EP2076771A2 - Biomarker - Google Patents

Biomarker

Info

Publication number
EP2076771A2
EP2076771A2 EP07824164A EP07824164A EP2076771A2 EP 2076771 A2 EP2076771 A2 EP 2076771A2 EP 07824164 A EP07824164 A EP 07824164A EP 07824164 A EP07824164 A EP 07824164A EP 2076771 A2 EP2076771 A2 EP 2076771A2
Authority
EP
European Patent Office
Prior art keywords
complement
precursor protein
pcos
haptoglobin
subject
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.)
Withdrawn
Application number
EP07824164A
Other languages
English (en)
French (fr)
Inventor
William Atiomo
Robert Layfield
Catherine Hughes
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.)
University of Nottingham
Original Assignee
University of Nottingham
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from GB0620467A external-priority patent/GB0620467D0/en
Priority claimed from GB0708064A external-priority patent/GB0708064D0/en
Application filed by University of Nottingham filed Critical University of Nottingham
Publication of EP2076771A2 publication Critical patent/EP2076771A2/de
Withdrawn legal-status Critical Current

Links

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
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation 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
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/361—Menstrual abnormalities or abnormal uterine bleeding, e.g. dysmenorrhea
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to novel biomarkers for Polycystic Ovarian Syndrome (PCOS) , and to methods of determining the PCOS status in a subject using these novel biomarkers.
  • PCOS Polycystic Ovarian Syndrome
  • PCOS Polycystic Ovary Syndrome
  • insulin resistance is understood to increase androgen production by inhibiting sex hormone-binding globulin (SHBG) synthesis and acting synergistically with LH to directly stimulate theca cell androgen production.
  • SHBG sex hormone-binding globulin
  • the lack of a precise aetiology for insulin resistance in PCOS and the presence of selective ovarian sensitivity in the presence of peripheral insulin resistance further reflects the challenges of PCOS. Hyperandrogenism appears to underlie all other manifestations of PCOS, again, the initial cause and molecular mechanisms of hyper androgenaemia are by no means clear.
  • PCOS cardiovascular disease
  • the current diagnosis of PCOS is one of exclusion of other androgenic- based diseases following clinical identification of at least two of the following three criteria: (i) chronic oligo-/anovulation; (ii) clinical and/or biochemical signs of hyperandrogenism; and (iii) ultrasound evidence of polycystic ovaries.
  • relative weightings of each criterion tend to vary depending on the clinician's medical background, again highlighting the clinical challenges of PCOS.
  • Treatment strategies also vary depending on the patient's presenting features and desired outcome, tending towards symptom-based strategies for gynaecological or cosmetic improvements or simple weight management and life-style changes.
  • the present invention provides novel biomarkers useful in the diagnosis of PCOS, as well as methods and kits for using the biomarkers to diagnose PCOS and determine the PCOS status of a subject.
  • the invention provides a method of determining the Polycystic Ovarian Syndrome (PCOS) status of a subject, comprising the steps of:
  • the method allows the diagnosis of PCOS in a subject from the analysis of the proteins in a sample provided by the subject.
  • the complement C4 precursor protein, cleavage fragments of the complement C4 precursor protein and haptoglobins have been identified as biomarkers for PCOS, and are referred to herein as the biomarkers or the biomarker proteins.
  • PCOS status includes any distinguishable manifestation of the PCOS disease, including diseased and non-diseased.
  • PCOS status includes, without limitation, the presence or absence of disease, the risk of developing the disease, the stage of the disease, the progression of the disease (e.g. , progress of disease or remission of disease over time) and the effectiveness or response of a subject to treatment of the disease
  • the C4 precursor protein is one component of the complement pathway, and is an important contributor to the immune response system.
  • C4 plays a central role in the classical pathway of the complement system.
  • the protein sequence of each isoform is given in Figures 8 and 9.
  • C4 protein and C4 precursor protein refer to the same protein, and are used interchangeably herein.
  • the one or more cleavage fragments of the complement C4 precursor protein may be selected from any of the group comprising the polypeptides: a) C4 ⁇ ; b) C4 ⁇ ; c) C4 ⁇ ; d) C4a anaphylatoxin; e) C4b; f) C4 ⁇ 3 c fragment; g) C4 ⁇ d; h) C4 ⁇ 4 c fragment; and i) a polypeptide with at least 75% amino acid sequence identity, more preferably at least 80%, 85%, 90% or 95% or more sequence homology, to the amino acid sequence of any of a) to h) .
  • At least one or the fragments a) to i) is detected in the method of the invention, more preferably at least 2, 3, 4, 5, 6, 7, 8, or 9 of the fragments a) to i) are detected.
  • the one or more haptoglobin proteins may be selected from the group comprising haptoglobin ⁇ and haptoglobin ⁇ .
  • the amino acid sequence of both haptoglobins is given in Figure 10.
  • the ability to differentiate between different forms of a protein depends upon the nature of the difference and the method used to detect or measure the protein level. For example, an immunoassay using a monoclonal antibody will detect all forms of a protein containing the epitope and will not distinguish between them.
  • a sandwich immunoassay that uses two antibodies directed against different epitopes on a protein will detect all forms of the protein that contain both epitopes and will not detect those forms that contain only one of the epitopes.
  • the assay method used to determine the level of one or more of the biomarker proteins preferably detects all forms of the specific biomarker protein. Preferably at least all biologically active forms of the specific biomarker protein are detected. Preferably, all forms of any of the biomarker proteins with at least 75% or more, preferably at least 80%, 85%, 90% or 95% or more, identity with the amino acid sequence of the biomarkers given in Figures 8, 9 and 10, will be detected in the method of the invention.
  • the PILEUP and BLAST algorithms can also be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S.F. (1993) J MoI Evol 36:290-300; Altschul, S, F et al (1990) J MoI Biol 215:403-10.
  • the method of the invention preferably will allow the level of biomarker proteins with polymorphisms common in the general population to be detected. Polymorphisms can occur that do not affect the function of a protein, it is intended that the method of the invention will allow these to be detected.
  • the sample material obtained from the subject may comprise whole blood, blood serum, blood plasma, urine, fat tissue (adipose) , endometrial tissue, ovary tissue or any other bodily fluid or tissue.
  • the level of the one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin may be determined by any suitable assay which may comprise the use of any of the group comprising immunoassays, spectrometry, mass spectrometry, Matrix Assisted Laser Desorption/Ionization Time-of- Flight (MALDI-TOF) Mass Spectrometry, microscopy, northern blot, western blot, southern blot, isoelectric focussing, SDS-PAGE, PCR 1 RT-PCR, gel electrophoresis, protein microarray, DNA microarray, and antibody microarray, or combinations thereof.
  • the level of the one or more biomarkers is determined using an immunoassay.
  • An immunoassay uses an antibody or antibodies to a specific antigen to determine the levels of the antigen.
  • an antibody or antibodies specific to one or more of the C4 precursor protein, or a cleavage fragment of the C4 precursor protein or a haptoglobin may be used.
  • the immunoassay may be an enzyme linked immunoassay (ELISA) , a sandwich assay, a radioimmunoassay, a Western Blot, an immunoassay using a biosensor, an immunoprecipitation assay, an agglutination assay, a turbidity assay or a nephelometric assay.
  • the one or more antibodies may be synthetic, monoclonal, polyclonal, bispecific, chimeric or humanised.
  • a chimeric antibody includes portions derived from different animals.
  • Humanised antibodies are antibodies from non-human species having one or more complementarity determining regions from the non-human species and a framework region from a human immunoglobulin molecule. Chimeric and humanised antibodies can be produced by recombinant techniques well known in the art.
  • the one or more antibodies may comprise a tag or a label selected from the group comprising a radioactive, a fluorescent, a chemiluminescent, a dye, an enzyme, or a histidine tag or label, or any other suitable label or tag known in the art.
  • the presence, and possibly even the level, of particular proteins in a sample may also be determined by using mass spectrometry techniques. Mass spectrometry techniques may be used to detect gas phase ions which correlate to specific proteins or parts of proteins, such as, trypsin peptides. Examples of mass spectrometers include time of flight, magnetic sector, quadruple filter, ion trap, ion cyclotron resonance, electrostatic sector analyser and hybrids of these.
  • the mass spectrometer may use laser desorption/ionisation.
  • MALDI Microx-assisted laser desorption/ionization
  • peptide mass fingerprinting may be used to identify the presence of particular biomarker proteins from 2D gel analysis and RPE/SDS PAGE analysis.
  • MALDI-MS and ANN analysis may be used to profile identified proteins and tryptic biomarker signatures for PCOS.
  • the reference values to which the determined levels of one or more of complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin, are compared are the levels observed in subjects who do not have any clinical symptoms of PCOS, so called "normal values" .
  • the reference values may be the previous values obtained for a specific subject. This kind of reference value may be used if the method is to monitor progression of a disease or to monitor the response of a subject to a particular treatment.
  • an increase or a decrease in the level of one or more of the biomarker proteins may be indicative of PCOS status of the subject.
  • an increase in the level of C4 ⁇ 3 c and/or the level of C4 ⁇ 4 c and/or the level of haptoglobin ⁇ and/or haptoglobin ⁇ may be indicative, or diagnostic, of PCOS.
  • a decrease in the level of the C4 precursor protein and/or C4 ⁇ and/or C4 ⁇ and/or C4 ⁇ and/or C4a anaphylatoxin may be indicative, or diagnostic, of PCOS.
  • the method of the invention may also be used to monitor disease progression and/or to monitor the efficacy of treatments administered to a subject. This may be achieved by analysing samples taken from a subject at various time points following initial diagnosis and monitoring the changes in the levels of biomarkers and comparing these levels to reference values.
  • reference levels may include the initial levels of the biomarkers in the subject, or the levels of the biomarkers in the subject when they were last tested, or both.
  • the method of the invention is carried out in vitro .
  • the subject may be mammal, and is preferably a human, but may alternatively be a monkey, ape, cat, dog, cow, horse, rabbit or rodent.
  • the invention provides one or more of the complement C4 precursor protein, a cleavage fragment of the complement
  • a panel of biomarkers comprises at least 2, 3, 4, 5 or more of the biomarkers selected from the list comprising C4 precursor protein, C4 ⁇ , C4 ⁇ , C4 ⁇ , C4a anaphylatoxin, C4b, C4 ⁇ 3 c fragment, C4 ⁇ d, C4cc 4 c fragment, haptoglobin ⁇ and haptoglobin ⁇ .
  • a method of determining the PCOS status in a subject comprising the steps of; (a) providing a sample of material obtained from a subject; (b) analysing proteins or tryptic peptides obtained from the sample using MALDI-MS;
  • the six ions m/z 8674, 8668, 1351 , 8727, 8673 and 6871 and the three ions m/z 2924, 3025 and 1977 are biomarkers for PCOS.
  • kits for use in determining the PCOS status of a subject comprising instructions to analyse a sample for the presence of the six ions m/z 8674, 8668, 1351, 8727, 8673 and 6871 in a protein sample and/or the presence of the three ions m/z 2924, 3025 and 1977 in a tryptic peptide sample.
  • kits for use in determining the PCOS status of a subject comprising at least one agent for determining the level of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein and a haptoglobin.
  • the agent may be an enzyme, an antibody, a nucleic acid, a protein probe or other suitable composition.
  • the agent for determining the level of the one or more PCOS biomarkers is preferably labelled.
  • the kit may also comprise means for detecting the label.
  • the kit may comprise one or more capture agents for capturing one or more biomarker proteins the level of which is to be determined.
  • the capture agent may be one or more antibodies.
  • the capture agent or the agent for determining the level of the one or more PCOS biomarkers may be attached to a solid support.
  • the solid support may be a chip, a micro titre plate, a bead or a resin.
  • the kit may also comprise a wash solution or instructions for making a wash solution.
  • the wash solution may, alone or in combination with the capture agent, allow efficient capture of a biomarker or biomarkers on a solid support for subsequent detection by, for example, mass spectrometry or immunoassay methods.
  • the kit may comprise instructions for suitable operational parameters in the form of a label or separate insert.
  • the instructions may inform a consumer about how to collect the sample, and/or how to wash a capture agent, and/or details of the particular biomarkers to be detected.
  • the kit may comprise one or more biomarker samples, to be used as standard(s) for calibration and comparison.
  • the invention provides the use of the determination of the levels of the C4 precursor protein and/or cleavage fragments of the C4 precursor protein and/or haptoglobin chains, with or without the detection of other PCOS biomarkers, as a means of assessing the PCOS status in an individual.
  • the invention provides a probe set capable of detecting one or more of the aforementioned PCOS biomarkers.
  • suitable probes which may, for example, be a synthetic molecule, an antibody, a nucleic acid or proteinacious in nature.
  • the probe may also carry one or more labels to facilitate its detection; the label may, for example, be a radioactive, a chemiluminescent or a florescent label, but is not limited to these examples.
  • the probe set contains at least two probes directed to different biomarker proteins.
  • the invention provides a method of treating PCOS in a subject comprising administering to the subject an agent capable of modulating the level of one or more PCOS biomarkers in a cell, wherein said one or more biomarkers are selected from the group comprising the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein and a haptoglobin.
  • the agent may be an antisense or an interfering RNA molecule designed to inhibit or reduce expression of a biomarker protein in the cell.
  • the agent may be an inhibitor of C4 precursor protein processing, or a composition which reduces complement activity.
  • the invention provides a method of determining the course of PCOS in a subject, or determining the efficacy of treatment administered to a subject with PCOS, comprising: (a) determining the level of one or more of the following proteins: the complement C4 precursor protein; a cleavage fragment of the complement C4 precursor protein; and a haptoglobin; in a sample obtained from a subject at a first time point;
  • This method may be used to compare sample taken at two or more time points.
  • the invention provides a method of identifying compounds for treating PCOS comprising screening for one or more compounds that modulate the level of one or more of the complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and a haptoglobin, in vitro or in vivo.
  • Compounds suitable for therapeutic testing may be screened initially by identifying compounds which interact with one or more of the PCOS biomarkers listed herein.
  • screening might include recombinantly expressing a biomarker, purifying the biomarker, and affixing the biomarker to a substrate.
  • Test compounds would then be contacted with the substrate, typically in aqueous conditions, and interactions between the test compound and the biomarker may be measured, for example, by measuring elution rates as a function of salt concentration.
  • Certain proteins may recognize and cleave one or more of the biomarkers, in which case the proteins may be detected by monitoring the digestion of one or more biomarkers in a standard assay, e.g. , by gel electrophoresis of the proteins .
  • the ability of a test compound to inhibit the activity of one or more of the biomarkers may be measured.
  • One of ordinary skill in the art will recognize that the techniques used to measure the activity of a particular biomarker will vary depending on the function and properties of the biomarker. For example, enzymatic activity of a biomarker may be assayed provided that an appropriate substrate is available and provided that the concentration of the substrate or the appearance of the reaction product is readily measurable.
  • the ability of potentially therapeutic test compounds to inhibit or enhance the activity of a given biomarker may be determined by measuring the rates of catalysis in the presence or absence of the test compounds.
  • Test compounds capable of modulating the activity of any of the PCOS biomarkers may be administered to patients who are suffering from or are at risk of developing PCOS.
  • the administration of a test compound which increases the activity of a particular biomarker may decrease the risk of PCOS in a patient if the activity of the particular biomarker in vivo prevents the accumulation of proteins which cause PCOS.
  • the administration of a test compound which decreases the activity of a particular biomarker may decrease the risk of PCOS in a patient if the increased activity of the biomarker is responsible, at least in part, for the onset of PCOS.
  • a yet further aspect provides the use of the C4 precursor protein, C4 ⁇ , C4 ⁇ , C4 ⁇ , C4a anaphylatoxin, C4b, C4 ⁇ 3 c fragment, C4 ⁇ d, C4 ⁇ 4 c fragment, haptoglobin ⁇ and haptoglobin ⁇ and/or other PCOS biomarkers as therapeutic targets for the treatment / alleviation of PCOS.
  • Figure 1 - shows the results of 2-dimensional gel electrophoresis illustrating the protein level changes between PCOS subjects and matched control subjects.
  • the upper 10 images are silver-stained 2D gels in which the protein spots of interest are circled, and the difference in the levels of protein between PCOS subjects and control age/BMI-matched subjects can be seen.
  • the lower 5 images are genereated using Delta 2D Image Analysis Software and are merged images of the corresponding gel pairs showing the same protein spot in a colorimetric display. Whilst the colours are not visible, the results show that the observed spot appears in four of the five gel pair merged images coloured orange indicating that it occurs only in the PCOS subjects.
  • the spot was subsequently identified as C4 ⁇ 3 c;
  • Figure 2 - shows a quantitative analysis of the intensity of the
  • Figure 3 - shows a western blot analysis of 10 depleted serum samples using an antibody raised against an unknown C4 ⁇ 4 c peptide sequence.
  • Figure 3 A shows western blot analysis of whole serum from the same sample set used in earlier 2DE studies. The blot was probed with a polyclonal anti-C4 ⁇ antibody raised against epitopes in the C4 ⁇ chain. The ⁇ 24 kDa band shown represents the C4 ⁇ 4 c fragment.
  • Figure 4 - shows a schematic representation of the primary structure of the complement component C4 protein.
  • the C4 precursor and a number of its cleavage fragments are shown.
  • the C4 protein is shown to consist of three chains, which can all be cleaved from the single C4 precursor and folded to form the protein's functional structure.
  • the ⁇ chain is the active element, when cleaved the C4a fragment is released to act as the anaphylatoxin.
  • the C4b fragment forms a complex with C2 to continue the enzymatic cascade of the classical complement pathway and can also act as an opsonin.
  • C4b is later cleaved into C4 ⁇ 3c, C4 ⁇ d and C4 ⁇ 4c fragments, this inactivates the complement cascade (based on Nagasawa et al. , (1980) J Immunol 125(2): 578-82 and Hessing, (1991) Biochem J
  • Figure 5 - shows RP-SPE analysis showing differences between
  • Figure 5 A shows a ⁇ 33kDa band present in 5/6 control samples and only very low level in 3/6 PCOS samples for fraction 12 only. There is potentially a similar sized band in fractions 4 and 5 but this is taken to be a different protein species.
  • Figure 5B shows a ⁇ 40kDa band present in all PCOS samples at an increased level than in the control samples. This difference was most pronounced in fractions 8-12.
  • Figure 5C shows a ⁇ 17kDa band present in 5/6 PCOS samples and only 2/6 control samples;
  • Figure 6 - shows a quantitative RP-SPE analysis showing differences between PCOS and matched controls.
  • Figure 6 A shows the results of densitometric quantification of the ⁇ 40Kda band seen on RP-SPE ( Figure 5B) .
  • P 0.001.
  • Figure 6B shows the results of densitometric quantification of the ⁇ 17Kda band seen on RP-SPE ( Figure 5C) ;
  • Figure 7 - illustrates representative MALDI-TOF mass spectra of:
  • Figure 7A serum peptides after tryptic digestion and from Figure 7B proteins after Ci 8 ZipTip clean-up from PCOS patients and control sera. This spectrum illustrates the observed differences between PCOS sera and control sera.
  • Figure 7C shows the predictive capability of ANNs to recognise tryptic peptide profiles based on a 3 ion model and
  • Figure 7D shows protein profiles based on a six ion ANNs model. The grey bars indicate control samples and the black bars indicate PCOS samples. A predicted value below 1.5 indicates a control sample, whilst a prediction greater than 1.5 indicates a PCOS sample;
  • Figure 8 (Sequence ID No: 1) - is the protein sequence of the C4 precursor protein of isoform A. More specifically, amino acids 20 to 675 are the C4 ⁇ cleavage fragment, amino acids 680 to 1446 are the C4 ⁇ cleavage fragment, amino acids 680 to 756 are the C4a anaphylatoxin cleavage fragment, amino acids 757 to 1446 are the C4b cleavage fragment, amino acids 757 to 956 are the C4 ⁇ 3 c cleavage fragment, amino acids 957 to 1333 are the C4 ⁇ d cleavage fragment, amino acids 1334 to 1446 are the C4 ⁇ 4 c cleavage fragment, and amino acids 1454 to 1744 are the C4 ⁇ cleavage fragment;
  • Figure 9 (Sequence ID No: 2) - is the protein sequence of the C4 precursor protein of isoform B. The amino acids different to isoform A are underlined; and
  • Figure 10 (Sequence ID No: 3) - is the protein sequence of haptoglobin. Amino acids 19 to 161 are haptoglobin ⁇ and amino acids 162 to 406 are haptoglobin ⁇ .
  • the study comprised eleven women (aged 18-40 years) diagnosed with PCOS, and ten age-BMI matched controls. N Women with PCOS were initially identified at the infertility, gynaecology or PCOS clinics run at the Queen's Medical Centre, Nottingham and were then approached verbally to participate in the study. Control women without PCOS were recruited by poster advertisements, from the same gynaecology clinics that women with PCOS were recruited from and also from female members of staff. Ethical approval for the study was granted by the Nottingham Local Research Ethics Committee and informed consent was obtained for each subject.
  • Demographic details along with menstrual, gynaecological, obstetric and other medical histories were collected for each subject during a clinical interview.
  • a regular menstrual cycle was defined as being between 21 and 35 days.
  • the presence of acne and hirsutism was assessed and hirsutism defined as a Ferriman Gallwey score of > 7 (Ferriman and Gallwey, (1961) J Clin Endocrinol Metab 21 : 1440-7) .
  • Subjects were excluded from the study if there was a history of thyroid disease, hyperprolactinaemia, recent delivery, miscarriage or surgery, a history of myocardial infarction, thrombosis or haematological disease or current use of sex steroid therapy.
  • BMI Body Mass Index
  • waist and hip circumferences were measured in centimetres and blood pressure was taken using an appropriately sized cuff with the patient in a sitting position.
  • blood pressure was taken using an appropriately sized cuff with the patient in a sitting position.
  • samples were collected for each woman.
  • samples were taken during the follicular phase of their menstrual cycle.
  • a transvaginal pelvic ultrasound was also performed. The sonographer performing the ultrasound scans and the clinical chemistry department were blind to the clinical diagnosis of each subject.
  • Blood samples were collected in BD vacutainer SST tubes for biochemical testing and BD vacutainer Fluoride tubes for glucose determination (both from BD Biosciences, Oxford, U.K.) .
  • the samples were assayed for endocrines to measure blood glucose, insulin, lipid profile (triglycerides, total cholesterol and HDL cholesterol) and serum hormone profile (testosterone, SHBG, luteinising hormone (LH) , follicle stimulating hormone (FSH) , 17-hydroxyprogesterone and prolactin) .
  • Serum samples were collected concurrently over a 12 month period from 21 subjects. The samples were separated within an hour of collection from coagulated whole blood in SST tubes (obtained in parallel with samples for biochemical testing) by centrifugation at 4,000 rpm for 10 min at 4 0 C and stored at -8O 0 C until required. Samples were arbitrarily labelled to maintain anonymity as P- for PCOS samples and C- for controls with consecutive numbering in each group. PCOS and control women with highly similar age and BMI were matched in sample pairs.
  • solubilisation buffer 7M urea (Sigma, Poole, U.K.), 2M thiourea (Sigma) , 4% w/v 3-(cyclohexylamino)-l-pro ⁇ anesulfonic acid (CHAPS; Amersham Biosciences) , 1OmM dithiothreitol (DTT; Fisher Scientific, Loughborough, Leics, U.K.) , 1% v/v BioLyte carrier ampholytes 3-10 (BioRad, Hercules, CA, U.S.A.)) , to a final concentration of 5 ⁇ g/ ⁇ l, as determined by modified Bradford assay (Quick Start Protein Assay; BioRad) .
  • IEF was performed using a PROTEAN IEF Cell (BioRad) .
  • ReadyStripTM 11 cm immobilised pH gradient (IPG) strips, pH 3-10NL (BioRad) were rehydrated for 16 hours at 20°C with 188 ⁇ l IEF buffer (7M urea, 2M thiourea, 4% w/v CHAPS, 10OmM bis(2-hydroxyethyl) disulfide (HED; DeStreak reagent, Amersham Biosciences) , 0.5% v/v BioLyte carrier ampholytes 3-10) .
  • HED Trihydroxyethyl
  • SDS-PAGE was performed on uniform 13% acrylamide or 8-20% acrylamide gradient gels with the equilibrated strips sealed onto the top of the gels using 1% w/v low-melt agarose (BioRad) made in Ix gel running buffer (0.25M Tris, 1.86M glycine (Fisher Scientific) , 0.1% w/v SDS) . Electrophoretic separation of the two strips were run in parallel, and was performed at 22 rtiA per gel for 3.5 h at room temperature. Proteins in the gels were stained using a modified silver staining protocol (Plus One Silver Stain Kit; Amersham Biosciences) compatible with mass spectrometry (Y an et al.
  • a modified silver staining protocol Plus One Silver Stain Kit; Amersham Biosciences
  • Equal amounts (50 ⁇ g) of diluted (1 :10 in dH 2 O) serum samples from the initial 10 samples were diluted 1:1 in gel loading buffer and denatured by heating at 95°C for 7 min. Samples were loaded on 5-20% acrylamide SDS-PAGE gels and separated by electrophoresis at 35 mA per gel for 2.5 h. Protein in the gels was then transferred to nitrocellulose membrane by blotting overnight at 40 mA.
  • the membrane was blocked using 5 % w/v skimmed milk (Marvel, Premier International Foods (UK) Ltd, Lines, U.K.) in TBS-T (1OmM Tris.HCl (pH 7.5) , 15OmM sodium chloride (Fisher Scientific) , 0.05 % (v/v) Tween-20 (Sigma)) and probed with a polyclonal primary antibody raised against the complement component C4 ⁇ chain (Santa Cruz, Heidelberg, Germany) followed by a horseradish peroxidase-conjugated secondary antibody (Dako, Ely, Cambs, U.K.) . Antibody binding was detected by chemiluminescence using an ECL detection kit (Amersham Biosciences) .
  • RP-SPE and SDS PAGE Serum Fractionation Serum fractionation of 6 PCOS and 6 control samples was performed using solid phase extraction (SPE) employing large pore (1000 A) polystyrenedivinylbenzene (PDVB) 25 mg resin (International Sorbent Technologies, mid-Glamorgan, U.K.) in reversed phase (RP) mode.
  • SPE solid phase extraction
  • PDVB polystyrenedivinylbenzene
  • RP reversed phase
  • Mass spectrometry grade mobile phase (Riedel de Haen, Sigma) was delivered, using an applied vacuum, at flow rates similar to lmL.min-1 and with the composition of ImL deliveries being adjusted volumetrically.
  • Resin was solvated using 70% v/v acetonitrile (MeCN, Sigma) 0.1% v/v trifluoroacetic acid (TFA, Sigma) and equilibrated with aqueous 0.1% TFA.
  • Serum samples 100 ⁇ l equating to ⁇ 7mg total protein
  • Binding of sample proteins was dependent upon molecular hydrophobicity (Badock et al. , 2001) . Columns were then washed with 0.1% v/v TFA and retained components were eluted in fractions using 5 to 100% v/v step-wise incrementing MeCN composition.
  • Eluates were directly compatible with analysis by electrospray or MALDI-TOF mass spectrometry but in this instance were subjected to electrophoretic analysis subsequent to drying by centrifugal vacuum evaporation (BETA- RVC, Christ Gefriertrocknungsanlagen Gmbh, Osterode am Harz, Germany) and resuspension in 100 ⁇ l gel loading buffer (0.15M Tris.HCl (pH 6.8), 8M urea, 2.5% w/v SDS, 20% v/v glycerol, 10% v/v 2- mercaptoethanol (Sigma) , 3% w/v DTT, 0.1% w/v bromophenol blue (Sigma)) at room temperature for 3 hours. Samples were stored in the gel loading buffer at -20 °C.
  • Protein in the gels was stained using filtered Coomassie blue (50% v/v methanol (Fisher Scientific) , 20% v/v acetic acid (Fisher Scientific) 0.12% w/v Brilliant blue-R250 (Sigma)) for 2.5 hr and then destained in 10% v/v acetic acid/10% v/v methanol solution to the desired intensity. Imaging of the stained gels was performed using Agfa Fotolook v3.0 and a Duoscan T1200 flatbed scanner followed by calibrated densitometric quantification (Gene Tools Quantification, SynGene) . Stained gels were stored in dH 2 O at room temperature.
  • Sample preparation randomization was carried out prior to sample handling and analysis of the full set of 11 PCOS and 10 control serum samples.
  • the same aliquot of serum diluted 1 in 10 with Q.I % TFA was used for protein and tryptic peptide analysis.
  • Diluted serum 25 ⁇ l was Ci 8 ZipTip fractionated according to manufacturer's instructions using the Xcise robotic system (Proteome systems, Shimadzu, UK) and the eluted proteins/peptides spotted together with SA, 10 mg/ml onto the MALDI- TOF MS target plate by the robotic system. The remaining elutate was carried forward for tryptic digestion.
  • fractionated serum sample was combined with ammonium bicarbonate (16.6 ⁇ L of 10OmM) , water (7.6 ⁇ L) , and trypsin (1.3 ⁇ L of 0.5 ⁇ g/ ⁇ L) and incubated at 37°C overnight.
  • the reaction was quenched and the sample cleaned using C 18 ZipTip according to the manufacturer's instructions and spotted onto the MALDI target using the dried droplet method with CHCA (10mg/ml solution in 50% ACN + 0.1% TFA, LaserBio Labs, Cedex, France) .
  • the target plate was analysed using the AXIMA-CFR + MALDI-TOF MS (Shimadzu, Manchester, UK) in linear mode using the raster option for proteins and reflectron and autoquality modes for tryptic peptides.
  • a bovine serum albumin (BSA) control was used to ensure the efficiency of the digestion procedure and 0.1% TFA blank to ensure there was no contamination from the reagents or plate.
  • Close external calibration was performed using protein calibration Proteomix3 and Proteomix2 for peptides (Laser Biolabs, Cedex, France) .
  • the raw mass spectral data (m/z, intensities) obtained from MALDI-MS were exported as ASCII files and smoothed to yield rounded masses and intensities for the mass range of interest (m/z 1000- 25000) . These intensity values were subsequently used as inputs to the ANN models, developed using Statistica 7.0 (StatSoft Inc. Tulsa, USA) . The models developed were used to predict membership of each sample to one of two output classes: control (1) or PCOS (2) .
  • Model Architecture Model architecture and network parameters are described in Lancashire et al (2005) Bioinformatics 21(10) :2191-9. Prior to training, samples were randomly divided into three subsets: training
  • PCOS polycystic ovary syndrome
  • BMI body mass index
  • WHR waist(cm)/hip(cm) ratio
  • F-G Ferriman & Gallwey
  • T testosterone
  • SHBG sex hormone-binding globulin
  • FAI free androgen index
  • LH luteinising hormone (IU/L)
  • FSH follicle stimulating hormone (IU/L).
  • the upregulated protein spot (Figure 1) was excised from the Pl, P3, P4 and P5 sample gels, pooled, trypsinised and the extracted peptides were analysed by MALDI-TOF MS.
  • the resultant 13 peptide ions when searched against the Human Swiss- Prot and TrEMBL databases, were consistent matches of the complement component-4, (Swiss-Prot accession number P01028) , ⁇ chain, c fragment (C4 ⁇ 3 c) with 5 matched peptide ions, covering 15.2% of the C4 ⁇ 3 c peptide sequence.
  • the predicted MW and pi of the C4 ⁇ 3 c sequence as calculated using the ExPASy primary structure analysis software, were 32.9 kDa and 5.1 respectively. This corresponds reasonably with the spot position in the gel ( ⁇ 35 kDa, pi ⁇ 7.0) .
  • control sample band was more intense than the PCOS sample band (C4-P1) is believed to relate to a control sample who was borderline PCOS thereby explaining this anomaly. It was not possible to source an antibody specific to the C4 ⁇ 3 c fragment but the increase in C4 ⁇ 4 c is indicative of the proteolytic degradation of the C4b polypeptide which produces C4 ⁇ 3 c and C4 ⁇ 4 c ( Figure 4) .
  • Samples from 6 PCOS and 6 control subjects were analysed be RP- SPE/SDS-PAGE.
  • the samples were from the 5 previous BMI and age matched PCOS and control subject pair and a further PCOS and control pair.
  • Aliquots of serum samples from the subjects were prefractionated using reversed-phase solid phase columns and sequential elution with a stepped gradient of incrementing MeCN concentration.
  • the resulting 12 samples for each elution fraction were then run side-by-side on SDS PAGE gels, one gel representing all 12 serum samples at the specified acetonitrile concentration, and stained with Coomassie blue. This enabled the protein composition of each fraction to be compared across the full set of age/BMI matched serum samples.
  • the data from a PCOS control pair (Pl and C4) was not taken into account in the quantitative data analysis.
  • the second difference between the two sample groups was one of abundance change in components present in all samples at ⁇ 4OkDa in the
  • the differential bands identified in the RP-SPE/SDS PAGE analysis were excised, pooled, trypsinised and analysed using MALDI-TOF MS. Interrogation of the Swiss-Prot and TrEMBL databases using measured peptide ions and derived sequences identified potential matches for all three observed differences.
  • the ⁇ 33 kDa band ( Figure 5B) extracted from the 70% MeCN fraction gel generated six peptide ions characteristic of the complement component C4 ⁇ chain, tandem MS/MS product ion analysis confirmed this match.
  • the predicted MW of the C4 ⁇ chain, as calculated using the ExPASy primary structure analysis software, is ⁇ 33.5 kDa, which corresponds well with the observed band position following SDS PAGE.
  • the - 40 kDa band ( Figure 5B and 6A) extracted from the 42% MeCN fraction gel yielded three peptide ions characteristic of the haptoglobin ⁇ chain, of which two were subsequently analysed by tandem LC-MS/MS product ion analysis to confirm this match. A further three peptide ions matched the ⁇ region of the haptoglobin precursor protein.
  • the ⁇ 17 kDa band ( Figure 5C and 6B) extracted from the 20 % MeCN fraction gel yielded six peptide ions characteristic of the haptoglobin ⁇ chain.
  • Theoretical sequence derived molecular masses for the haptoglobin ⁇ chain sequence is 15.9 kDa, which corresponds well with the observed molecular weight following SDS PAGE, and for the ⁇ chain sequence is 27.3 kDa, with the precursor having a molecular weight of 43.6 kDa, which is a better match for the observed ⁇ 40 kDa band in the RP-SPE analysis.
  • the presence of these bands in multiple fractions from the RP- SPE columns suggests that the protein can be differentially modified (e.g. by glycosylation) and so exhibit different affinities for the column matrix.
  • ANN analysis identified an optimal subset of three and six biomarker ions for protein and peptide datasets respectively.
  • the three biomarker ions (2924, 3025 and 1977) identified from the digested peptide data successfully classified the independent validation subset of samples to an accuracy of 100 % ( Figure 7D) .

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Optics & Photonics (AREA)
  • Biophysics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
EP07824164A 2006-10-16 2007-10-15 Biomarker Withdrawn EP2076771A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB0620467A GB0620467D0 (en) 2006-10-16 2006-10-16 Biomarker
GB0708064A GB0708064D0 (en) 2007-04-26 2007-04-26 Biomarker
PCT/GB2007/003913 WO2008047086A2 (en) 2006-10-16 2007-10-15 Biomarker

Publications (1)

Publication Number Publication Date
EP2076771A2 true EP2076771A2 (de) 2009-07-08

Family

ID=38834488

Family Applications (1)

Application Number Title Priority Date Filing Date
EP07824164A Withdrawn EP2076771A2 (de) 2006-10-16 2007-10-15 Biomarker

Country Status (3)

Country Link
EP (1) EP2076771A2 (de)
JP (1) JP2010507093A (de)
WO (1) WO2008047086A2 (de)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2153234A2 (de) 2007-05-11 2010-02-17 The Institutes for Pharmaceutical Discovery, LLC Verfahren zur frühdiagnose einer nierenerkrankung
WO2013079560A1 (en) 2011-11-29 2013-06-06 Westfälische Wilhelms-Universität Münster Genetic marker for polycystic ovary syndrome (pcos)
JP5924587B2 (ja) * 2012-09-18 2016-05-25 国立大学法人 鹿児島大学 血中ｈｃｖ検出方法及び抗ｈｃｖ治療の効果判定方法
CN106442764B (zh) * 2016-08-31 2019-01-22 王义明 多囊卵巢综合征的诊断和/或分型标志物及制备试剂的用途
CN111148844A (zh) 2017-09-01 2020-05-12 韦恩生物科技股份公司 鉴定和使用糖肽作为诊断和治疗监测的生物标记物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2008047086A3 *

Also Published As

Publication number Publication date
WO2008047086A3 (en) 2008-07-17
JP2010507093A (ja) 2010-03-04
WO2008047086A2 (en) 2008-04-24

Similar Documents

Publication Publication Date Title
US12422433B2 (en) Blood biomarker that predicts persistent cognitive dysfunction after concussion
CN110286234B (zh) 尿液中妊娠糖尿病的蛋白质标志物及其在早期诊断中的用途
EP2657706B1 (de) Biomarker für krankheiten im zusammenhang mit kognitiver dysfunktion und verfahren zur erkennung von krankheiten im zusammenhang mit kognitiver dysfunktion mittels dieses biomarkers
SG173310A1 (en) Apolipoprotein fingerprinting technique
AU2010200352A1 (en) Proteomic analysis of biological fluids
CN101680884A (zh) 检测先兆子痫的方法
Roverso et al. A preliminary study on human placental tissue impaired by gestational diabetes: a comparison of gel-based versus gel-free proteomics approaches
Bradford et al. Analytical validation of protein biomarkers for risk of spontaneous preterm birth
US11740245B2 (en) Mass spectrometry-based methods for the detection of circulating histones H3 and H2B in plasma from sepsis or septic shock (SS) patients
EP2076771A2 (de) Biomarker
US8673644B2 (en) Serum markers for type II diabetes mellitus
WO2021009074A1 (en) Novel markers as early predictors of alzheimer's pathology
WO2014160237A2 (en) Methods of prognosing preeclampsia
EP2664923A2 (de) Biomarker für Erkrankungen des Zentralnervensystems
KR102475926B1 (ko) 전신 홍반성 루푸스 진단용 바이오마커 조성물 및 이를 이용한 전신 홍반성 루푸스 진단에 필요한 정보를 제공하는 방법
Yin et al. Analysis of the peptides detected in atopic dermatitis and various inflammatory diseases patients-derived sera
ST Lourenco et al. Proteomics-based technologies in the discovery of biomarkers for multiple sclerosis in the cerebrospinal fluid
RU2742801C1 (ru) Способ выявления диабетической фетопатии плода
US20060046276A1 (en) Acetyl-LDL receptor as a biomarker for breast cancer
Roverso et al. A preliminary study on human placental tissue impaired by gestational diabetes: a comparison of gel-based vs. gel-free proteomics approaches
Pulinx et al. Biomarker discovery in MScl

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20090514

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20100125

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20100605