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
  • G01N33/6893—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
  • 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
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/32—Cardiovascular disorders

Definitions

• 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 disease (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 disease
• Vascular disorders are diseases that affect the vessels of an organism and, subsequently, organs such as the heart, brain, kidney, etc. These are z. As arteriosclerosis, circulatory disorders, hypertension, and cardiac arrhythmias.
• 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 components, fatty substances, metabolic waste products and calcium salts accumulate. It forms so-called plaques, area calcifications; which makes the vessel wall harder and tighter.
• the artery loses its elasticity and blood transport from the heart to the individual parts of the body is made more difficult.
• Secondary diseases include, for example, angina pectoris, myocardial infarction, circulatory collapse, stroke, deep-seated leg vein thrombosis, embolisms.
• Circulatory disorders usually affect the lower body, 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 the vessels close in such a way 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. High blood pressure damages the vessel walls, so there is a risk of tearing or occlusion. Vascular disease can be prevented by prevention because it is associated with an unhealthy lifestyle. A radical reversal in lifestyle may arrest arteriosclerosis in the early stages, e.g.
• vascular diseases By lowering blood pressure and blood lipid levels.
• drug therapies eg, acetylsalicylic acid, beta-receptor blockers, ACE inhibitors, etc.
• damaged vessels are irreparable, the process at an advanced stage is irreversible. This makes early detection of vascular diseases particularly important.
• 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 HbAlc.
• LDL cholesterol, HDL cholesterol, triglycerides lipid status
• HbAlc lipid status
• X-ray contrast agents are used.
• the indication for coronary angiography is a low or medium pretest probability, whereas non-invasive diagnostics have not yielded reliable results.
• the coronary angiography can only be performed if, in addition to the previously mentioned preliminary investigations, various complications such. As a thyroid hyperfunction or a contrast agent allergy are excluded. Because the contrast agent is excreted via the kidney, it must also have sufficient renal function. This shows that there is a need for a non-invasive option for the early and reliable diagnosis of vascular disease. It is therefore the object of the present invention to provide methods and means for the diagnosis of vascular diseases.
• the object is achieved by a method for the diagnosis of vascular diseases, comprising the step of determining a presence or absence or amplitude of at least three polypeptide markers in a urine sample, wherein the polypeptide markers are selected from the markers shown in Table 1 by values for the molecular masses, the migration time characterizes and possibly their peptide sequence are characterized.
• the evaluation of the measured polypeptides can be based on the presence or absence or amplitude of the markers taking into account the following limits:
• 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 will recognizes persons as healthy, ie. 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.
• the markers according to the invention make it possible to achieve 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% for vascular diseases.
• the markers according to the invention make it possible to achieve 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% for vascular diseases.
• the migration time is determined by capillary electrophoresis (CE) - such.
• 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, for. Example, the polypeptides given in the examples be (see example point 3).
• 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 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% or 0.005%.
• the polypeptide markers according to the invention are proteins or peptides or degradation products of proteins or peptides. They can be chemically modified, for. By post-translational modifications such as glycolization, phosphorylation, alkylation or disulfide bridging, or by other reactions, e.g. B. in the context of degradation, be changed. In addition, the polypeptide markers can also be chemically altered during the purification of the samples, for. B. oxidized, be. Based on the parameters that the poly- peptide 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 vascular diseases. Diagnosis is the process of gaining knowledge 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.
• 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 vascular disease.
• polypeptide markers that are typically present in individuals with vascular disease, but are less common or non-existent in individuals without vascular disease.
• polypeptide markers which are present in patients with vascular diseases, but are not or only rarely present in patients without vascular diseases.
• amplitude markers can also be used for diagnosis.
• 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.
• There are Two nomination procedures are possible to achieve comparability between differently concentrated samples or different measurement methods.
• all peptide signals of a sample are normalized to a total amplitude of 1 million counts.
• the respective average amplitudes of the individual markers are therefore given as parts per million (ppm).
• 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 presence of a vascular disease, it corresponds more to the mean amplitudes of the control group, is not to be assumed by a vascular disease.
• 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.
• a frequency marker is a variant of the amplitude marker, where in some samples the amplitude is so low that it is below the detection limit. 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 - is 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 vascular disease. Preferably it is the individual is a mammal, most preferably a human.
• 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.
• 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 a stool sample ,
• it is a liquid sample.
• the sample is a urine sample.
• Urine samples may be known as known in the art.
• 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 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. Basically, the presence or absence of a polypeptide marker by direct methods, such as. Mass spectrometry, or indirect methods, e.g. by ligands.
• the sample of the individual may be analyzed prior to measuring the presence or absence of the or peptide marker pretreated by any suitable means and z.
• 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 a single analysis. Any type of mass spectrometer can be used. With mass pectrometry it is possible to routinely measure 10 fmoles of a polypeptide marker, ie 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
• quadrupoles, ion traps or time-of-flight (TOF) analyzers can be used to analyze the resulting ions.
• electrospray ionization the molecules present in solution are inter alia. sprayed under the influence of high voltage (eg 1-8 kV) to form charged droplets, which become smaller due to evaporation of the solvent. Finally, so-called Coulomb explosions lead to the formation of free ions, which can then be analyzed and detected.
• high voltage eg 1-8 kV
• 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, 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 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 measuring a sample 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 vascular diseases. More preferred is a CE-MS method which includes CE coupled online to an ESI-TOF-MS.
• solvents include acetonitrile, methanol and the like.
• the solvents may be diluted with water and an acid (e.g., 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.
• fused silica 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, ie show their hydrophilic groups on the inside, 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.
• 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.
• Urine was used to detect polypeptide markers for diagnosis. Urine was withdrawn from healthy donors (peer group) as well as patients suffering from vascular disease. For the subsequent CE-MS measurement, proteins such as albumin and immunoglobulins, which are also present in urine in higher concentrations, had to be separated by ultrafiltration. For this purpose, 700 .mu.l of urine were removed and treated with 700 .mu.m filtration buffer (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.
• 700 .mu.l of urine were removed and treated with 700 .mu.m filtration buffer (2M urea, lOmM ammonia, 0.02% SDS). These 1.4 ml sample volumes were ultrafiltered (20 kDa, Sartorius, Göttingen,
• 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 Beckman Coulter, having an ID / OD of 50/360 pm and a length of 90 cm.
• the mobile phase for the CE separation consisted of 20% acetonitrile and 0.25% formic acid in water.
• An IM NH 3 solution is injected for 7 seconds (at 1 psi) prior to sample injection, and after injection of the sample for 5 seconds, a 2M formic acid solution is injected. After applying the separation voltage (30 kV), the analytes are automatically concentrated between these solutions.
• the following CE separation was performed with a pressure method: 0 psi for 40 minutes, 0.1 psi for 2 min, 0.2 psi for 2 min, 0.3 psi for 2 min, 0.4 psi for 2 min, and 0.5 psi for 32 min. 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.
• the proteins / polypeptides are each used in a concentration of 10 pmol / ⁇ in water.
• REV amino acid sequence
• ELM amino acid sequence polypeptide
• KINCON amino acid sequence polypeptides
• 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, you can find several markers with a corresponding mass, the most likely one is Assignment of those in which there is a substantially linear relationship between the shift for the peptide 1 and for the peptide 2.
• further proteins from his sample for the assignment, for example ten proteins.
• 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.

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• 2010
  • 2010-09-14 EP EP10775725A patent/EP2478377A2/de not_active Withdrawn
  • 2010-09-14 WO PCT/EP2010/063475 patent/WO2011029954A2/de active Application Filing
  • 2010-09-14 JP JP2012528391A patent/JP2013504751A/ja active Pending
  • 2010-09-14 US US13/395,757 patent/US20120241321A1/en not_active Abandoned
• 2014
  • 2014-09-26 US US14/497,613 patent/US20150018246A1/en not_active Abandoned

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