EP1977250A1 - Méthode et marqueurs pour le diagnostic de maladies rénales - Google Patents

Méthode et marqueurs pour le diagnostic de maladies rénales

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Publication number
EP1977250A1
EP1977250A1 EP06703568A EP06703568A EP1977250A1 EP 1977250 A1 EP1977250 A1 EP 1977250A1 EP 06703568 A EP06703568 A EP 06703568A EP 06703568 A EP06703568 A EP 06703568A EP 1977250 A1 EP1977250 A1 EP 1977250A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
marker
disease
markers
diagnosis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP06703568A
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German (de)
English (en)
Inventor
Harald Mischak
Thorsten Kaiser
Stefan Wittke
Michael Walden
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
Original Assignee
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
Publication of EP1977250A1 publication Critical patent/EP1977250A1/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
    • G01N33/6893—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

Definitions

  • the present invention relates to the diagnosis, particularly differential diagnosis, of renal diseases.
  • renal diseases present an increasing problem to the health system.
  • Many renal diseases are irreversible, therefore an early diagnosis and/or a differential diagnosis of renal diseases is important.
  • Early diagnosis and a therapy precisely tailored to each particular disease could reduce the number of patients requiring dialysis and could also reduce the high cardiovascular risk of the patients.
  • kidney biopsies Currently, precise diagnosis and/or differential diagnosis relies mostly on kidney biopsies.
  • biopsies serve as the current "gold standard" in renal diagnostics, biopsies have the disadvantage of being invasive and therefore being conducted only on selected patients.
  • Urine analysis is a different approach to diagnose renal diseases.
  • currently only few parameters of urine are routinely measured, for example creatinin, urea, albumin, blood cells (such as leukocytes and erythrocytes), bacteria, sugar, urobilinogen, bilirubin and pH value.
  • the diagnostic value of these analyses is limited, as they lack sufficient sensitivity and/or selectivity, particularly for differential diagnosis.
  • V. Thongboonkerd et al. have used two-dimensional polyacrylamide gel electrophoresis (2D-P AGE) in combination with matrix-assisted laser desorption ionization-time-of-fiight (MALDI-TOF) mass spectrometry followed by mass fingerprinting to investigate normal human urinary proteins.
  • 2D-P AGE two-dimensional polyacrylamide gel electrophoresis
  • MALDI-TOF matrix-assisted laser desorption ionization-time-of-fiight
  • CS. Spahr et al. have digested the proteins contained in urine samples with trypsin and identified 751 peptides from 124 proteins by means of liquid chromatography-tandem mass spectrometry (CS. Spahr et al. (2001). Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest. Proteomics vol. 1, p. 93-107).
  • the object the present invention is to provide methods and means for the diagnosis of renal diseases, particularly for differential diagnosis of renal diseases. It is an particular object of the present invention to provide methods and means for the diagnosis and/or differential diagnosis of IgA-nephropathy, which is the most common glomerulopathy.
  • the problem is solved by the use of the presence of at least one polypeptide marker in a urine sample for the diagnosis, preferably the differential diagnosis, of a renal disease, wherein the polypeptide marker is selected from the group of polypeptide markers as shown in table 1 to 22.
  • the polypeptide marker is selected from the group of polypeptide markers as shown in table 1 to 22.
  • the present invention has numerous advantages compared to the state of the art.
  • the presence of the polypeptide markers according to the invention can be determined in urine samples. Therefore, there is no need to take biopsies.
  • the present invention allows a simplified and fast diagnosis of renal diseases, allowing to screen patients regularly for the presence of renal diseases and to diagnose renal diseases at early stages.
  • the polypeptide markers according to the invention can be used for differential diagnosis between different renal diseases.
  • the high number of markers identified according to the present invention allows to increase both specificity and sensitivity of diagnosis as compared to the use of only a single or a small number of markers.
  • the present invention provides methods which allow to measure said polypeptide markes without the use of specific ligands such as antibodies or aptamers.
  • the polypeptide markers as shown in the tables have been identified by a method named capillary electrophoresis-mass spectrometry (CE-MS), which will be described further below. Furthermore, the method has been described in detail in von Neuhoff et al. (2004) (Mass Spectrometry for the Detection of Differentially Expressed Proteins: A Comparison of Surface-Enhanced Laser Desorption/Ionization and Capillary Electrophoresis/Mass Spectrometry. Rapid Communications in Mass Spectrometry, vol. 18: 149-156). Starting from the parameters defining the polypeptide markers, it is possible by methods known in the art to identify the sequence of the corresponding polypeptides and then to synthesize or produce the corresponding polypeptides, e.g. with the help of protein synthesis or expression of the corresponding gene in appropriate cells.
  • CE-MS capillary electrophoresis-mass spectrometry
  • the markers are defined by there mass and their migration time in capillary electrophoresis (CE), particularly mass and their migration time obtained according to Example 1. It is known that CE migration times can vary, typically in the range of 5 min, more typically in the range of 3 minutes. However, the sequence of markers being eluted is typically the same or very similar for each CE system applied.
  • the system can be calibrated by use of polypeptides which are present in almost any urine sample, e.g. by the polypeptides given in tables 23 or 24. Furthermore, the polypeptides given in SEQ ID NO: 1 to SEQ ID NO: 5 can serve for calibration. Variation of the masses between measurements or between different mass spectrometers is relatively small, typically it is in the range of plus or minus 0.05%.
  • polypeptide markers are listed which are preferred for the discrimination between healthy individuals and individuals suffering from a renal disease, particularly from a glomerulonephritis or glomerulopathy.
  • polypeptide markers are listed, which are preferred for a discrimination between FSGS and the healthy condition.
  • polypeptide markers are listed, which can be used for differential diagnosis between FSGS and MCD.
  • polypeptide markers are listed, which are preferred for a differential diagnosis of FSGS and MGN.
  • polypeptide markers are listed, which are preferred for a differential diagnosis between FSGS on the one hand, and MCD or MGN on the other hand.
  • polypeptide markers are listed, which are preferred for diagnosis of MCD as compared to the healthy condition.
  • polypeptide markers are listed, which are preferred for differential diagnosis between MCD and MGN.
  • polypeptide markers are listed, which are preferred for differential diagnosis between MCD on the one hand, and FSGS or MGN on the other hand.
  • polypeptide markers are listed, which are preferred for diagnosis of MGN as compared to the healthy condition.
  • polypeptide markers are listed, which are preferred for differential diagnosis between MGN on the one hand, and FSGS or MCD on the other hand.
  • polypeptide markers are listed, which are preferred for diagnosis of IgA- nephropathy or MGN on the one hand as compared to the healthy condition.
  • polypeptide markers are listed, which are preferred for diagnosis of IgA- nephropathy as compared to the healthy condition.
  • polypeptide markers are listed, which are preferred for differential diagnosis between IgA-nephropathy and MGN. -
  • polypeptides are listed with their respective frequency in healthy, FSGS, MCD 5 and MGN patients.
  • polypeptides are listed which have been used for differential diagnosis between healthy individuals and renal patients using support vector machines according to Example 1.
  • polypeptides are listed which have been used for differential diagnosis between healthy, FSGS, MCD, and MGN patients using random forest analysis according to Example 1.
  • polypeptides are listed which have been used for differential diagnosis between MCD and MGN patients using using support vector machines according to Example 1.
  • polypeptides are listed which have been used for differential diagnosis between MCD and FSGS patients using using support vector machines according to Example 1.
  • polypeptides are listed which have been used for differential diagnosis between MGN and FSGS patients using using support vector machines according to Example 1.
  • polypeptides are listed which can be used for diagnosis of diabetes and/or diabetic nephropathy.
  • polypeptides are listed, which are preferred as internal standards to standardize the CE-time.
  • polypeptides are listed, which are preferred as internal standards to standardize the CE-time if the pressure method (0.3 to 1 psi) according to Example 1 is used. These standards are e.g. preferred as internal standards in diagnosis of igA-nephropathy.
  • the polypeptide markers used according to the present invention can be identified and their presence can be measured in urine samples.
  • Urine samples can be taken as known in the state of the art.
  • midstream urine is used in the context of the present invention.
  • the polypeptide markers used according to the present invention can be gene expression products such as proteins, peptides, and fragments or other degradation products of proteins or peptides. They can be modified by posttranslational modifications, e.g. by glycosylation, phoshorylation, alkylation or disulfide bond. It is known that fragments and degradation products can have a different diagnostic value and/or physiological role than the protein or peptide they have been derived from. For example, in different diseases, different proteolytic degradation products or fragments can be found. It is also considered to be within the scope of the present invention if the urine sample is pretreated to chemically modify the polypeptide markers contained in the urine and to measure these chemically modified polypeptide markers.
  • the polypeptide markers according to the present invention have a molecular mass between 400 and 20 000 Da, particularly between 700 and 14000 Da, more particularly between 800 and 11000 Da.
  • Preferred polypeptide markers according to the present invention are listed in tables 1 to 4.
  • polypeptide markers which are listed in table 1, but not in table 14 and/or 15 and/or 16 and/or 17 and/or 18 and/or 19 and/or 20 and/or 21 and/or 22.
  • polypeptide markers which are listed in table 5, but not in table 14 and/or 16 and/or 18 and/or 19. Preferred are also polypeptide markers which are listed in table 6, but not in table 14 and/or 16.
  • polypeptide markers which are listed in table 7, but not in table 14 and/or 16 and/or 17.
  • polypeptide markers which are listed in table 8, but not in table 14 and/or 16.
  • polypeptide markers which are listed in table 9, but not in table 14 and/or 16 and/or 20 and/or 21.
  • polypeptide markers which are listed in table 10, but not in table 14 and/or 16.
  • polypeptide markers which are listed in table 11, but not in table 14 and/or 16.
  • Renal disease relates to any kind of renal disease or kidney dysfunction known to the person skilled in the art, for example IgA-nephropathy, MGN (membranous glomerulonephritis), MCD (minimal-change disease), FSGS (focal- segmental glomerulosclerosis), or diabetic nephropathy.
  • renal disease relates to a glomerulopathy such as IgA-nephropathy, MGN, MCD, or FSGS.
  • a glomerulopathy such as IgA-nephropathy, MGN, MCD, or FSGS.
  • IgA-nephropathy MCD
  • FSGS renal- segmental glomerulosclerosis
  • renal disease relates to IgA-nephropathy
  • the glomerulopathies are a subgroup of renal diseases. Glomerulopathies comprise a several diseases of different etiology. Glomerulopathies are characterized by pathomorphological changes in malpighian corpuscles, glomerulus, and Bowman's capsule. As a consequence of these changes, further pathomorphological changes may appear in other parts of the nephron and interstice.
  • IgA-nephropathy is also known as Berger-Nephritis. IgA-nephropathy is the most common glomerulopathy. It may be a specific, kidney-limited, form of purpura Schoenlein-Henoch (also known as anaphylactoid purpura) with increased plasma concentration of IgA. The histopathology includes all forms of glomerular lesions and deposits of IgA in the mesangium. Clinically, IgA nephropathy presents as micro- and macro-hematouria. Therapy may be attempted with ACE inhibitors and omega-3 fatty acids. Progression of the disease occurs over the course of several years and includes transition into progressive renal insufficiency.
  • MGN is characterized by thickening of the basal membrane and granular subepithelial IgG deposits. MGN becomes frequently manifest in the between the age of 40 and 50. It is frequently caused by medicaments, e.g. gold, D-penicillamine, or ACE inhibitors. Therapy of MGN may be attempted with glucocorticoids or cyclophosphamide. MGN is a nephrotic syndrome, a transition into progressive renal insufficiency may take several years.
  • MCD is also known as lipoid nephrosis. MCD is the most common cause of a nephrotic syndrome in children. The etiology of the disease is unknown. Histologically, no or only very discrete changes can be found. Therapy of MCD may include treatment with glucocorticoids, cyclosporin A, or cyclophosphamide. In children, the disease spontaneously heals in 90% of the cases, in adults in 50% of the cases. A transition into FSGS is possible.
  • FSGS is also known as IgM-nephropathy.
  • FSGS is typically characterized by deposits of IgM and C3 in the mesangium. Clinically, it becomes manifest as a nephrotic syndrome.
  • Therapy of FSGS may include treatment with glucocorticoids, cyclosporin A, or cyclophosphamide. Prognosis is poor and includes transition into progressive renal insufficiency.
  • Diabetic nephropathy is also known as diabetic glomerulosclerosis. Diabetic nephropathy is the most common cause for requirement of dialysis treatment.
  • renal diseases include a variety of diseases which may show quite similar histology.
  • etiology, treatment, and prognosis can be quite different for each disease.
  • IgA-nephropathy requires different treatment from any other glomerulopathy described above:
  • IgA-nephropathy treatment with ACE inhibitors may be attempted, which would not be recommendable in the case of MGN. Therefore, fast and reliable diagnosis is of great importance for treatment.
  • diagnosing or diagnosis means that, for an individual patient, the probability of having the respective disease is determined.
  • Diagnosis may also include confirming a preliminary diagnosis, particularly a preliminary diagnosis established by a different method.
  • diagnosis according to the present invention particularly relates to "differential diagnosis".
  • the term "differential diagnosis” relates to distinguishing between two different diseases, i.e. to determining for an individual patient the probability of having a certain first disease as compared to having a certain second disease. More particularly, differential diagnosis according to the present invention relates to distinguishing between at least two renal diseases chosen from the group consisting of IgA -nephropathy, MGN, MCD, FSGS, and diabetic nephropathy.
  • the present invention relates to a method for the differential diagnosis of a renal disease, the method comprising:
  • the individual probabilities according to step b) are as indicated in the tables.
  • measuring relates to determining the presence of a polypeptide or other substance of interest.
  • the decision whether a polypeptide marker is present or absent may depend on definition of a suitable threshold value.
  • the threshold value can either be defined through the sensitivity of the method of measurement, or it can be defined at will.
  • the threshold in the context of the present invention is 25 fmol/ ⁇ l in a sample which has been injected into a mass spectrometer according to Example 1. However, this threshold may be the same when other methods are used. This threshold coincides with the detection threshold of a typical mass spectrometer. This threshold corresponds approximately to a concentration of the polypeptide marker in the urine sample of 50-5000 pmol/1. If different thresholds are to be used (e.g. when using another detection method), the corresponding probabilities may differ, but can easily be established by the person skilled in the art.
  • the “disease patient” according to the present invention is suffering from a renal disease.
  • the disease is at least one from the group consisting of IgA-nephropathy, MGN, MCD, FSGS, and diabetic nephropathy.
  • control patient can either be healthy or suffering from a disease different from the one the disease patient is suffering from, i.e. the control patient can either represent the healthy condition or a disease or group of diseases.
  • the represented disease is at least one from the group consisting of IgA-nephropathy, MGN, MCD, FSGS, and diabetic nephropathy.
  • Tables 1 to 14, 16, 20, 21, and 22 list the probability (also designated as "frequency") of a given polypeptide marker being present in a urine sample of a healthy control patient or a control patient suffering from a certain disease.
  • the discrimination factor indicates the difference between the probability of presence in the disease as compared to a given control condition.
  • the discrimination factor can easily be calculated from the respective probabilities. The higher the discrimination factor, the better is the potential of the given marker to distinguish between the disease and the control condition. An absolute value of the discrimination factor of 0.40 or higher is preferred.
  • the person skilled in the art is able to establish similar tables for the polypeptide markers by himself and/or to refine the data contained in the tables, e.g. based on further patient data and/or according to different thresholds for the presence of the polypeptide marker.
  • the probability of the presence of the polypeptide marker in a disease patient is compared to the probability of the presence of this marker in a control patient, wherein the individual probabilities are as indicated in the tables. If the probability of the presence of this marker in a disease patient is higher than the probability of the presence of this marker in a control patient, then the presence of this marker in the sample is indicative that the patient from whom the sample originates has a higher probability of having the disease rather than the control condition. If the probability of the presence of this marker in a disease patient is lower than the probability of the presence of this marker in a control patient, then the absence of this marker in the sample is indicative that the patient from whom the sample originates has a higher probability of having the disease rather than the control condition.
  • a given marker may have a probability of 73% of being present in a control representing IgA-nephropathy but a probability of 0% of being present in a control representing the healthy condition. If this marker is present in the sample, then the individual is diagnosed as having a 73% probability of suffering from IgA-nephropathy as compared to being healthy. If this marker is not present in the sample, then the individual is diagnosed as having a 73% probability of being healthy instead of suffering from IgA- nephropathy.
  • diagnosis can be established according to statistical methods familiar to the person skilled in the art.
  • the invention can be carried out using only one of the polypeptide markers or using a plurality of the polypeptide markers.
  • presence of a plurality of polypeptide markers is measured.
  • at least 3 of the markers, more preferably at least 10 of the markers, even more preferably at least 20, most preferred at least 50 of the markers according to the present invention are measured.
  • An advantage of the present invention is that it provides a multitude of suitable markers. Measuring a plurality of markes can increase both sensitivity and selectivity of diagnosis. Therefore, also markers which show low discrimination factors between the disease and control can be used for diagnosis if they are combined with other markers.
  • a "pattern" is be generated which contains the information about the presence for each marker measured. This pattern can then be compared to the pattern of probabilities of presence of the polypeptide markers in a disease or control patient. Each table represents a pattern of probabilities of finding given polypeptide markers in certain disease and control patients.
  • the present invention relates to a method for the differential diagnosis of a renal disease, the method comprising: a) establishing a pattern of presence or absence for a plurality of polypeptide markers in a urine sample, wherein at least one polypeptide marker is selected from the group of polypeptide markers shown in table 1 to 22, and
  • the individual probability for the at least one polypeptide marker according to step b) is as indicated in the tables.
  • Comparison of the found pattern with the probability of finding the pattern in a disease or control patient can be performed according to statistical methods known in the art.
  • automated methods are employed, e.g. CART-analysis, random forest analysis, and support vector machines (SVM, see e.g. Xiong. M., et al. (2001). Biomarker identification by feature wrappers. Genome Research vol. 11, p. 1878-1887). Comparison can also be performed simultaneously for several different patterns and the probability of finding them.
  • SVM support vector machines
  • the measured pattern is typically compared to the probability of finding the pattern in at least two different conditions.
  • An example for diagnosis and differential diagnosis of renal diseases according to this method is shown in Fig. 3.
  • the urine samples may be pre-treated before measurement of the polypeptide marker.
  • lipids, nucleic acids or polypeptides may be purified from the sample according to methods known in the art, including filtration, centrifugation, or extraction methods such as chloroform/phenol extraction. Measuring the presence of a polypeptide marker can be done by any method known in the art.
  • Preferred methods include gas phase ion spectrometry, such as laser desorption/ionization mass spectrometry, surface enhanced laser desorption/ionization time-of flight mass spectrometry (SELDI-TOF MS) and CE-MS. These spectrometry methods allow to measure the polypeptide markers without the need for ligands such as antibodies or aptamers.
  • gas phase ion spectrometry such as laser desorption/ionization mass spectrometry, surface enhanced laser desorption/ionization time-of flight mass spectrometry (SELDI-TOF MS) and CE-MS.
  • Urine sample generally are highly complex, i.e. they contain numerous polypeptides. In case of high complexity, a spectrometric analysis becomes difficult.
  • the polypeptides contained in the sample may be separated by any suitable means, e.g. by electrophoretic separation, affinity-based separation, or separation based on ion exchange chromatography.
  • Particular examples include gel electrophoresis, two-dimensional polyacrylamide gel electrophoresis (2D-P AGE), capillary electrophoresis, metal-affinity chromatography, immobilized metal-affinity chromatography (IMAC) 5 affinity chromatography based on lectins, liquid chromatography, high pressure liquid chromatography (HPLC), and reversed-phase HPLC, cation exchange chromatography, and selectively binding surfaces (such as the surfaces used in SELDI-TOF, see below).
  • IMAC immobilized metal-affinity chromatography
  • 2D-PAGE is commonly used for polypeptide separation and can be combined with mass spectrometry (MS) yielding identification of individual polypeptides. Over 1000 protein spots can be discerned with 2D-P AGE. However, each single spot must be analysed separately by MS/MS for identification.
  • MS mass spectrometry
  • SELDI surface enhanced laser desorption/ionization
  • the ProteinChip Arrays are the most important component. They are narrow metal strips carrying 8 or 16 spots in a row on the surface. Samples to be analyzed are directly applied to the spots, either as a standing drop or in volumes up to 500 ⁇ l, by using sample holders called “bioprocessors" as supporting units. They are placed onto the arrays during incubation and washing steps and removed again afterwards.
  • bioprocessors sample holders
  • the different types of arrays belong to two main series: chromatographic arrays, presenting hydrophobic, hydrophilic, cation-exchanging, anion- exchanging or immobilized metal ion affinity-surfaces, and preactivated arrays with chemical groups to allow the covalent coupling of proteins.
  • chromatographic arrays presenting hydrophobic, hydrophilic, cation-exchanging, anion- exchanging or immobilized metal ion affinity-surfaces
  • preactivated arrays with chemical groups to allow the covalent coupling of proteins Preferably, a chip with cation-exchange surfaces is used.
  • the composition of the analyte depends on the array type used and the washing conditions applied. This explains why the SELDI-process can be defined as a further development of the traditional MALDI (matrix assisted laser desorption / ionization)-technique. In the SELDI-process, only on those polypeptides are measured that actually bind to the chip surface.
  • MALDI matrix assisted laser desorption / ionization
  • the energy absorbing matrix After binding of sample proteins, the energy absorbing matrix is applied to each spot.
  • the matrix rapidly crystallizes and the analysis can start immediately.
  • the ProteinChip Arrays are placed into the ProteinChip Reader for analysis.
  • the reader is a TOF (time-of-flight) mass spectrometer in which the proteins are desorbed and ionized with the help of a laser beam. As the crystallized proteins are equally distributed on the spot surface, the ionizing laser beam always hits a representative average of the molecules in the analyte, allowing quantitative calculations. After ionization, the proteins are accelerated by an electric field to fly down the flight tube, before reaching the detector.
  • TOF time-of-flight
  • CE-MS capillary electrophoresis
  • MS mass spectrometry
  • glas capillaries comprising an outer sheath as mechanical support and to improve mechanical flexibility, e.g. a sheath made of thermoplastic material.
  • the capillary is untreated, i.e. it shows hydroxy-groups on its inside.
  • the capillary may also be coated on the inside. E.g., hydrophobic coating can be used to improve discriminatory power.
  • pressure may be applied, which is typically in the range of 0 to 1 psi. The pressure can also be applied or increased during the run.
  • a stacking protocol can be applied when loading the sample: Before loading of the sample, a base is loaded, then the sample is loaded, then an acid. The principle is to capture the analyte ions between a base and an acid. If voltage is applied, the positively charges analyte ions move towards the base. There, they get negatively charged and move into the opposite direction towards the acid, where they get positively charged. This stacking repeats itself until acid and base are neutralized. Then, the separation starts from a well concentrated sample.
  • the sample is contained in an appropriate buffer in which polypeptides are soluble, e.g. phosphate buffer.
  • phosphate buffer e.g. phosphate buffer.
  • volatile solvents e.g. acetonitrile, isopropanol, methanol, and the like.
  • the solvents can also be combined with water and a weak acid (e.g. 0.1% formic acid), the latter to protonate the analyte.
  • the polypeptides in the sample are separated according to size and charge, which determine the run-time in the capillary.
  • CE is characterized by high separating power and short time of analysis.
  • either fractions collected from the CE can be analyzed as separate batches or, preferably, the CE system can be coupled via a suitable interface to the mass spectrometer to allow continous flow analysis.
  • the flow from the CE may be used to generate continuous "separation tracks", which can be analyzed separately.
  • ions generated from the sample are analyzed according to the mass/charge (m/z) quotient.
  • mass spectrometry it is possible to routinely analyze 10 frnol (i.e. 0.1. ng of a 10 kDa polypeptide) with a precision of ⁇ 0.01%. Experimentally, it is possible to analyze even less than 0.1 fmol.
  • mass spectrometer any type of mass spectrometer can be used.
  • an ion-generating device is coupled with an suitable analyzer.
  • the electrospray ionization (ESI) interfaces are most commonly used to produce ions from liquid samples, whereas MALDI is most commonly used to produce ions from individually processed samples.
  • ESI electrospray ionization
  • MALDI is most commonly used to produce ions from individually processed samples.
  • analyzers e.g. ion trap analyzers or time-of-flight (TOF) analyzers.
  • TOF time-of-flight
  • a preferred CE-MS method according to the present invention includes capillary electrophoresis coupled online via ESI to a TOF analyzer.
  • the CE-MS technique permits to measure the presence of several hundred polypeptide markers simultaneously in a short time in a small volume with high sensitivity. Once the presence of the polypeptide markers has been measured, a pattern of the measured polypeptide markers is generated and can be compared to a disease pattern by any of the methods described further above. However, in many cases it will be sufficient for diagnosis to measure only one or a limited number of the markers.
  • polypeptide sequences can be determined according to methods well-known to the person skilled in the art (see e.g. CS. Spahr et al. (2001). Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest. Proteomics vol. 1, p. 93-107).
  • polypeptide marker it is possible to measure its presence or absence by further means. For example, if the polypeptide is biologically active, its presence may be determined by cellular or enzymatic assays. Presence of a polypeptide can also be determined by use of ligands binding to the polypeptide of interest. Binding according to the present invention includes both covalent and non-covalent binding.
  • a ligand according to the present invention can be any peptide, polypeptide, nucleic acid, or other substance binding to the polypeptide of interest. It is well known that polypeptides, if obtained or purified from the human or animal body, can be modified, e.g. by glycosylation. A suitable ligand according to the present invention may bind the polypeptide also via such sites.
  • Preferred ligands include antibodies, nucleic acids, peptides or polypeptides, and aptamers, e.g. nucleic acid or peptide aptamers.
  • suitable ligands are commercially available.
  • methods to generate suitable ligands are well-known in the art. For example, identification and production of suitable antibodies or aptamers is also offered by commercial suppliers.
  • antibody includes both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab) 2 fragments that are capable of binding antigen or hapten.
  • the ligand should bind specifically to the polypeptide to be measured.
  • “Specific binding” means that the ligand should not bind substantially to ("cross-react” with) another polypeptide or substance present in the sample investigated.
  • the specifically bound protein or isoform should be bound with at least 3 times higher, more preferably at least 10 times higher and even more preferably at least 50 times higher affinity than any other relevant polypeptide.
  • Non-specific binding may be tolerable, particularly if the investigated peptide or polypeptide can still be distinguished and measured unequivocally, e.g. according to its size on a Western Blot, or by its relatively higher abundance in the sample.
  • a method for measuring the presence of a polypeptide of interest may comprise the steps of (a) contacting a polypeptide with a specifically binding ligand, (b) (optionally) removing non-bound ligand, (c) measuring the presence or amount of bound ligand. Binding of the ligand can be measured by any method known in the art. First, binding of a ligand may be measured directly, e.g. by NMR or surface plasmon resonance. Second, the ligand also serves as a substrate of an enzymatic activity of the peptide or polypeptide of interest, an enzymatic reaction product may be measured (e.g. the presence of a protease can be measured by measuring the amount of cleaved substrate, e.g. by Western Blot). Third, the ligand may be coupled covalently or non-covalently to a label allowing detection and measurement of the ligand.
  • Labeling may be done by direct or indirect methods.
  • Direct labeling involves coupling of the label directly (covalently or non-covalently) to the ligand.
  • Indirect labeling involves binding (covalently or non-covalently) of a secondary ligand to the first ligand.
  • the secondary ligand should specifically bind to the first ligand.
  • Said secondary ligand may be coupled with a suitable label and/or be the target (receptor) of tertiary ligand binding to the secondary ligand.
  • the use of secondary, tertiary or even higher order ligands is often used to increase the signal.
  • Suitable secondary and higher order ligands may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.)
  • the ligand or substrate may also be "tagged" with one or more tags as known in the art. Such tags may then be targets for higher order ligands. Suitable tags include biotin, digoxygenin, His-Tag, Glutathion- S -Transferase, FLAG, GFP, myc-tag, influenza A virus haemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, the tag is preferably at the N-terminus and/or C-terminus.
  • Suitable labels are any labels detectable by an appropriate detection method.
  • Typical labels include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g. magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels.
  • Enzymatically active labels include e.g. horseradish peroxidase, alkaline phosphatase, beta-Galactosidase, Luciferase, and derivatives thereof.
  • Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidme, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, available as ready- made stock solution from Roche Diagnostics), CDP-StarTM (Amersham Biosciences), ECFTM (Amersham Biosciences).
  • a suitable enzyme-substrate combination may result in a colored reaction product, fluorescence or chemoluminescence, which can be measured according to methods known in the art.
  • Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, the Alexa dyes (e.g. Alexa 568), and quantum dots.
  • Typical radioactive labels include 35 S, 125 1, 32 P, 33 P, and the like.
  • suitable measurement methods also include precipitation (particularly immunoprecipitation), electrochemilurninescence (electro- generated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immune tests, electrochemiluminescence sandwich immunoassays (ECLIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA) 5 scintillation proximity assay (SPA) 5 turbidimetry, nephelometry, latex- enhanced turbidimetry or nephelometry, or solid phase immune tests.
  • precipitation particularly immunoprecipitation
  • electrochemilurninescence electrochemilurninescence
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • sandwich enzyme immune tests sandwich enzyme immune tests
  • electrochemiluminescence sandwich immunoassays ECLIA
  • DELFIA dissociation-enh
  • the ligand may also be present on an array.
  • Said array contains at least one additional ligand, which may be directed against a peptide, polypeptide or a nucleic acid of interest.
  • Said additional ligand may also be directed against a peptide, polypeptide or a nucleic acid of no particular interest in the context of the present invention.
  • ligands for at least five, more preferably at least 10, even more preferably at least 20 polypeptide markers according to the present invention are contained on the array.
  • array refers to a solid-phase or gel-like carrier upon which at least two compounds are attached or bound in one-, two- or three- dimensional arrangement.
  • arrays including “gene chips”, “protein chips”, antibody arrays and the like) are generally known to the person skilled in the art and typically generated on glass microscope slides, specially coated glass slides such as polycation-, nitrocellulose- or biotin-coated slides, cover slips, and membranes such as, for example, membranes based on nitrocellulose or nylon.
  • the array may include a bound ligand or at least two cells expressing each at least one ligand.
  • suspension arrays as arrays according to the present invention (Nolan JP, Sklar LA. (2002). Suspension array technology: evolution of the flat- array paradigm. Trends Biotechnol. vol. 20(1), p. 9-12).
  • the carrier e.g. a microbead or microsphere
  • the array consists of different microbeads or microspheres, possibly labeled, carrying different ligands.
  • the invention further relates to a method of producing arrays as defined above, wherein at least one ligand is bound to the carrier material in addition to other ligands.
  • arrays for example based on solid-phase chemistry and photo- labile protective groups, are generally known (US 5,744,305). Such arrays can also be brought into contact with substances or substance libraries and tested for interaction, for example for binding or change of conformation. Therefore, arrays comprising a polypeptide marker according to the present invention may be used for identifying ligands binding specifically to said peptides or polypeptides.
  • polypeptide To determine the sequence of a polypeptide, it should be purified to the highest level achievable. However, the polypeptide does not need to be completely isolated. For example, it is enough to have the polypeptide detectable as a coomassie-stained band in a polyacrylamide gel. The corresponding gel piece can then be cut out and used for the next identification steps. After purification of the polypeptide, it can be enzymatically digested with trypsin and the molecular weights of the resulting fragments determined using any suitable method, for example mass spectrometry. Using mass spectrometry, each polypeptide displays a characteristic "fingerprint" of fragments allowing its identification by database searches. In case that the polypeptide to be identified is not present in the database or if the researcher wants to have a closer characterization for any reasons, the polypeptide fragments can also be sequenced according to methods known in the art.
  • CE-MS allows particularly easy determination of the polypeptide sequences.
  • the capillary electrophoresis elution time for each marker is listed in the tables. Thus, it is possible to collect the fraction containing the polypeptide at relatively high purity. If a single fraction contains insufficient material, fractions of more than one experiment may be pooled.
  • Fig. 1 Depiction of the information from a crude CE-MS analysis (A) as a three dimensional contour plot (left side). Here a contour plot of urine from a healthy volunteer is shown, mass per charge on the Y-axis against the retention time in min (X-axis), signal intensity colour coded. Next, the signal to noise is calculated and the noise removed, thus leaving only actual signals (B). The software calculates the actual mass (C) based on both isotopic distribution and conjugated masses. This results in a table of up to 1500 polypeptides defined via their mass and retention time. As an example, bottom right shows 17 polypeptides found in the sample. CE- t, CE-time (migration time) ; int., intensity; m.p.c, mass per charge, cal. m., calculated mass.
  • CE- t CE-time (migration time) ; int., intensity; m.p.c, mass per charge, cal. m., calculated mass.
  • Fig. 2 Contour plots of polypeptides (actual masses) for healthy subjects (NC) and for patients with focal-segmental glomerulosclerosis (FSGS), minimal-change disease (MCD) and membranous glomerulonephritis (MGN) are shown.
  • the upper mass limit for each plot i.e. the maximum value along the X-axis
  • the contour plots differ significantly between the healthy subjects and the renal disease groups.
  • Fig. 3 Flow sheet for diagnosis and differential diagnosis of renal diseases (example).
  • Samp. sample; MS-dat, MS-data; Disea., disease; Y, yes; N, no; n.d., no disease; d.n., diabetic nephropathy, FSGS, FSGS; MGN, MGN; MCD, MCD; IgA, IgA- nephropathy, diff, differential diagnosis
  • the mobile phase used contained 30% methanol and 0.5% formic acid in water. The same liquid was used for the sheath flow, which was applied at 2 ⁇ l/min. Sample injection was performed with pressure: 1 psi for 20 sec. Under these conditions about 100 nl of sample could be injected. For sample stacking, the following protocol was applied: injection of IM NH 3 for 7 sec, injection of sample, injection of 2M formic acid for 5 sec. The subsequent CE-MS run was performed at +30 kV with the sequence of the following pressures: 40 min at 0 psi, 2 min at 0.1 psi, 2 min at 0.2 psi, 2 min at 0.3 psi, 2 min at 0.4 psi, 80 min at 0.5 psi.
  • the following pressure sequence was used: 40 min at 0.3 psi, 2 min at 0.4 psi, 2 min at 0.6 psi, 2 min at 0.8 psi, 80 min at 1 psi. After each run, the CE capillary was rinsed for 5 min with 0.1 M NaOH, followed by 5 min with water and 5 min with running buffer.
  • Random Forests For discrimination between healthy subjects and different groups of patients with renal diseases we used the method of Random Forests and the corresponding S-PIus program version 6/2002 Breiman L: Random Forests.
  • Fig. 1 A graphical depiction (contour plot) of a typical sample is presented in Fig. 1 (raw data).
  • Fig. 1 raw data
  • PP polypeptides with molecular weights from 800 up to 30.000 Dalton
  • FIG. 1 A list of polypeptides present with high probability that were chosen as internal standards to assure sample comparability is shown in table 23.
  • Table 24 For analysis of protein-rich samples, such as samples from suspected IgA- nephropathy patients, higher pressure was applied and the polypeptides according to table 24 were preferred as internal standards. Repeated analyses of identical samples did not reveal any significant differences under identical conditions of the CE-MS run for an individual sample.
  • Fig. 1 The subsequent electronic data manipulation for one example is summarised in Fig. 1.
  • Each run results in the cnide spectrum depicted in the upper part of Fig. 1 and is composed of single spectra (blow up Fig. 1) generated every 3 seconds.
  • CE-MS peaks were identified in the first data analysis run (Fig. IA).
  • the charge of each peak was ascertained utilising both isotopic distribution and conjugated peaks (Fig. IB).
  • conjugated peaks were summarised in one single peak and the real mass was calculated, as shown in Fig. 1C.
  • the samples were spiked with external standards of known mass. This allowed subsequent definition of internal standards of PP present with high probability in the urine samples.
  • the CE-time could be normalized to the internal standards.

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Abstract

La présente invention concerne des méthodes pour le diagnostic d'une maladie rénale, en particulier un diagnostic différentiel. Des maladies rénales présentant un intérêt particulier dans le contexte de l'invention sont une hématurie idiopathique bénigne, une glomérulonéphrite à dépôts extra-membraneux (MNG), un syndrome néphrotique à lésions glomérulaires minimes (MCD), une glomérulosclérose segmentaire focale (FSGS), et une néphropathie diabétique.
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