JP2010507093A - Biomarker - Google Patents

Abstract

Following steps;
(i) provide a sample of material obtained from the subject;
(ii) one or more postscript proteins in the sample:
Complement C4 precursor protein complement,
A cleavage fragment of the C4 precursor protein, and haptoglobin,
Determining the level of
(iii) comparing the determined level to one or more reference values;
A method of determining a subject's polycystic ovary syndrome (PCOS) status comprising.

Description

  The present invention relates to novel biomarkers for polycystic ovary syndrome (PCOS) and methods of determining a subject's PCOS status using these novel biomarkers.

  Women with polycystic ovary syndrome (PCOS) often show a wide range of signs to clinicians, the most common symptoms being irregular menstruation and / or acne, hirsutism and androgenic alopecia Difficult pregnancy / infertility with problems associated with hyperandrogenism including Many of these women are obese and may increase the risk of diabetes, heart disease and endometrial cancer in later years. PCOS is thought to affect 5-10% women of reproductive age in U.K. Stein and Leventhal et al. First characterized the phenotype of PCOS expression in 1935, and since then significant advances have gained an understanding of pathophysiology, diagnosis, clinical presentation and treatment. Despite these advances, there are still no diagnostic markers available consistently, and it remains a challenge to develop effective treatments for this multifaceted syndrome.

  There are currently three common and updated hypotheses involved in the pathogenesis of PCOS, which relate to the following: (i) the hypothalamus-pituitary-adrenocortical system, (ii) insulin resistance and (iii) androgens level. For example, the endocrinological basis for PCOS may be attributed to the hypothalamus that increases the pulse frequency of luteinizing hormone (LH) released from the pituitary gland due to increased release of gonadotropin-releasing hormone. Elevated LH levels are thought to increase androgen production by ovarian capsular cells, ie increase circulating levels of free testosterone in affected women. However, LH hypersecretion is not common in all subjects diagnosed with PCOS. In other cases, insulin resistance inhibits the synthesis of sex hormone binding globulin (SHBG) and acts synergistically with LH to directly stimulate androgen production in capsular cells, thereby producing androgen production. Is understood to increase. However, the lack of precise etiology for insulin resistance in PCOS and the presence of selective ovarian sensitivity in the presence of peripheral insulin resistance further poses a challenge for PCOS. Hyperandrogenemia has been found to be the basis for all other signs of PCOS, and the primary cause and molecular mechanism for hyperandrogenemia is not clear at all.

  Current diagnosis of PCOS excludes other androgenemia-based diseases and follows clinical identification of at least two of the following three criteria: (i) chronic under-ovulation / anovulation (ii) high Clinical and / or biochemical signs of androgenemia and (iii) ultrasound evidence of polycystic ovaries. However, the relative specific gravity of each criterion tends to change depending on the medical background of the clinician and also by emphasizing the clinical challenges of PCOS. The treatment methods also vary depending on the characteristics presented by the subject and the desired outcome, and change to symptom-based methods for gynecological or cosmetic improvements or simply weight management and lifestyle changes.

  Thus, there remains a need for a simple, rapid, reliable and reproducible method for diagnosing PCOS and a suitable new therapeutic target for the treatment / mitigation of the disease.

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 a subject's PCOS status.

According to a first aspect, the present invention provides the following steps:
(a) provide a sample of material obtained from the subject;
(b) one or more postscript proteins in the sample;
Determining the levels of complement C4 precursor protein, cleavage fragments of complement C4 precursor protein and haptoglobin, and
(c) comparing the determined level to one or more reference values;
A method of determining a subject's polycystic ovary syndrome (PCOS) status.

  Preferably, the method can diagnose a subject's PCOS from an analysis of proteins in a sample provided by the subject.

  Complement C4 precursor protein, cleavage fragments of complement C4 precursor protein and haptoglobin are identified as biomarkers for PCOS and are considered herein as the biomarker or the biomarker protein.

  These biomarkers can be used to determine a subject's PCOS status, eg, to diagnose PCOS. The term “PCOS condition” includes any distinguishable symptom of PCOS disease, including disease and non-disease. For example, a PCOS condition includes the presence or absence of a disease, the risk of disease progression, the stage of the disease, the progression of the disease (e.g., disease progression over time or disease alleviation) and the subject's effect on treatment of the disease or Including, but not limited to, responses.

  Usually, the method of the invention is used in combination with the evaluation of clinical signs.

C4 precursor protein is a component of the complement pathway and an important participant in the immune response system. C4 plays a central role in the classical pathway of the complement system. The DNA sequence of C4 can be found as two isoforms in the GenBank database, these are isoform A (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotid&val=67190747 ) For accession number NM007293 and isoform B (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=50345295) are available under accession number NM001002029. Corresponding proteins are found at accession numbers POCOL4 and POCOL5 (protein sequence as C4 precursor is http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotid & val = 67190747 The corresponding DNA sequence can be found at http://www.ebi.ac.uk/cgi-bin/expasyfetch?K02403). The protein sequence for each isoform is shown in FIGS.

  The terms “C4 protein” and “C4 precursor protein” refer to the same protein and are used interchangeably herein.

One or more cleavage fragments of the complement C4 precursor protein can include the following polypeptides:
a) C4α
b) C4β
c) C4γ
d) C4a Anaphylatoxin
e) C4b
f) C4α ₃ c fragment
g) C4αd
h) C4α ₄ c fragment and
i) at least 75% amino acid sequence identity, more preferably at least 80%, 85%, 90% or 95% or more than 95% sequence homology to any amino acid sequence of a) to h) Polypeptide,
Can be selected from any of the groups comprising

  Preferably at least one or said fragment a) to i) is detected in the method of the invention, more preferably at least 2, 3, 4, 5, 6, 7, 8, or 9 fragment a) to i. ) Is detected.

  The amino acid sequences of cleavage fragments a) to h) can be found with reference to FIGS.

  The one or more haptoglobin proteins can be selected from the group comprising haptoglobin alpha and haptoglobin beta. The amino acid sequences of both haptoglobins are shown in FIG.

  Proteins are frequently present in the body and in samples obtained from the body in several different forms. These forms can be obtained from either or both pre- and post-translational modifications. The ability to distinguish between different forms of a protein when detecting or determining the level of the protein in a sample depends on the nature of the difference and the method used to detect or measure the protein level. . For example, an immunoassay using a monoclonal antibody detects all forms of the protein containing the epitope, but does not distinguish between them. However, sandwich immunoassays that use two antibodies against different epitopes on the protein detect all forms of the protein that contain both epitopes, but not those forms that contain only one of the epitopes.

  In the methods of the invention, the assay method used to determine the level of one or more biomarker proteins preferably detects all forms of a specific biomarker protein. Preferably, at least all biologically active forms of the specific biomarker protein are detected. Preferably all of the biomarker proteins having at least 75% identity, preferably at least 80%, 85%, 90%, 95% identity or more with the amino acid sequences of the biomarkers shown in FIGS. Are detected in the method of the present invention.

  Methods for determining polypeptide / protein identity are well known to those of skill in the art. For example, the UWGCG Package provides a BESTFIT program that can be used to calculate identity (eg, used with its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).

  PILEUP and BLAST algorithms can also be used to calculate homology or align sequences (usually against its default settings), for example, Altschul SF (1993) J Mol Evol 36: 290-300 Altschul , S, F et al (1990) J Mol Biol 215: 403-10.

  Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

  The method of the present invention preferably enables detection of biomarker protein levels having polymorphisms common to the general population. Polymorphisms may occur that do not affect the function of the protein, and the method of the present invention is intended to allow these to be detected.

  Sample material obtained from a subject may include whole blood, serum, plasma, urine, adipose tissue (fat), endometrial tissue, ovarian tissue or other body fluid or tissue.

  Levels of one or more complement C4 precursor proteins, cleavage fragments of complement C4 precursor proteins, and haptoglobin are determined by immunoassay, spectroscopy, mass spectrometry, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectroscopy Analysis, microscope, Northern blot, Western blot, Southern blot, isoelectric focusing, SDS-PAGE, PCR, RT-PCR, electrophoresis, protein microarray, DNA microarray and antibody microarray, or any combination thereof Can be determined by any suitable assay. Preferably, the level of the one or more biomarkers is determined using an immunoassay. An immunoassay uses an antibody or antibodies against a specific antigen to determine the level of the antigen. In this case, one or more antibodies specific for one or more C4 precursor proteins, cleavage fragments of C4 precursor proteins or haptoglobin can 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 scattered light assay.

  The one or more antibodies may be synthetic monoclonal, polyclonal, bispecific, chimeric or humanized antibodies. Chimeric antibodies can include portions obtained from various animals. A humanized antibody is an antibody derived from a non-human species having one or more complementarity determining regions derived from a non-human species and a framework region derived from a human immunoglobulin molecule. Chimeric and humanized antibodies can be produced by recombinant techniques known to those skilled in the art.

  The one or more antibodies comprise a tag or label selected from the group comprising radioactive, fluorescent, chemiluminescent, dye, enzyme or histidine tags or labels, or other suitable labels or tags known to those skilled in the art. obtain.

  The presence and optionally the level of a particular protein in a sample can also be determined by using mass spectrometry techniques. Mass spectrometry techniques can be used to detect gas phase ions that correlate with specific proteins or portions of proteins, such as tryptic peptides. Examples of mass spectrometers include time-of-flight, magnetic sector, quadruple filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and combinations thereof.

  The mass spectrometer may use laser desorption / ionization.

  MALDI (Matrix Assisted Laser Desorption / Ionization) peptide mass fingerprints can be used to identify the presence of specific biomarker proteins from 2D gel analysis and RPE / SDS PAGE analysis.

  MALDI-MS and ANN analysis can be used to profile protein and trypsin biomarker characteristics identified for PCOS.

  Preferably, a reference value comparing the determined level of one or more complement C4 precursor protein, a cleavage fragment of complement C4 precursor protein and haptoglobin was observed in a subject without any clinical symptoms of PCOS Level, so-called “normal value”.

  Alternatively, the reference value may be an early value obtained for the specific object. This type of reference value can be used if the method monitors disease progression or the subject's response to a particular treatment.

  When comparing the determined levels of one or more complement C4 precursor proteins, cleaved fragments of complement C4 precursor proteins, and haptoglobin to a reference value, an increase or decrease in the level of one or more biomarker proteins is the target PCOS It can be an indicator of the condition.

More specifically, increase of C4 [alpha] ₃ c levels and / or C4 [alpha] ₄ c levels and / or haptoglobin α and / or levels of haptoglobin β may be characterized for use in or diagnosis is indicative of PCOS.

  Reduced levels of C4 precursor protein and / or C4α and / or C4β and / or C4γ and / or C4a anaphylatoxin may be an indicator of PCOS or a characteristic used for diagnosis.

  The methods of the invention can also be used to monitor disease progression and / or monitor the effectiveness of treatment given to a subject. This can be accomplished by analyzing samples taken from the subject at various time points after the initial diagnosis, monitoring changes in biomarker levels, and comparing these levels to reference values.

  In this case, the reference level may include the first level of the biomarker in the subject, or the level of the biomarker in the subject when last tested, or both.

  Preferably, the method of the present invention is performed in vitro.

  The subject may be a mammal, preferably a human, but may be a monkey, ape, cat, dog, cow, horse, rabbit or rodent.

According to other embodiments, the present invention provides one or more complement C4 precursor proteins for use as a biomarker or as part of a panel of biomarkers, to determine a subject's PCOS status, Cleaved fragments of complement C4 precursor protein and haptoglobin are provided. Preferably, the panel of biomarkers is selected from the group comprising C4 precursor protein, C4α, C4β, C4γ, C4a anaphylatoxin, C4b, C4α ₃ c fragment, C4αd, C4α ₄ c fragment, haptoglobin α and haptoglobin β Contains at least 2, 3, 4, 5 or 5 or more biomarkers.

According to another aspect of the present invention, the following steps:
(a) provide a sample of material obtained from the subject;
(b) analyzing the protein or trypsin peptide obtained from the sample using MALDI-MS,
(c) The presence of six ions m / z 8674, 8668, 1351, 8727, 8673, and 6871 in a protein sample and / or the presence of three ions m / z 2924, 3025, and 1977 in a tryptic peptide sample Determining that it is a feature used in the diagnosis of PCOS in
A method for determining a PCOS status in a subject, comprising:

  Six ions m / z 8674, 8668, 1351, 8727, 8673 and 6871, and three ions m / z 2924, 3025 and 1977 are biomarkers for PCOS.

  According to another embodiment of the present invention, the presence of six ions m / z 8674, 8668, 1351, 8727, 8673 and 6871 in a protein sample and / or three ions m / z 2924, 3025 and 1977 in a tryptic peptide sample A kit is provided for use in determining a subject's PCOS status, including instructions for analyzing a sample for the presence of.

  In accordance with another aspect of the invention, determining a subject's PCOS status comprising one or more complement C4 precursor proteins, cleavage fragments of complement C4 precursor proteins, and at least one reagent for determining the level of haptoglobin. Provide kits for use in cases.

  The reagent can be an enzyme, antibody, nucleic acid, protein probe or other suitable composition.

  Preferably, the reagent for determining the level of one or more PCOS biomarkers is labeled. The kit can also include a means for detecting the label.

  The kit can include one or more capture reagents for capturing one or more biomarker protein levels to be determined. The capture reagent may be one or more antibodies.

  A capture reagent or reagent for determining the level of one or more PCOS biomarkers may be bound to the solid support. The solid support can be a chip, microtiter plate, bead or resin.

  The kit can also include a wash solution or instructions for making the wash solution. The wash solution allows effective capture of a biomarker or multiple biomarkers on a solid support for continuous detection, for example by mass spectrometry or immunoassay methods, alone or in combination with a capture reagent Become.

  The kit may include instructions for suitable operational parameters in the form of a label or another insert. The instructions may provide the purchaser with information about how to collect the sample and / or how to wash the capture reagent and / or details of a particular biomarker to be detected.

  The kit may contain one or more biomarker samples to be used as standards for calculations and comparisons.

  In accordance with yet another aspect, the present invention relates to C4 precursor protein and / or C4 precursor protein cleavage fragments and with or without detection of other PCOS biomarkers as a means of assessing an individual's PCOS status. The use of haptoglobin chain level determination is provided.

  According to a further aspect, the present invention provides a probe pair capable of detecting one or more of the PCOS biomarkers. Those skilled in the art will fully understand how to design suitable probes (eg, which may be synthetic molecules, antibodies, nucleic acids or natural protein analogs). The probe may carry one or more labels to facilitate its detection; the label can be, for example, a radioactive, chemiluminescent or fluorescent label, but is not limited to these examples. Absent. Preferably, the probe pair comprises at least two probes for different biomarker proteins.

  In accordance with another aspect, the present invention provides a method of treating a PCOS subject, comprising administering to the subject a reagent capable of modulating the level of one or more PCOS biomarkers in a cell. Wherein the one or more biomarkers are selected from the group comprising complement C4 precursor protein, a cleavage fragment of complement C4 precursor protein, and haptoglobin. The reagent can be an antisense or interfering RNA molecule designed to inhibit or reduce the expression of a biomarker protein in a cell.

  Alternatively, the reagent can be an inhibitor of C4 precursor protein processing or a composition that reduces complement activity.

According to a further embodiment, the present invention comprises the following steps
(a) one or more of the following proteins in the sample obtained from the subject at the first time point: complement C4 precursor protein, cleavage fragment of complement C4 precursor protein and haptoglobin,
Determining the level of
(b) determining the level of one or more of the following proteins: complement C4 precursor protein, cleaved fragment of complement C4 precursor protein and haptoglobin in a sample taken from the subject at a later second time point; and
(c) comparing the first measured value and the second measured value, where the comparative measurement indicates the course of PCOS;
Comprising
A method of determining the progression of PCOS in a subject or determining the effect of treatment given to a PCOS subject is provided.

  Using this method, samples taken at two or more time points can be compared.

  According to a further aspect, the invention screens one or more complement C4 precursor proteins, cleavage fragments of complement C4 precursor proteins and one or more compounds that alter the level of haptoglobin in vitro or in vivo. A method of identifying a compound for treating PCOS.

  Compounds suitable for therapeutic testing can be initially screened by identifying compounds that interact with one or more PCOS biomarkers listed herein. As an example, the screening can include recombinantly expressing the biomarker, purifying the biomarker, and attaching the biomarker to a substrate. Usually, the test compound is then contacted with the substrate under aqueous conditions, and the interaction of the test compound and biomarker can be measured, for example, by measuring the elution rate as a function of salt concentration. A particular protein can recognize and cleave one or more biomarkers, in which case the protein monitors the digestion of one or more biomarkers by standard assays (eg, protein electrophoresis). Can be detected.

  In related embodiments, the ability of a test compound to inhibit the activity of one or more biomarkers can be measured. As will be appreciated by those skilled in the art, the technique used to measure the activity of a particular biomarker will vary depending on the function and properties of the biomarker. For example, the enzymatic activity of a biomarker can be assayed under conditions where an appropriate substrate is available and where the concentration of the substrate or the generation of reaction products can be readily measured. The ability of a therapeutic test compound that may inhibit or enhance the activity of a given biomarker can be determined by measuring the rate of the catalyst in the presence or absence of the test compound.

  A test compound that can alter the activity of any PCOS biomarker can be administered to a subject with or at risk of developing PCOS. For example, if the activity of a specific biomarker in vivo inhibits the accumulation of a protein that causes PCOS, reducing the risk of PCOS in the subject by administering a test compound that increases the activity of the specific biomarker Can do. Conversely, if the increased activity of the biomarker is at least partially involved in the pathogenesis of PCOS, administration of a test compound that reduces the activity of a particular biomarker can reduce the risk of PCOS in the subject. .

Still further embodiments include C4 precursor protein, C4α, C4β, C4γ, C4a anaphylatoxin, C4b, C4α ₃ c fragment, C4αd, C4α ₄ c fragment, haptoglobin α and haptoglobin as therapeutic targets for treatment / relief of PCOS Use of β and / or other PCOS biomarkers is provided.

  One skilled in the art will recognize that one preferred feature in any of the embodiments and / or aspects of the invention may be applied to all other embodiments and / or aspects of the invention.

  The invention will be further described in more detail, by way of example only, with reference to the following figures.

FIG. 1 shows the results of two-dimensional gel electrophoresis showing the change in protein level between PCOS subjects and corresponding control subjects. The top 10 images are silver-stained 2D gels, with the protein spots of interest in the gels marked in circles, and the protein between the PCOS target and the control age / BMI target. Differences in levels can be seen. The five images at the bottom were created using DELTA 2D image Analysis software and are overlays of corresponding pairs of gel images showing the same protein spot in the colorimetric display. Although no color development is seen, the results show that this observed spot appeared in 4 out of 5 gel pairs superimposed with an orange colored image indicating that it only occurs in PCOS objects. The spot was subsequently identified as C4α ₃ c.

FIG. 2 shows a quantitative analysis of the intensity of the C4α ₃ c spot of an age-weight matched PCOS subject-control subject gel pair. Data was calculated using DELTA 2D image analysis software.

FIG. 3 shows a Western blot analysis of 10 serum-depleted samples using antibodies raised against the unknown C4α ₄ c peptide sequence. FIG. 3A shows a Western blot analysis of whole sera from the same sample pair used in the 2DE test. The blot was probed with a polyclonal anti-C4α antibody raised against an epitope within the C4α chain. The ~24kDa band shown, illustrating the C4 [alpha] ₄ c fragment. FIG. 3B shows a quantitative analysis of the band intensity of FIG. 3A determined using SynGene Gene Tools densitometry software. * P = 0.03.

FIG. 4 shows a diagrammatic representation of the primary structure for complement component C4 protein. The C4 precursor and many of its cleavage fragments are shown. C4 protein has been shown to be composed of three chains, which can all be cleaved from one C4 precursor and folded to form the functional structure of the protein. The α chain is an active element when the cleaved C4a fragment is released and acts as an anaphylatoxin. The C4b fragment forms a complex with C2 to continue the classical complement pathway enzymatic cascade and may also act as an opsonin. C4b is later cleaved into C4α3c, C4αd and C4α4c fragments, which inactivate the complement cascade (Nagasawa et al., (1980) J Immunol 125 (2): 578-82 and Hessing, (1991) Biochem J 277: 581 92). Note: † C4α ₃ c fragment identified by 2DE analysis as being up-regulated in PCOS sample. ‡ C4α ₄ c fragment shown by Western blot to be upregulated in PCOS. * C4γ chain identified by SDS PAGE analysis of the RP-SPE fraction as being down-regulated in PCOS.

  FIG. 5 shows an RP-SPE analysis showing the difference between PCOS and the corresponding control. FIG. 5A shows the ~ 33 kDa band present in the 5/6 control sample, and very low levels in the 3/6 PCOS sample versus fraction 12 only. There may be similar sized bands in fractions 4 and 5, but this is thought to be a different protein type. FIG. 5B shows the ˜40 kDa band present in all PCOS samples at an increased level than is present in the control sample. This difference was most noticeable in fractions 8-12. FIG. 5C shows the ˜17 kDa band present in the 5/6 PCOS sample and the only 2/6 control sample.

  FIG. 6 shows a quantitative RP-SPE analysis showing the difference between PCOS and the corresponding control. FIG. 6A shows the results of densitometry quantification of the ˜40 Kda band seen with RP-SPE (FIG. 5B). P = 0.001. FIG. 6B shows the results of quantification of the ˜17 Kda band seen with RP-SPE (FIG. 5C) by densitometry.

FIG. 7 shows a representative MALDI-TOF mass spectrum:
FIG. 7A is the serum peptide after trypsin digestion, and FIG. 7B is the C ₁₈ ZipTip cleanup protein from PCOS subject and control sera. This spectrum shows the differences observed between PCOS serum and control serum. FIG. 7C shows ANN's predictive ability to evaluate tryptic peptide profiles based on three ion models, and FIG. 7D shows protein profiles based on six ion ANN models. Gray bars represent control samples and black bars represent PCOS samples. A predicted value of 1.5 or less indicates a control sample, while a predicted value of 1.5 or more indicates a PCOS sample.

FIG. 8 (SEQ ID NO: 1) is the protein sequence of the isoform A C4 precursor protein. More specifically, amino acids 20-675 are C4β cleavage fragments, amino acids 680-1446 are C4α cleavage fragments, amino acids 680-756 are C4a anaphylatoxin cleavage fragments, and amino acids 757-1446 are C4b cleavage fragments. And amino acids 757-956 are C4α ₃ c cleavage fragments, amino acids 957-1333 are C4αd cleavage fragments, amino acids 1334-1446 are C4α ₄ c cleavage fragments, and amino acids 1445-1744 are C4γ cleavage fragments. .

  FIG. 9 (SEQ ID NO: 2) is the protein sequence of the isoform B C4 precursor protein. Amino acid differences from isoform A are underlined.

  FIG. 10 (SEQ ID NO: 3) is the protein sequence of haptoglobin. Amino acids 19 to 161 are haptoglobin α, and amino acids 162 to 406 are haptoglobin β.

Materials and Methods Subjects This study consisted of 11 women diagnosed with PCOS (age 18-40) and 10 controls matched by their age-BMI. Infertility, gynecology or PCOS clinics operated by the Queen's Medical Center, Nottingham, first identified women with PCOS and then orally invited them to participate in the study. Control women without PCOS were recruited from the same gynecological clinic as women with PCOS and from female staff in poster advertisements. Ethical approval of this study was granted by the Nottingham Local Research Ethics Committee and informed consent was obtained from each subject.

Clinical evaluation In addition to menstruation, gynecology, obstetrics and other medical history, details of the subject group formation layer were collected for each subject during the clinical interview. A regular menstrual cycle was defined as between 21 and 35 days. The presence of acne and hirsutism was assessed and hirsutism was defined as a Ferriman Gallwey Score> 7 (Ferriman and Gallwey, (1961) J Clin Endocrinol Metab 21: 1440-7). The subject was excluded from this study if there was a history of thyroid disease, hyperprolactinemia, recent birth, miscarriage or surgery, history of myocardial infarction, thrombus or blood disease, or current use of sex steroid therapy . Various body measurements were also made during this interview. Height and weight measurements were taken, body mass index (BMI) was calculated, waist and hip cm were measured, and blood pressure was measured using a sphygmomanometer band of the appropriate size for the subject in the sitting position.

  Following clinical interviews, fasting blood samples were collected for each woman. For all control women, samples were taken during the follicular phase of the menstrual cycle, if possible, in women with PCOS. Transvaginal pelvic ultrasound was also performed. Ultrasound scanning and clinical chemistry departments are not informed of each subject's clinical diagnosis. 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 endocrine to determine blood glucose, insulin, lipid profiles (triglycerides, total cholesterol and HDL cholesterol), and serum hormone profiles (testosterone, SHBG, luteinizing hormone (LH), follicle stimulating hormone (FSH) , 17-hydroxyprogesterone and prolactin).

  Clinical use of Rotterdam criteria (Rotterdam ESHRE / ASRM Sponsored PCOS Consensus Workshop (2004) Fertil Steil 81 (1): 19-25) for more than 2 hypoovulation / anovulation, polycystic ovary and hyperandrogenemia A subject is diagnosed with PCOS when experimental and / or biochemical signs are presented. Hyperandrogenemia is clinically defined by hirsutism (Ferriman Gallwey score> 7) and biochemically elevated testosterone (> 2 nmol / L) and / or elevated free androgen index (> 10) (Testosterone / SHBG x 100) Control subjects have a regular 21-35 day menstrual cycle and no multicystic ovarian ultrasound evidence or evidence of hyperandrogenemia.

Sample Preparation Serum samples were collected simultaneously from 21 subjects over a 12 month period. The sample is separated from whole blood coagulated in SST tubes by centrifugation at 4,000 rpm for 10 minutes at 4 ° C within 1 hour of collection (obtained in parallel with the sample for biochemical testing) Stored at −80 ° C. until To keep the samples anonymous, they were numbered sequentially within each group and labeled appropriately as P- for PCOS samples and C- for controls. PCOS and control women with highly similar ages and BMI were matched in sample pairs.

Serum depletion and sample purification For initial testing, serum samples (15 μl) from PCOS-control pairs with 5 age-BMI were obtained from the manufacturer's protocol (Alubumin & IgG Removal Kit Amersham Biosciences, Little Chalfont, Albumin and immunoglobulin G (IgG) were simultaneously depleted using antibody-based columns according to Bucks, UK). The resulting sample (˜500 μl) was subjected to protein precipitation according to the manufacturer's protocol (2D clean-up kit Amersham Biosciences). The protein precipitate was then lysed with a solubilization buffer [7M urea (Sigma, Poole, UK) at room temperature to a final concentration of 5 μg / μl as determined by a modified Bradford assay (Quick Start Protein Assay BioRad). , 2M thiourea (Sigma), 4% w / v 3- (cyclohexylamino) -1-propanesulfonic acid (CHAPS Amersham Biosciences), 10 mM dithiothreitol (DTT Fisher Scientific, Loughborough, Leics, UK), 1% v / v BioLyte carrier ampholytes 3-10 (BioRad, Hercules, CA, USA)].

Isoelectric focusing (IEF)
IEF was performed using PROTEAN IEF cells (BioRad). ReadyStrip® ⁽ 11 cm) immobilized pH gradient (IPG) strip pH 3-10 NL (BioRad), IEF buffer (188 μl) [7M urea, 2M thiourea, 4% w / v CHAPS, 100 mM Rehydration was performed at 20 ° C. for 16 hours using bis (2-hydroxyethyl) disulfide (HED DeStreak reagent, Amersham Biosciences), 0.5% v / v BioLyte carrier ampholytes 3-10]. After strip rehydration, a sample (60 μg total protein) (12 μl) was placed at the anode end of each strip. The optimized IEF program was then applied at 20 ° C. for 15 minutes at a voltage of 250 V, linearly raised to 8,000 V over 2.5 hours and focused at 8,000 V as 50 kVh. All 10 serum-depleted and purified samples were fractionated simultaneously and the strips were stored individually at -20 ° C until needed.

Two-dimensional SDS-polyacrylamide electrophoresis (SDS-PAGE)
Dissolve each age and BMI-matched PCOS-control pair sample strip generated by IEF and equilibrate with 0.3% Tris.HCl (pH 8.8) containing 2% w / v DTT for the first 15 minutes at room temperature. Invitrogen, Paisley, UK), 6M urea, 2% w / v sodium dodecyl sulfate (SDS Fisher Scientific), 30% v / v glycerol (Sigma)], then 2.5% w / v iodoacetamide (15% for 15 minutes) Equilibrated in equilibration buffer containing (Sigma). SDS-PAGE was performed at the top of the gel using 1% w / v low melting agarose (BioRad) made with 1x gel running buffer [0.25M Tris, 1.86M glycine (Fisher Scientific), 0.1% w / v SDS]. Were performed on a uniform 13% acrylamide gel or 8-20% acrylamide gradient gel with a sealed equilibration strip. Electrophoretic separation of the two strips was performed equilibrated and performed at room temperature for 3.5 hours at 22 mA per gel. Using a modified silver staining method (Plus One Silver Stain Kit Amersham Biosciences) that is compatible with mass spectrometry (Yan et al., 2000) with a color development step performed at 4 ° C to improve the spot analysis of proteins in the gel And stained. Stained gel imaging was performed using Agfa Fotolook v3.0 and Duoscan T1200 flatbed scanner followed by qualitative and quantitative analysis using DELTA 2D image analysis software (v3.3 Decodon, Greifswald, Germany). Stained gels were stored in dH ₂ O at room temperature.

Western blotting Equal volume (50 μg) of diluted serum sample (1:10 in dH ₂ O) from the first 10 samples is diluted 1: 1 with gel running buffer and at 95 ° C for 7 min Denatured by heating. Samples were run on a 5-20% acrylamide SDS-PAGE gel and separated by electrophoresis at 35 mA per gel for 2.5 hours. The gel protein was then blotted overnight at 40 mA and transferred to a nitroserose membrane. The membrane was washed with 5% w / v skim milk (Marvel, 10 mM Tris.HCl (pH 7.5), 150 mM sodium chloride (Fisher Scientific), 0.05% (v / v) Tween-20 (Sigma)). Premier International Foods (UK) Ltd, Lincs, UK) and polyclonal primary antibody raised against the complement component C4α chain (Santa Cruz, Heidelberg, Germany) followed by horseradish peroxidase-conjugated secondary antibody ( Dako, Ely, Cambs, UK). Antibody binding was detected by chemiluminescence using an ECL detection kit (Amersham Biosciences).

RP-SPE and SDS PAGE
Serum fraction
Serum fractionation of 6 PCOS and 6 control samples was performed in reverse phase mode (RP) with large pore (1000 Å) polystyrene divinylbenzene (PDVB) resin (International Sorbent Technologies, mid-Glamorgan, UK) (25 mg ) Using solid phase extraction (SPE). A mass spectrometry grade mobile phase (Riedel de Haean, Sigma) is flowed with a volume adjusted delivery composition (1 mL) using a compatible vacuum at a flow rate on the order of 1 mL / min. The resin was solvated using 70% v / v acetonitrile (MeCN, Sigma), 0.1% v / v trifluoroacetic acid (TFA, Sigma) and equilibrated with 0.1% aqueous TFA. Serum samples from PCOS-control pairs to which age-BMI corresponds (100 μl corresponds to ˜7 mg total protein) were layered on a solid phase column. Sample protein binding depends on the hydrophobicity of the molecule (Badock et al., 2001). The column was then washed with 0.1% v / v TFA and the retentate was eluted in multiple fractions with a step increase in MeCN composition from 5 to 100% v / v. The eluate can be directly applied for analysis by electrospray or MALDI-TOF mass spectrometry, but in this example it was dried by centrifugal vacuum evaporation after electrophoretic analysis (BETA-RVC, Christ Gefriertrocknungsanlagen Gmbh, Osterode am Harz, Germany) 3 hours at room temperature, gel running 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)] (100 μl). Samples were stored at −20 ° C. in gel running buffer.

SDS-PAGE
An equal volume (5 μl) of each serum sample fraction in gel running buffer was denatured by heating at 95 ° C. for 7 minutes. Samples were layered onto pre-formed 4-12% acrylamide bis-Tris SDS-PAGE gels (Invitrogen) and electrophoresed in 3-morpholinopropane sulfonic acid (MOPS Invitrogen) at 200 mA per gel for 2.5 hours. separated. Proteins in the gel were filtered for 2.5 hours with filtered Komaji blue (50% v / v methanol (Fisher Scientific), 20% v / v acetic acid (Fisher Scientific), 0.12% w / v brilliant blue R250 (Sigma)). And then decolorized to the desired intensity in a 10% v / v acetic acid / 10% v / v methanol solution. The stained gel was imaged 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.

Peptide Mass Fingerprints and Database Search Individual spots with significantly different expression profiles in PCOS samples when compared to controls were excised from the stained gel and destained. The protein was trypsin digested in the gel and subjected to matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) to obtain a mass fingerprint of the peptide. Submit a single isotope m / z value for trypsin peptide ions to Aldente software (http://www.expasy.org/tools/aldente/), search in the Human Swiss-Prot and TrEMBL databases, The protein could be identified.

Statistical analysis Differences between control and PCOS groups were assessed using paired sample Student's t test. A two-sided p-value < 0.05 was considered statistically significant.

MALDI Mass Spectrometry Sample Preparation Protein and Peptide Analysis Sample preparations were randomized prior to sample handling and a complete set of 11 PCOS and 10 control serum samples were analyzed. An identical aliquot of serum diluted 1/10 with 0.1% TFA was used for protein and tryptic peptide analysis. Diluted serum (25 μl) was C ₁₈ ZipTip fractionated according to the manufacturer's instructions using the Xcise robotic system (Proteome Systems, Shimadzu, UK), and the eluted protein / peptide was separated from the robotic system (robotic system) was spotted with SA (10 mg / ml) on MALDI-TOF MS target plates. Residual eluate was recommended for trypsin digestion. Briefly, fractionated serum samples were incubated overnight at 37 ° C. in combination with ammonium bicarbonate (100 mM 16.6 μL), water (7.6 μL) and trypsin (1.3 μL of 0.5 μg / μL). The reaction was stopped and the sample was cleaned using a C ₁₈ ZipTip according to the manufacturer's instructions and dry droplets with CHCA (10 mg / ml solution in 50% ACN + 0.1% TFA, LaserBio Labs, Cedex, France). Method was used to spot on MALDI target. Target plates were analyzed using AXIMA-CFR + MALDI-TOF MS (Shimadzu, Manchester, UK) in linear mode with the raster option for proteins and in reflectron and autoquality modes for tryptic peptides. Bovine serum albumin (BSA) control was used to verify the efficiency of the digestion procedure and 0.1% TFA blank was used to ensure there was no contamination from reagents and plates. Close external calibration was performed on peptides (Laser Biolabs, Cedex, France) using protein calibration Proteomix3 and Proteomix2.

  The resulting mass spectra were all visually examined and rejected outliers (with a weak signal to noise) were removed from the data set prior to bioinformatics processing.

ANN (Artificial Neural Network) analysis Data processing: Rough mass spectrum data (m / z, intensity) obtained from MALDI-MS is saved as ASCII file, and target mass range (m / z 1000-25000) Smoothed and rounded mass and strength were obtained. These intensity values were then generated using Statistica 7.0 (Stat Soft Inc. Tulsa, USA) as input data for the ANN model. This model was used to predict the constituents of each sample for one of two output classes: control (1) or PCOS (2).

  Model building: Model building and network parameters are described in Lancashire et al (2005) Bioinformatics 21 (10): 2191-9. Prior to training, the samples were randomly divided into three subsets: training (60%, n = 14), trial (20%, n = 5) and validation (20%, n = 5). Training was performed using 50 randomly sampled data partitions, along with a validation subset used to independently evaluate this model against blind data at each time point to ensure that no overfitting occurred. did. Sensitivity analysis was performed to identify the input values that had the greatest impact on the model system for classification performance. These input values were then subjected to further training. This involves using a stepwise method that allows identification of the optimal subset of biomarkers for class discrimination. The procedure was discontinued if there were no further significant improvements for the 3 steps (independent addition of ions to the optimal subset) for an independent validation set of data (p = <0.001).

Results The 21 women analyzed in this study were recruited from the same hospital clinic and were defined as PCOS or controls using the Rotterdam criteria. Although there were differences in both the control and PCOS groups, age and BMI range corresponded similarly. The biological, clinical and biochemical profiles for women with PCOS were consistent with the current consensus for PCOS subjects in the literature. Table 1 shows elevated hirsutism mean score, testosterone level, free androgen index (FAI) and LH, along with a low mean level of SHBG, in a sample pair of 5 PCOS affected women compared to 5 control subjects. Indicates the / FSH ratio. Consistent with the consensus, PCOS women in this study on average had higher follicle counts and increased ovarian volume compared to control women. However, the variability between individuals within each group is large, which reflects the variability of the phenotypic population in both normal and PCOS women. The only significant difference (P <0.05) was found at SHBG levels, which was found to be significantly lower in PCOS women than in control women, and FAI levels were increased in PCOS women. However, when studying a larger subject cohort from the same clinic (11 PCOS and 10 controls) (data not shown), increased testosterone levels, LH / FSH ratio, hirsutism score, Significant differences were observed for follicle number and ovary volume.

注：PCOS -多嚢胞性卵巣症候群; BMI =ボディマス指数; WHR = ウエスト(cm)/ヒップ(cm)割合; F-G = Ferriman & Gallwey; T = テルトステロン; SHBG = 性ホルモン結合グロブリン; FAI = 遊離アンドロゲンインデックス; LH = 黄体形成ホルモン(IU/L); FSH = 卵胞刺激ホルモン (IU/L).
・＝ PCOSおよびコントロール群間の統計学的有意差
・一人のコントロール女性は、14のFerriman Galweyスコアを示したが、コントロールと一致した規則的な月経周期および超音波の特徴を有した。相違するコントロールは、正常なアンドロゲンおよび規則的な月経周期を持つが、卵巣超音波にて増加した卵胞を摘出していた。 Table 1
Demographic, anthropometric, biochemical and reproductive parameters in the two test groups used in the RP-SPE test

Note: PCOS-polycystic ovary syndrome; BMI = body mass index; WHR = waist (cm) / hip (cm) ratio; FG = Ferriman &Gallwey; T = tertosterone; SHBG = sex hormone-binding globulin; FAI = free androgen Index; LH = luteinizing hormone (IU / L); FSH = follicle stimulating hormone (IU / L).
• Statistically significant difference between PCOS and control group • One control woman showed a Ferriman Galwey score of 14, but had regular menstrual cycle and ultrasound characteristics consistent with the control. Dissimilar controls had normal androgens and regular menstrual cycles but removed increased follicles with ovarian ultrasound.

  During this test, it was noted that one of the control subjects (C4) showed some aspect of the PCOS borderline using some of the Rotterdam (2004) criteria. Therefore, this C4 control and corresponding PCOS pair was excluded from most tests. For the reverse phase mode-solid phase extraction test (RP-SPE) performed after the 2DE test, the excluded pair was replaced with a new pair consisting of one PCOS subject and one distinct control.

FIG. 1 shows the results of two-dimensional gel electrophoresis showing the change in protein level between PCOS subjects and corresponding control subjects. The top 10 images are silver-stained 2D gels, with the protein spots of interest in the gels marked in circles, and the protein between the PCOS target and the control age / BMI target. Differences in levels can be seen. The five images at the bottom were created using DELTA 2D image Analysis software and are overlays of corresponding pairs of gel images showing the same protein spot in the colorimetric display. Although no color development is seen, the results show that this observed spot appeared in 4 out of 5 gel pairs superimposed with an orange colored image indicating that it only occurs in PCOS objects. The spot was subsequently identified as C4α ₃ c. FIG. 2 shows a quantitative analysis of the intensity of the C4α ₃ c spot of an age-weight matched PCOS subject-control subject gel pair. Data was calculated using DELTA 2D image analysis software. FIG. 3 shows a Western blot analysis of 10 serum-depleted samples using antibodies raised against the unknown C4α ₄ c peptide sequence. FIG. 3A shows a Western blot analysis of whole sera from the same sample pair used in the 2DE test. The blot was probed with a polyclonal anti-C4α antibody raised against an epitope within the C4α chain. The ~24kDa band shown, illustrating the C4 [alpha] ₄ c fragment. FIG. 3B shows a quantitative analysis of the band intensity of FIG. 3A determined using SynGene Gene Tools densitometry software. * P = 0.03. FIG. 4 shows a diagrammatic representation of the primary structure for complement component C4 protein. The C4 precursor and many of its cleavage fragments are shown. C4 protein has been shown to be composed of three chains, which can all be cleaved from one C4 precursor and folded to form the functional structure of the protein. The α chain is an active element when the cleaved C4a fragment is released and acts as an anaphylatoxin. The C4b fragment forms a complex with C2 to continue the classical complement pathway enzymatic cascade and may also act as an opsonin. C4b is later cleaved into C4α3c, C4αd and C4α4c fragments, which inactivate the complement cascade (Nagasawa et al., (1980) J Immunol 125 (2): 578-82 and Hessing, (1991) Biochem J 277: 581 92). Note: † C4α ₃ c fragment identified by 2DE analysis as being up-regulated in PCOS sample. ‡ C4α ₄ c fragment shown by Western blot to be upregulated in PCOS. * C4γ chain identified by SDS PAGE analysis of the RP-SPE fraction as being down-regulated in PCOS. FIG. 5 shows an RP-SPE analysis showing the difference between PCOS and the corresponding control. FIG. 5A shows the ~ 33 kDa band present in the 5/6 control sample, and very low levels in the 3/6 PCOS sample versus fraction 12 only. There may be similar sized bands in fractions 4 and 5, but this is thought to be a different protein type. FIG. 5B shows the ˜40 kDa band present in all PCOS samples at an increased level than is present in the control sample. This difference was most noticeable in fractions 8-12. FIG. 5C shows the ˜17 kDa band present in the 5/6 PCOS sample and the only 2/6 control sample. FIG. 6 shows a quantitative RP-SPE analysis showing the difference between PCOS and the corresponding control. FIG. 6A shows the results of densitometry quantification of the ˜40 Kda band seen with RP-SPE (FIG. 5B). P = 0.001. FIG. 6B shows the results of quantification of the ˜17 Kda band seen with RP-SPE (FIG. 5C) by densitometry. Figure 7 shows a representative MALDI-TOF mass spectrum: Figure 7A is a serum peptide after trypsin digestion, and Figure 7B is a protein after C ₁₈ ZipTip cleanup from PCOS subject and control sera. is there. This spectrum shows the differences observed between PCOS serum and control serum. FIG. 7C shows ANN's predictive ability to evaluate tryptic peptide profiles based on three ion models, and FIG. 7D shows protein profiles based on six ion ANN models. Gray bars represent control samples and black bars represent PCOS samples. A predicted value of 1.5 or less indicates a control sample, while a predicted value of 1.5 or more indicates a PCOS sample. FIG. 8 (SEQ ID NO: 1) is the protein sequence of the isoform A C4 precursor protein. More specifically, amino acids 20-675 are C4β cleavage fragments, amino acids 680-1446 are C4α cleavage fragments, amino acids 680-756 are C4a anaphylatoxin cleavage fragments, and amino acids 757-1446 are C4b cleavage fragments. And amino acids 757-956 are C4α ₃ c cleavage fragments, amino acids 957-1333 are C4αd cleavage fragments, amino acids 1334-1446 are C4α ₄ c cleavage fragments, and amino acids 1445-1744 are C4γ cleavage fragments. . FIG. 9 (SEQ ID NO: 2) is the protein sequence of the isoform B C4 precursor protein. Amino acid differences from isoform A are underlined. FIG. 10 (SEQ ID NO: 3) is the protein sequence of haptoglobin. Amino acids 19 to 161 are haptoglobin α, and amino acids 162 to 406 are haptoglobin β.

Example 1-C4α3C as a biomarker for PCOS
2D SDS PAGE separation was performed on the first cohort of 5 PCOS and 5 control serum-depleted samples and the results are shown in FIG. When comparing stained gels, it was noted that one protein spot was visually present in all PCOS sample gels as well as the C1 control sample gel (FIG. 1).

  The association of this protein is shown to be equal for the corresponding pair of gels that pseudo-stain the control gel spot as blue and the PCOS gel spot as orange so that the evenly expressed spot appears black in the superimposed image. Confirmation was performed using the DELTA 2D image analysis software that created the superimposed image. In most of the analyzed gel areas, the spot patterns were identical and black. However, the circled spots (bottom 5 images) in FIG. 5 were found only in multiple PCOS samples and one control sample (C1).

  Image analysis software could then be used to quantify the spot volume as a percentage of the total spot volume for the analyzed gel area, thus allowing all gels to be directly compared. This was confirmed by visual evaluation. FIG. 2 shows the quantification in graphical form. Maximum differential expression was seen with the C2-P3 sample pair, compared to C2, the spot intensity in the P3 gel increased by ˜10-fold. The mean quantification value revealed an overall upregulation of spot intensity in the PCOS sample 4 times as much as the control sample.

Protein Identification Up-regulated protein spots (FIG. 1) were excised from P1, P3, P4 and P5 sample gels, pooled and trypsin digested, and the extracted peptides were analyzed by MALDI-TOF MS.

The resulting 13 peptide ions, when searched against the Human Swiss-Prot and TrEMBL databases, included 5 corresponding peptide ions covering 15.2% C4α ₃ c peptide sequences and complement component-4 ( There was a certain correspondence with Swiss-Prot accession number P01028), α chain, c fragment (C4α ₃ c). The predicted MW and pI of the C4α ₃ c sequence were 32.9 kDa and 5.1 kDa, respectively, as calculated using ExPASy primary structure analysis software. This corresponds appropriately with the spot position in the gel (˜35 kDa, pI˜7.0).

Example 2-C4α ₄ C as a biomarker for PCOS
Western blot analysis of samples from PCOS and control subjects shows differential expression of C4 derived proteins. Whole serum from PCOS and control samples was probed with polyclonal antibodies raised against αd and α ₄ c fragments of complement C4 precursor protein (FIG. 3). alpha ₄ c fragment, i.e. one C4b degradation products, the bands obtained in the to 24 kDa indicating a, but was present in all samples (Fig. 3A), from Densitometric analysis of the band intensities (Figure 3B), It was found that the intensity was significantly higher in the PCOS sample (P = 0.03) compared to the control. In age-BMI pairs, band intensity was higher in 4 out of 5 pairs. This is described as an exception because the pair in which the control sample band was higher in intensity than the PCOS sample band (C4-P1) would be related to the control sample that was on the border line of the PCOS. Can not be procured antibodies specific for C4 [alpha] ₃ c fragment, but an increase of C4 [alpha] ₄ c is indicative of proteolytic degradation of C4b polypeptide (FIG. 4) for generating a C4 [alpha] ₃ c and C4 [alpha] ₄ c.

Example 3-C4γ and haptoglobin α and β chains as biomarkers for PCOS
Analysis of samples from 6 PCOS and 6 control subjects is RP-SPE / SDS-PAGE. Samples were from the five said BMI and age-matched PCOS and control pairs and another PCOS and control pair. An aliquot of a serum sample from the subject was pre-fractionated using a reverse phase solid phase column and serial elution with a stepwise concentration gradient increasing the MeCN concentration. The 12 samples obtained for each elution fraction are then run side by side on an SDS PAGE gel, a gel displaying all 12 serum samples at a specific acetonitrile concentration, and stained with coma blue. did. This allows the protein composition of each fraction to be compared to be compared across all pairs of age / BMI matched serum samples. For reasons outlined above, the data obtained from the PCOS-control pair (P1 and C4) was not taken into account for quantitative data analysis.

  Visual analysis of the stained gel showed three obvious differences in the band pattern between PCOS and control samples (FIGS. 5A, 5B and 5C). The first difference detected in the 70% MeCN fraction (Figure 5A) is present in all control samples, but in a small amount in only one of the five PCOS samples. Regarding the observed component of ˜33 kDa. Due to the low signal intensity, densitometric quantification of these components was not possible.

  The second difference between the two sample groups is a change in the abundance of one of the components present at ~ 40 kDa in all samples in the 42-70% MeCN fraction (Figure 5B). In the continuous elution fraction, all PCOS samples increased compared to the control sample. A further illustration of the upregulation of potential proteins in PCOS serum was found throughout the gel analysis, although most notably in the 10-20% MeCN fraction. In this case, the component at ˜17 kDa was present in only 4 PCOS samples and 2 control samples (FIG. 5C). Densitometric quantification for these two changes in FIGS. 5B and 5C is illustrated in FIGS. 6A and 6B, with differential expression at ˜40 kDa (FIG. 6A) with a significance value of 0.001.

Identification of differentially expressed proteins
Differential bands identified by RP-SPE / SDS PAGE analysis were excised, collected, trypsinized, and analyzed using MALDI-TOF MS. From a Swiss plot using the measured peptide ions and the resulting sequences and a search of the TrEMBL database, the possibility of a match for all three observed differences was identified. The ˜33 kDa band extracted from the 70% MeCN fraction gel (FIG. 5B) resulted in the characteristics of the six peptide ions of the complement component C4γ chain, confirming this agreement from tandem MS / MS product ion analysis. This predicted MW of the C4 γ chain is ˜33.5 kDa as calculated using ExPASy primary structure analysis software, which is in good agreement with the band position observed after SDS PAGE.

  The ~ 40 kDa band (Figures 5B and 6A) extracted from a 42% MeCN fractionated gel gives three peptide ions characteristic of the haptoglobin β chain, after which two of these are tandem LC-MS / MS products Analysis was confirmed by ion analysis to confirm this agreement. Three additional peptide ions were consistent with the alpha region of the haptoglobin precursor protein. The ˜17 kDa band extracted from the 20% MeCN fraction gel (FIGS. 5C and 6B) gave six peptide ions characteristic of the haptoglobin α chain. The molecular weight obtained from the theoretical sequence for the haptoglobin α-chain sequence is 15.9 kDa, which corresponds considerably to the molecular weight observed after SDS PAGE, and for the β-chain sequence is 27.3 kDa, the precursor is 43.6 kDa. It has a molecular weight of kDa, which is in good agreement with the ˜40 kDa band observed in RP-SPE analysis. The presence of these bands in multiple fractions from the RP-SPE column suggests that the protein may be differentially modified (eg, due to glycosylation) and thus exhibit various affinities for the column matrix.

Example 4 MALDI-MS Serum Protein / Peptide Biomarker Characteristics for PCOS This analysis was performed on 21 serum samples from 11 PCOS subjects and 10 controls.

  Compared to unaffected individuals, the most prominent spectral discrimination patterns in PCOS are illustrated in FIGS. 7A and 7B, respectively. However, a comprehensive bioinformatic ANN analysis was performed to investigate all data pairs for both proteins and tryptic peptides.

  From the ANN analysis, optimal subsets of 3 and 6 biomarker ions were identified for each protein and peptide data pair. Six ions (m / z 8674, 8668, 1351, 8727, 8673, and 6871) allow an independent validation set of 90.4% samples at a threshold of 0.5 based on whether they are controls or PCOS Classified correctly (Figure 7C). These three biomarker ions (2924, 3025, and 1977) identified from digested peptide data successfully classified an independent validation subset of samples to 100% accuracy (Figure 7D)

Claims (34)

A method of determining a subject's polycystic ovary syndrome (PCOS) status, comprising:
Following steps:
(a) providing a sample of material obtained from the subject;
(b) one or more postscript proteins in the sample;
Complement C4 precursor protein,
Determining the cleavage fragment of the complement C4 precursor protein, and the level of haptoglobin,
(c) comparing the determined level with one or more reference values;
Including a method. A cleavage fragment of the complement C4 precursor protein is
a) C4α
b) C4β
c) C4γ
d) C4a anaphylatoxin
e) C4b
f) C4α ₃ c fragment
g) C4αd
j) h) C4α ₄ c fragment, and
i) a polypeptide having at least 75% amino acid sequence identity to any amino acid sequence of a) to h),
2. The method of claim 1, wherein the method is selected from the group comprising:   3. The method of claim 1 or 2, wherein the one or more haptoglobin proteins are selected from the group comprising haptoglobin alpha and haptoglobin beta.   The method according to any one of claims 1 to 3, wherein the sample material is whole blood, serum, plasma, urine, adipose tissue (adipose), endometrial tissue, ovarian tissue or other body fluid or tissue.   One or more levels of complement C4 precursor protein, cleavage fragment of complement C4 precursor protein, and haptoglobin are immunoassay, spectroscopic, mass spectrometric, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass Select from the group including spectroscopic analysis, microscopy, Northern blot, Western blot, Southern blot, isoelectric focusing, SDS-PAGE, PCR, RT-PCR, gel electrophoresis, protein microarray, DNA microarray, antibody microarray and combinations thereof The method according to any one of claims 1 to 4, which is determined using an assay to be performed.   One or more reference values were observed in one or more subjects without any clinical signs of PCOS, at the level of one or more complement C4 precursor proteins, complement fragment of complement C4 precursor proteins and haptoglobin 6. The method according to any one of claims 1 to 5, wherein:   The one or more reference values are early levels of one or more complement C4 precursor protein, a cleavage fragment of complement C4 precursor protein and haptoglobin as determined in the same subject that provided the sample. 6. The method according to any one of 5 above.   The increase or decrease in one or more levels of one or more complement C4 precursor proteins, cleaved fragments of complement C4 precursor proteins, and haptoglobin is an indicator of a subject's PCOS status. The method according to item. An increase in the level of C4α ₃ c and / or C4α ₄ c and / or haptoglobin α and / or haptoglobin β in the sample compared to the reference value is an indicator of PCOS or a diagnostic feature 9. The method of claim 8, wherein   9. The method of claim 8, wherein a reduction in the level of C4 precursor protein and / or C4α and / or C4β and / or C4γ and / or C4a anaphylatoxin is a characteristic used for PCOS indicators or diagnosis.   11. A method according to any one of claims 1 to 10, wherein the method is performed in vitro.   12. The method according to any one of claims 1 to 11, wherein the subject is a mammal.   13. The method of claim 12, wherein the mammal is a human. Following steps;
(a) provide a sample of material obtained from the subject;
(b) analyzing the protein or trypsin peptide obtained from the sample using MALDI-MS;
(c) the presence of 6 ions m / z 8674, 8668, 1351, 8727, 8673, and 6871 in the protein sample and / or the presence of 3 ions m / z 2924, 3025, and 1977 in the tryptic peptide sample Determining that it is a feature used in the diagnosis of PCOS in
A method for determining PCOS status in a subject comprising:   A kit for use in determining a subject's PCOS status comprising at least one complement C4 precursor protein, a cleavage fragment of the complement C4 precursor protein, and at least one reagent for determining the level of haptoglobin. A cleavage fragment of the complement C4 precursor protein is
a) C4α
b) C4β
c) C4γ
d) C4a Anaphylatoxin
e) C4b
f) C4α ₃ c fragment
g) C4αd
k) h) C4α ₄ c fragment, and
i) a polypeptide having at least 75% amino acid sequence identity with the amino acid sequence of any of a) to h),
16. The kit of claim 15, wherein the kit is selected from the group comprising:   17. The kit of claim 15 or 16, wherein the one or more haptoglobin proteins are selected from the group comprising haptoglobin alpha and haptoglobin beta.   18. A kit according to claim 15, 16 or 17 wherein the reagent is selected from the group comprising enzymes, antibodies, nucleic acids, protein probes and other suitable compositions.   The kit according to any one of claims 15 to 18, wherein the reagent is labeled.   21. The kit of claim 19, further comprising means for detecting the label.   21. A kit according to any one of claims 15 to 20, further comprising instructions for suitable operational parameters in the form of a label or another insert.   23. Any of claims 15-21, further comprising one or more complement C4 precursor proteins, cleaved fragments of complement C4 precursor proteins, and / or haptoglobin samples to be used as standards for calibration and comparison Or the kit according to item 1.   A method of treating PCOS in a subject comprising administering to the subject one or more complement C4 precursor protein, a cleavage fragment of complement C4 precursor protein and a reagent capable of modulating the level of haptoglobin.   24. The method of claim 23, wherein the reagent is an antisense or interfering RNA molecule designed to inhibit or reduce expression of complement C4 precursor protein, a cleavage fragment of complement C4 precursor protein and haptoglobin. .   24. The method of claim 23, wherein the reagent is an inhibitor of C4 precursor protein processing.   24. The method of claim 23, wherein the reagent is a composition that reduces complement activity. (a) in the sample obtained from the subject at the first time point,
One or more postscript proteins:
Complement C4 precursor protein,
A cleavage fragment of the complement C4 precursor protein, and haptoglobin,
Determining the level of
(b) one or more of the following proteins in a sample taken from the subject at a later second time point:
Complement C4 precursor protein,
Determining the cleavage fragment of complement C4 precursor protein, and the level of haptoglobin, and
(c) comparing the first measured value and the second measured value, wherein the course of the PCOS is determined by the comparative measured value;
Determining the progression of PCOS in a subject, or determining the effect of a treatment given to a subject suffering from PCOS.   Treat PCOS, including screening in vitro or in vivo for one or more complement C4 precursor proteins, cleavage fragments of complement C4 precursor proteins and one or more compounds that modulate haptoglobin levels A method of identifying compounds for C4 precursor protein, C4α, C4β, C4γ, C4a anaphylatoxin, C4b, C4α ₃ c fragment, C4αd, C4α ₄ c fragment, haptoglobin α and / or haptoglobin β as therapeutic targets for the treatment / mitigation of PCOS use.   A probe pair comprising one or more complement C4 precursor proteins, a cleavage fragment of the complement C4 precursor protein and one or more probes capable of detecting haptoglobin.   32. A probe pair according to claim 30, wherein the probes are selected from the group comprising antibodies, nucleic acids or peptides.   32. The probe pair of claim 30 or 31, wherein the probe is labeled.   33. The probe pair of claim 30, 31 or 32 comprising two probes for two different proteins selected from the group comprising complement C4 precursor protein, a cleavage fragment of complement C4 precursor protein and haptoglobin.   Analyze the sample for the presence of 6 ions m / z 8674, 8668, 1351, 8727, 8673 and 6871 in the protein sample and / or the presence of 3 ions m / z 2924, 3025 and 1977 in the tryptic peptide sample Kit for use in determining the target PCOS status, including instructions for.

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