose, the polypeptide markers of the invention are determined in a sample of an individual, their presence or absence suggesting the degree of VE. 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.
  • 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 the degree of VE (frequency marker).
  • VE degree of VE
  • polypeptide markers which are typically present in individuals with VE, but are more rare or non-existent in individuals without VE, eg, 1-24 (Table 2).
  • polypeptide markers present in patients with VE such as polypeptide markers Nos. 25 to 106, but are not or only rarely present in patients without VE.
  • the amplitude markers indicated in Table 3 can also be used for the diagnosis of CE (number 107-526). Amplitude markers are used in a manner that does not determine the presence or absence, but decides the magnitude of the signal (amplitude) in the presence of the signal in both groups.
  • Tables 3 and 4 the mean amplitudes of the respective signals (characterized by mass and migration time) over all measured samples are given. Two nomination procedures are possible to achieve comparability between differently concentrated samples or different measurement methods. In the first approach, all peptide signals of a sample are normalized to a total amplitude of 1 million counts. The respective mean amplitudes of the single markers are therefore given as parts per million (ppm). The amplitude markers resulting from this procedure are shown in Table 3 (number 107-413).
  • All groups used consist of at least 20 individual patient or control samples to obtain a reliable mean amplitude.
  • 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 VE group. If the value is close to the mean amplitude of the VE group, it can be assumed that the presence of a vascular disease is more likely to correspond to the mean amplitudes of the VE group Control group, does not assume a VE.
  • the distance to the mean amplitude can be interpreted as a probability of belonging to a group. An exemplary explanation shall be given by means of marker No. 137 (Table 3).
  • the mean amplitude of the marker is markedly increased at one VE (12044 ppm vs 5726 ppm in the control group). If, in a patient sample, the value for this marker is 0 to 5726 ppm, or a maximum of 20% above this, ie 0 to 6871 ppm, this sample belongs to the control group. If the value is 12044 ppm, or 20% lower, or higher, ie between 9635 ppm and very high values, this is to be regarded as an indication of a vascular disease.
  • the distance between the measured value and the mean amplitude may be considered as a probability of belonging to a group.
  • a frequency marker is a variant of the amplitude marker, in which the amplitude is low in some samples. It is possible to convert such frequency markers into amplitude markers in which, in the calculation of the amplitude, the corresponding samples in which the marker is not found, with a very small amplitude - in the range of the detection limit - are included in the calculation.
  • the individual from whom the sample is derived, in which the presence or absence of one or more polypeptide markers is determined can be any individual who may be suffering from PE.
  • the subject is a mammal, most preferably a human.
  • a polypeptide marker not only a polypeptide marker, but a combination of markers is used to determine the degree of VE. It is concluded by their presence or absence on the degree of VE. By comparing a plurality of polypeptide markers, the falsification 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 (VE or not).
  • it may be blood, urine, synovial fluid, tissue fluid, body secretions, sweat, cerebrospinal fluid, lymph, intestinal, gastric, pancreatic, bile, tears, tissue, sperm, vaginal fluid, or a stool sample.
  • it is a liquid sample.
  • the sample is a urine sample or a blood sample, where a blood sample may be a serum (blood) or (blood) plasma sample.
  • Urine samples may be known as known in the art.
  • a mid-jet urine sample is used.
  • the urine sample can be removed, for example, by means of a catheter or else by means of a urination apparatus as described in WO 01/74275.
  • Blood samples may be taken by methods known in the art, for example from a vein, artery or capillary.
  • a blood sample is obtained by removing venous blood from an individual, for example, from the arm by means of a syringe.
  • the term blood sample also includes samples obtained from blood by further purification and separation techniques known in the art, such as blood plasma or blood serum.
  • the presence or absence 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, e.g. by ligands.
  • the sample of the subject eg, the urine or blood sample
  • the treatment may include, for example, purification, separation, dilution or concentration.
  • the methods may be, for example, centrifugation, filtration, ultrafiltration, dialysis, precipitation or chromatographic methods such as affinity separation or separation by ion exchange chromatography, or electrophoretic separation.
  • the sample is separated by electrophoresis prior to its measurement, purified by ultracentrifugation and / or separated by ultrafiltration into fractions containing polypeptide labels of a specific molecular size.
  • a mass spectrometric method is used to determine the presence or absence 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 means of 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.
  • electrospray ionization (ESI) interfaces are mostly 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.
  • MALDI matrix assisted laser desorption / ionization
  • For analysis of the resulting ions e.g. Quadrupoles, ion traps or time-of-flight (TOF) analyzers are used.
  • electrospray ionization the molecules present in solution i.a. under the influence of high voltage (e.g., 1-8 kV) to form charged droplets which become smaller by evaporation of the solvent.
  • high voltage e.g. 1-8 kV
  • Coulomb explosions lead to the formation of free ions, which can then be analyzed and detected.
  • 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.
  • 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.
  • CE-MS in which capillary electrophoresis is coupled with mass spectrometry. This process is described in detail, for example, in German patent application DE 10021737, in Kaiser et al. (J. Chromatogr. A 1 2003, Vol. 1013: 157-171, and Electrophoresis, 2004, 25: 2044-2055) and in Wittke et al. (J. Chromatogr. A 1 2003, 1013: 173-181).
  • 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. 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 UAS. More preferred is a CE-MS method which includes CE coupled online to an ESI-TOF-MS.
  • the use of volatile solvents is preferred, and it is best to work under essentially salt-free conditions.
  • suitable solvents include acetonitrile, methanol and the like.
  • the solvents may be diluted with water and treated with a weak acid (eg 0.1% to 1% formic acid) 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.
  • 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.
  • quartz glass capillaries with internal diameters of typically 50 to 75 ⁇ m are normally used. The used lengths are 30-100 cm.
  • 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.
  • 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.
  • the markers of the sample are separated by capillary electrophoresis, then directly ionized and transferred online to a mass spectrometer coupled thereto for detection.
  • polypeptide markers can advantageously be used for the diagnosis of VE.
  • at least three polypeptide markers may be used, for example, markers 1, 2 and 3; 1, 2 and 4; etc.
  • markers 1 to 11 are preferred. Most preferred is the use of all 526 markers listed in Tables 1-4.
  • Urine was used to detect the polypeptide markers for the diagnosis of VE. Urine was withdrawn from healthy donors (peer group) and from patients with severe PU.
  • proteins such as albumin and immunoglobulins, which are also present in urine of patients in higher concentrations, had to be separated by ultrafiltration.
  • 700 .mu.l of urine were removed and treated with 700 .mu.m filtration buffer (2M urea, 1OmM ammonia, 0.02% SDS).
  • 700 .mu.m filtration buffer (2M urea, 1OmM ammonia, 0.02% SDS).
  • sample volumes were ultrafiltered (20 kDa, Sartorius, Gottingen, DE).
  • the UF was carried out at 3000 rpm in a centrifuge until 1.1 ml of ultrafiltrate were obtained.
  • CE-MS measurements were performed with a capillary electrophoresis system from Beckman Coulter (P / ACE MDQ System, Beckman Coulter Ine, Fullerton, USA) and a Bruker's ESI-TOF mass spectrometer (micro-TOF MS, Bruker Daltonik, Bremen, Germany).
  • the CE capillaries were purchased from Beckman Coulter, having an ID / OD of 50/360 ⁇ m 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 2 ⁇ l / min.
  • the coupling of CE and MS was performed by a CE-ESI-MS Sprayer Kit (Agilent Technologies , Waldbronn, DE).
  • the duration of the injection was 99 seconds. With these parameters about 150 nl of the sample were injected into the capillary, this corresponds to about 10% of the capillary volume.
  • a "stacking" technique was used, injecting an IM NH 3 solution for 7 sec (at 1 psi) before injecting the sample, and then injecting a 2M formic acid solution for 5 sec after sample injection the separation voltage (30 kV), the analytes are automatically concentrated between these solutions.
  • CE separation was performed with a pressure method: 0 psi for 40 minutes, 0.1 psi for 2 minutes, 0.2 psi for 2 minutes, 0.3 psi for 2 minutes, 0.4 psi for 2 minutes, finally 32 min at 0.5 psi.
  • the total duration of a separation run was thus 80 minutes.
  • the "Nebulizer gas" was set to the lowest possible value.
  • the voltage applied to the spray needle to generate the electrospray was 3700 - 4100 V.
  • the remaining settings on the mass spectrometer were optimized according to the manufacturer's instructions for peptide detection.
  • the spectra were recorded over a mass range of m / z 400 to m / z 3000 and accumulated every 3 seconds. 3.
  • ELM sequence: ELMTGELPYSHINNRDQIIFMVGR 23.4 min
  • the proteins / polypeptides are each used in a concentration of 10 pmol / ⁇ l in water.
  • REV "REV”, "ELM”, “KINCON” and “GIVLY” represent synthetic peptides.
  • 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.

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Abstract

L'invention concerne un procédé permettant de diagnostiquer des maladies vasculaires (VE), selon lequel il est prévu de déterminer la présence ou l'absence d'au moins un marqueur polypeptidique dans un échantillon. Le marqueur polypeptidique est sélectionné parmi les marqueurs 1 à 526, qui sont caractérisés par les valeurs des masses moléculaires et le temps de migration (temps CE).
EP06819861A 2005-11-30 2006-11-30 Marqueur polypeptidique pour diagnostiquer et evaluer des maladies vasculaires Ceased EP1955075A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP06819861A EP1955075A2 (fr) 2005-11-30 2006-11-30 Marqueur polypeptidique pour diagnostiquer et evaluer des maladies vasculaires

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE102005057382 2005-11-30
EP06120879 2006-09-19
EP06819861A EP1955075A2 (fr) 2005-11-30 2006-11-30 Marqueur polypeptidique pour diagnostiquer et evaluer des maladies vasculaires
PCT/EP2006/069096 WO2007063089A2 (fr) 2005-11-30 2006-11-30 Marqueur polypeptidique pour diagnostiquer et evaluer des maladies vasculaires

Publications (1)

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EP1955075A2 true EP1955075A2 (fr) 2008-08-13

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EP06819861A Ceased EP1955075A2 (fr) 2005-11-30 2006-11-30 Marqueur polypeptidique pour diagnostiquer et evaluer des maladies vasculaires

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US (2) US20100126861A1 (fr)
EP (1) EP1955075A2 (fr)
JP (1) JP5165580B2 (fr)
KR (1) KR20080074190A (fr)
AU (1) AU2006319138B2 (fr)
BR (1) BRPI0619248A2 (fr)
CA (1) CA2631602A1 (fr)
EA (1) EA014529B1 (fr)
IL (1) IL191605A0 (fr)
NO (1) NO20082394L (fr)
SG (1) SG170009A1 (fr)
WO (1) WO2007063089A2 (fr)

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EP2478377A2 (fr) * 2009-09-14 2012-07-25 Mosaiques Diagnostics And Therapeutics AG Polypeptide marqueur pour le diagnostic et l'évaluation de maladies vasculaires

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WO2004089184A2 (fr) * 2003-04-01 2004-10-21 Diadexus, Inc. Nouvelles utilisations de la lp-pla2 en combinaison pour evaluer le risque coronaire
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BRPI0619248A2 (pt) 2011-09-20
KR20080074190A (ko) 2008-08-12
US20100126861A1 (en) 2010-05-27
WO2007063089A2 (fr) 2007-06-07
CA2631602A1 (fr) 2007-06-07
IL191605A0 (en) 2008-12-29
US20150247865A1 (en) 2015-09-03
AU2006319138A1 (en) 2007-06-07
EA014529B1 (ru) 2010-12-30
WO2007063089A3 (fr) 2008-01-17
JP2009521670A (ja) 2009-06-04
SG170009A1 (en) 2011-04-29
JP5165580B2 (ja) 2013-03-21
EA200801475A1 (ru) 2009-02-27
AU2006319138B2 (en) 2013-05-16
NO20082394L (no) 2008-08-26

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