WO2018176808A1 - 与重度少弱精子症相关的生物标志物的筛选与应用 - Google Patents

与重度少弱精子症相关的生物标志物的筛选与应用 Download PDF

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WO2018176808A1
WO2018176808A1 PCT/CN2017/106915 CN2017106915W WO2018176808A1 WO 2018176808 A1 WO2018176808 A1 WO 2018176808A1 CN 2017106915 W CN2017106915 W CN 2017106915W WO 2018176808 A1 WO2018176808 A1 WO 2018176808A1
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protein
severe
sperm
weak
mass
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French (fr)
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杨静华
陈新骏
吕鑫
赵涵
李翠玲
毕白斌
巩晶
王凤芹
孙胜楠
王兴元
陈子江
杨静鸣
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Shandong University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins

Definitions

  • the invention relates to the technical field of medicine and molecular diagnosis, in particular to screening and application of biomarkers related to severe oligozoospermia.
  • Infertility has become a reproductive health problem worldwide, with infertility due to male factors accounting for about 50%, and has shown an upward trend in recent years.
  • the main cause of male infertility is less azoospermia.
  • azoospermia According to the World Health Organization standards, if the spermatozoa level is ⁇ 25%, the sperm count of (a+b) grade is ⁇ 50%, and the sperm motility rate is less than 60%, it can be diagnosed as weak sperm disease.
  • Less azoospermia is a condition in which the number of sperm in semen is lower than that of a normal fertile male. When a male sperm is less than 20 million per milliliter, it is oligozoospermia.
  • Non-coding amino acids including post-translational modifications and amino acid mutations, are important ways to regulate protein function and structure, so non-coding amino acids with abnormal or quantitative changes in disease states are used as biomarkers for disease, and thus for disease diagnosis. The process is important. However, there are no reports of non-coding amino acids as biomarkers associated with severely weak sperm diseases.
  • the object of the present invention is to provide a screening and application of biomarkers associated with severely weak sperm disease.
  • the present invention first utilizes the NanoHPLC-MS/MS mass spectrometry system and the non-labeled quantitative proteomics method to perform deep mass spectrometry analysis on the sperm protein non-coding amino acids of multiple groups of severe oligospermia diseases; The mass spectrometry data was searched, and then the multivariate Gaussian mixture distribution cluster analysis was used to identify the non-encoded amino acids in the sperm protein group as much as possible. Finally, the comparison with the non-coding amino acids in the normal and patient sperm protein groups was obtained. The savvy-associated protein does not encode an amino acid site, making it a molecular marker for severe oligospermia.
  • the present invention adopts the following technical solutions:
  • a screening method for a biomarker associated with severe oligozoospermia comprising the steps of:
  • step (3) The sample of step (2) is separated by nanoflow liquid chromatography, and the sample separated by nanoflow liquid chromatography is subjected to mass spectrometry detection to collect mass spectrometry data;
  • the whole protein of the sperm cells is extracted by washing the sperm sample with DPBS, adding RIPA lysate for 1 to 2 minutes, immersing on ice for 30 min, lysing, centrifuging, and taking the supernatant.
  • centrifugation is carried out at 4 ° C, the centrifugation speed is 14,000 g, and the centrifugation time is 20 min.
  • step (2) preferably, the protein is separated by 10% polyacrylamide gel electrophoresis (SDS-PAGE).
  • the digested peptide is desalted using a ziptip.
  • step (3) the chromatographic conditions for nanoflow liquid chromatography are: mobile phase A: water containing 0.1% formic acid, mobile phase B: acetonitrile containing 0.1% formic acid; nanoflow liquid chromatography mass spectrometry system is Orbitrap Elite ( Thermo Scientific)
  • the elution conditions were: 0-100 min, 95-68% mobile phase A, 5-32% mobile phase B; 100-120 min, 68-20% mobile phase A, 32-80% mobile phase B; 120-150 min, 20 % mobile phase A, 80% mobile phase B;
  • the flow rate was 300 nL/min.
  • the conditions for mass spectrometry detection are: full scan of 350-1800 m/z, and the resolution is 60,000 (m/z 200).
  • the activation time is 10ms and the isolation width is 2m/z;
  • the fragmentation method is collision-induced dissociation (CID), the normalized collision energy is set to 35%, and the dynamic discharge time is 90s.
  • step (4) the parameters for searching the mass spectrometry data are set as: protease is trypsin, the missed cut site is set to 2, the parent ion mass deviation is 10 ppm, the fragment ion mass deviation is 0.6 Da, and the blind search upper limit is set to 1000, the lower limit of blind search is set to -200, and the protein FDR is 0.01;
  • Select peptide segment score> 200 peptide and FDR ⁇ 0.01 searched data as modified unknown Wildcard Search TM, composed of one-dimensional data matrix mass change (-200Da-400Da), then the data in the range of 1Da, 0.5Da For the boundary, it is divided into 601 data windows.
  • modified unknown Wildcard Search TM composed of one-dimensional data matrix mass change (-200Da-400Da)
  • the multivariate Gaussian mixture distribution clustering method is: for each data window, the Gaussian mixture distribution cluster analysis is performed by using the mclust package in the R language, and the optimal value is obtained according to the BIC, and then A peak is combined and analyzed, then each peak is fitted with a Gaussian distribution to determine the peak; the peptide site data contained in each peak after clustering is selected as a distribution of data greater than 5% according to the amino acid distribution of the site. Class non-coding amino acids.
  • the non-coding amino acids of the normal individual and the severely weak and weak individual are screened according to the T test (p ⁇ 0.05) and the ratio (ratio>2) of the detection frequency, thereby obtaining the differential non-coding amino acid.
  • the above screening method is for obtaining biomarkers, and is not for obtaining the diagnosis and treatment results of the diseases; the biomarkers obtained by the above screening methods can be used for theoretical research or development of new drugs for severe oligozoospermia.
  • biomarker associated with severe oligozoospermia selected according to the above screening method, the biomarker including but not limited to:
  • AKAP3 protein 208 position +79.96685 mass shift of serine (labeled S + 79.96685; according to the mass shift value, determine the position of the serine phosphorylation);
  • AKAP4 protein 186 position occurs -113.05347 mass shift of asparagine (labeled N-113.05347);
  • AKAP4 protein at position 186 occurs -114.04278 mass shift of asparagine (labeled N-114.04278);
  • AKAP4 protein 617 occurs with a -17.62660 mass shift of glutamine (labeled Q-17.02660);
  • ATP5A1 protein 531 occurs +42.01108 mass shift of lysine (labeled K + 42.01108; according to the mass offset value, it is determined that lysine at this position is acetylated);
  • the GAPDHS protein has a +79.96685 mass shift of threonine at position 64 (labeled T+79.96685; it is determined by the mass shift value that the threonine at this position is phosphorylated);
  • a use of serine at a 208 position of the AKAP3 protein at the 208 position of a mass loss of 79.96685 as a biomarker for the preparation of a diagnostic agent for severe oligospermia.
  • the serine of +79.96685 mass shift at position 208 of the AKAP3 protein can also be used as a target for treatment of severe oligospermia, thereby being used for the treatment of severe oligospermia.
  • the present invention also provides the use of serine at a 208 position of the AKAP3 protein at the 208 position and having a mass shift of 99.96685 as a biomarker for the preparation of a therapeutic drug for severe oligospermia.
  • the present invention also provides a kit for the diagnosis of severe oligospermia, which comprises a reagent for specifically detecting the above biomarker (serine having a mass shift of +79.96685 in the 208 position of AKAP3 protein).
  • the present invention also provides a medicament for treating severe oligospermia, which comprises a component capable of phosphorylating a serine at position 208 of the AKAP3 protein.
  • the invention also provides a diagnostic method for severely weak and weak, the steps are: detecting the frequency of the +79.96685 mass shift of the serine at position 208 of the AKAP3 protein of the sample to be tested, and if the frequency of detection is less than 1.5, it is judged to be a weak and weak patient.
  • asparagine which has a -113.05347 mass shift of AKAP4 protein at position 186, as a biomarker for the preparation of a diagnostic reagent for severe oligospermia.
  • the present invention also provides a kit for the diagnosis of severe oligospermia, which comprises an agent for specifically detecting the above biomarker (asparagine having a mass shift of -113.05347 at position 186 of AKAP4 protein).
  • the invention also provides a diagnostic method for severely weak and weak sperm, the steps are: detecting the frequency of the -113.05347 mass deviation of the 186 amino acid asparagine of the AKAP4 protein to be tested, and the frequency of detection is less than 0.5, and the patient is judged to be weak and weak. .
  • asparagine which has a -114.04278 mass shift of the AKAP4 protein at position 186, as a biomarker for the preparation of a diagnostic reagent for severe oligospermia.
  • the present invention also provides a kit for the diagnosis of severe oligospermia, which comprises an agent for specifically detecting the above biomarker (asparagine of AKAP4 protein at position 186-114.04278 mass shift).
  • the invention also provides a diagnostic method for severely weak and weak, the steps are: detecting the frequency of the mass shift of the 186-position asparagine at the 186-position of the AKAP4 protein of the sample to be tested, and detecting the frequency of less than 0.5, the patient is judged to be weak and weak. .
  • glutamine which is a -17.02660 mass shift of AKAP4 protein at position 617 as a biomarker for the preparation of a diagnostic reagent for severe oligospermia.
  • the present invention also provides a kit for the diagnosis of severe oligospermia, which comprises a reagent for specifically detecting the above biomarker (the AKAP4 protein at position 617, which has a mass loss of 1.7.26260 glutamine).
  • the invention also provides a diagnostic method for severely weak and weak, the steps are as follows: detecting the frequency of the -17.06860 mass shift of the AKAP4 protein 617 in the sample to be tested, and the frequency of the detection is less than 0.5, and the patient is judged to be weak and weak. .
  • a lysine having a 211.09682 mass shift of the 733 position of the AKAP4 protein as a biomarker for the preparation of a diagnostic reagent for severe oligospermia.
  • the present invention also provides a kit for the diagnosis of severe oligospermia, which comprises a reagent for specifically detecting the above biomarker (the AKAP4 protein 733 occurs +211.09682 mass shift of lysine).
  • the invention also provides a diagnosis method of severely weak and weak, the steps are as follows: detecting the frequency of the 733 lysine of the test sample AKAP4 protein +211.09682 mass deviation, and the detection frequency is less than 3.5, the patient is judged to be weak and weak .
  • a lysine having a +42.01108 mass shift of the ATP5A1 protein at position 531 as a biomarker for the preparation of a diagnostic reagent for severe oligospermia.
  • the lysine with a mass loss of +42.01108 in the 531 position of the ATP5A1 protein can also be used as a target for the treatment of severe oligospermia, thereby being used for the treatment of severe oligospermia.
  • the present invention also provides the use of lysine having a mass shift of 42.01108 at the 531 position of the ATP5A1 protein as a biomarker for the preparation of a therapeutic drug for severe oligospermia.
  • the present invention also provides a kit for the diagnosis of severe oligospermia, which comprises a reagent for specifically detecting the above biomarker (ATP5A1 protein 531 position + 42.01108 mass shift lysine).
  • the present invention also provides a medicament for treating severe oligospermia, which comprises a component capable of acetylating ATP5A1 protein lysine 531.
  • the invention also provides a diagnostic method for severely weak and weak, the steps are: detecting the frequency of the 531 lysine of the sample ATP5A1 protein to be +42.01108 mass deviation, and if the detection frequency is less than 0.5, it is judged to be weak patient.
  • a lysine having a +42.01108 mass shift at the 87 position of the COX4I1 protein as a biomarker for the preparation of a diagnostic reagent for severe oligospermia.
  • the lysine with a mass loss of +42.01108 in the 87 position of the COX4I1 protein can also be used as a target for treatment of severe oligospermia, thereby being used for the treatment of severe oligospermia.
  • the present invention also provides the use of lysine having a mass shift of 42.01108 at the 87 position of the COX4I1 protein as a biomarker for the preparation of a therapeutic drug for severe oligospermia.
  • the present invention also provides a kit for the diagnosis of severe oligospermia, which comprises a reagent for specifically detecting the above biomarker (the lysine of the COX4I1 protein 87 position + 42.01108 mass shift).
  • the present invention also provides a medicament for treating severe oligospermia, which comprises a component capable of acetylating a lysine at position 87 of the COX4I1 protein.
  • the invention also provides a diagnosis method of severely weak and weak, the steps are: detecting the frequency of the +42.01108 mass shift of the 87th lysine of the COX4I1 protein of the sample to be tested, and if the detection frequency is less than 0.5, it is judged to be weak and weak. patient.
  • a threonine at a 64 position of the GAPDHS protein at a position of +79.96685 mass shift as a biomarker for the preparation of a diagnostic reagent for severe oligospermia.
  • the threonine with a mass loss of +79.96685 at the 64 position of the GAPDHS protein can also be used as a target for the treatment of severe oligospermia, thereby being used for the treatment of severe oligospermia.
  • the present invention also provides the use of threonine at the 64 position of the GAPDHS protein at a position of +79.96685 mass shift as a biomarker for the preparation of a therapeutic drug for severe oligospermia.
  • the present invention also provides a kit for the diagnosis of severe oligospermia, which comprises a reagent for specifically detecting the above biomarker (the threonine of the GAPDHS protein at position 64 +79.96685 mass shift).
  • the present invention also provides a medicament for treating severe oligospermia, which comprises a component capable of phosphorylating a threonine at position 64 of the GAPDHS protein.
  • the invention also provides a diagnostic method for severely weak and weak, the steps are: detecting the frequency of the loss of the hyalin of the 64th position of the GAPDHS protein of the test sample by +79.96685, and if the detection frequency is less than 3.5, it is judged to be weak. patient.
  • the serine of +79.96685 mass shift at position 692 of KIAA1683 protein can also be used as a target for treatment of severe oligospermia, thereby being used for the treatment of severe oligospermia.
  • the present invention also provides the use of serine at the 692 position of the KIAA1683 protein at the 692 position as a biomarker for the preparation of a therapeutic drug for severe oligospermia.
  • the present invention also provides a kit for the diagnosis of severe oligospermia, which comprises a reagent for specifically detecting the above biomarker (serine which has a mass shift of +79.96685 in the 692 position of KIAA1683 protein).
  • the present invention also provides a medicament for treating severe oligospermia, which comprises a component capable of phosphorylating a serine at position 692 of KIAA1683 protein.
  • the invention also provides a diagnostic method for severely weak and weak, the steps are: detecting the frequency of the loss of +79.96685 mass of the serine at the 692 position of the KIAA1683 protein to be tested, and if the frequency of detection is less than 0.5, it is judged to be a weak and weak patient.
  • the present invention establishes for the first time a screening method for biomarkers associated with severe oligozoospermia.
  • a screening method for biomarkers associated with severe oligozoospermia By analyzing the mass spectrometric data of a large number of sample sperm proteins, the non-coding of the sperm protein group is identified as much as possible. Amino acid; finally, by comparing the non-coding amino acids in the normal and patient sperm protein groups, the protein associated with severe oligospermia is obtained.
  • the amino acid site is encoded to serve as a molecular marker for severe oligospermia.
  • the present invention further studies the biomarkers obtained by the above screening method, and finds that the diagnosis of severe oligozoospermia can be diagnosed by the frequency of the above-mentioned biomarkers, and provides new diagnosis and treatment for severe oligospermia. Target.
  • Figure 1 ROC curve of the phosphorylation of S+79.96685 detection frequency on serine at position 208 of the AKAP3 protein.
  • Figure 2 Comparison of the detection frequency of phosphorylation of S+79.96685 on serine at position 208 of AKAP3 protein in healthy and oligospermia samples.
  • Figure 3 ROC curve of the detection frequency of the 186 non-coding amino acid N-113.05347 of the AKAP4 protein.
  • Figure 4 Comparison of detection frequencies of 186 non-coding amino acids N-113.05347 of AKAP4 protein in healthy and oligospermia samples.
  • Figure 5 ROC curve of the detection frequency of the 186 non-coding amino acid N-114.04278 of the AKAP4 protein.
  • Figure 6 Comparison of detection frequencies of 186 non-coding amino acids N-114.04278 of AKAP4 protein in healthy and oligospermia samples.
  • Figure 7 ROC curve of the detection frequency of the 617 non-coding amino acid Q-17.02660 of the AKAP4 protein.
  • Figure 8 Comparison of detection frequencies of 617 non-coding amino acids Q-17.02660 of AKAP4 protein in healthy and oligospermia samples.
  • Figure 9 ROC curve of the detection frequency of the 733 non-coding amino acid K+211.09682 of the AKAP4 protein.
  • Figure 10 Comparison of the detection frequencies of non-coding amino acids K+211.09682 for healthy and weak samples.
  • Figure 11 ROC curve of the detection frequency of ATP5A1 protein lysine acetylation modification K+42.01108.
  • Figure 12 Comparison of detection frequency of ATP5A1 protein 531 lysine acetylation modification K+42.01108 for healthy and oligospermia samples.
  • Figure 13 ROC curve of the detection frequency of KX42.01108 for lysine acetylation at position 87 of COX4I1 protein.
  • Figure 14 Comparison of the detection frequency of the lysine acetylation modification of KX42.01108 for COX4I1 protein in healthy and oligospermic samples.
  • Figure 15 ROC curve of the phosphorylation of the GAPDHS protein on the threonine at the 64th position of the T+79.96685 detection frequency.
  • Figure 16 Comparison of the frequency of detection of phosphorylated T+79.96685 on the 64th position of GAPDHS protein in healthy and weak samples.
  • Figure 17 ROC curve of the detection frequency of serine phosphorylation modification S+79.96685 of KIAA1683 protein.
  • Figure 18 Comparison of the detection frequency of serine phosphorylation modification S+79.96685 of KIAA1683 protein in healthy and weak samples.
  • the present invention proposes a screening method and application of biomarkers related to severe oligozoospermia.
  • a screening method for biomarkers associated with severe oligozoospermia comprising the steps of:
  • step (3) The sample of step (2) is separated by nanoflow liquid chromatography, and the sample separated by nanoflow liquid chromatography is subjected to mass spectrometry detection to collect mass spectrometry data;
  • This application first uses the NanoHPLC-MS/MS mass spectrometry system and non-labeling. Quantitative proteomics method was used for deep mass spectrometry analysis of sperm protein non-coding amino acids in multiple groups of severe oligospermia diseases; then the mass spectrometry data was searched by non-limiting amino acid protein modification analysis method, and then multivariate Gaussian mixture distribution clustering Analysis, the largest number of non-coding amino acids in the sperm protein group were identified.
  • the non-coding amino acids of the normal and diseased groups were screened according to the T test (p ⁇ 0.05) and the ratio (ratio>2) of the detection frequency, thereby obtaining differential non-coding amino acids. Then the SPSS software was used to make the difference non-coding amino acid ROC curve, and the area under the curve (AUC) was calculated to judge its diagnostic value.
  • AKAP3 protein 208 position +79.96685 mass shift of serine (labeled S + 79.96685; according to the mass shift value, determine the position of the serine phosphorylation);
  • AKAP4 protein 186 position occurs -113.05347 mass shift of asparagine (labeled N-113.05347);
  • AKAP4 protein at position 186 occurs -114.04278 mass shift of asparagine (labeled N-114.04278);
  • AKAP4 protein 617 occurs with a -17.62660 mass shift of glutamine (labeled Q-17.02660);
  • ATP5A1 protein 531 occurs +42.01108 mass shift of lysine (labeled K + 42.01108; according to the mass offset value, it is determined that lysine at this position is acetylated);
  • the GAPDHS protein has a +79.96685 mass shift of threonine at position 64 (labeled T+79.96685; it is determined by the mass shift value that the threonine at this position is phosphorylated);
  • kits for the diagnosis of severe oligospermia comprising reagents for specifically detecting the above biomarkers.
  • a medicament for treating severe oligospermia which comprises a serine at position 208 of AKAP3 protein, a threonine at position 64 of GAPDHS protein or a serine at position 692 of KIAA1683 protein.
  • test materials used in the embodiments of the present invention are all conventional test materials in the art, and can be purchased through commercial channels. To.
  • Example 1 Screening of biomarkers associated with severe oligozoospermia
  • Proteolysis Take about 150 ⁇ g of sperm protein in each of the severely weak and normal sperm samples, and separate the proteins by 10% polyacrylamide gel electrophoresis (SDS-PAGE), and divide into 5 parts for gelatinization. The peptide was desalted using a ziptip.
  • Mass spectrometry analysis nanoflow liquid chromatography separation: phase A: water containing 0.1% formic acid; phase B: acetonitrile containing 0.1% formic acid
  • Pre-columns and analytical columns were prepared by balancing 4 ⁇ L of Phase A before separation of the samples.
  • the specifications of the precolumn and the analytical column are: precolumn (4cm ⁇ 150 ⁇ m ID, C18 filler particle size 5 ⁇ m, ), analytical column (30cm ⁇ 75 ⁇ m ID, C18 filler filled, particle size 3 ⁇ m, Dr. Maisch GmbH, Germany).
  • precolumn 4cm ⁇ 150 ⁇ m ID, C18 filler particle size 5 ⁇ m,
  • analytical column (30cm ⁇ 75 ⁇ m ID, C18 filler filled, particle size 3 ⁇ m, Dr. Maisch GmbH, Germany).
  • the 150 min chromatographic gradient was changed as follows: 5-32% mobile phase B 100 min; 32-80% mobile phase B, 20 min; 80% mobile phase B, 30 min.
  • the flow rate is always maintained at 300 nL/min.
  • Samples separated by nanoflow liquid phase directly enter the ESI ion spray source and enter the Orbitrap Elite mass spectrometer for mass spectrometry.
  • Mass spectrometry data acquisition full scan at 350-1800 m/z with a resolution of 60,000 (m/z 200).
  • the activation time was 10 ms and the isolation width was 2 m/z.
  • the fragmentation method is collision-induced dissociation (CID), the normalized collision energy is set to 35%, and the dynamic discharge time is 90s.
  • the non-coding amino acids of the normal and diseased groups were screened according to the T test (p ⁇ 0.05) and the ratio (ratio>2) of the detection frequency, thereby obtaining differential non-coding amino acids. Then the SPSS software was used to make the difference non-coding amino acid ROC curve, and the area under the curve (AUC) was calculated to judge its diagnostic value.
  • the mass spectrometry data was analyzed and compared with the normal non-coding amino acids of the diseased group to obtain 9 differential non-coding amino acids, which can be used as biomarkers related to severe oligozoospermia, as follows:
  • AKAP3 protein 208 position +79.96685 mass shift of serine (labeled S + 79.96685; according to the mass offset value, determine the position of the serine phosphorylation modification)
  • the present invention uses ROC curve analysis, and AUC is the area under the ROC curve, which is the most commonly used evaluation feature of ROC curve.
  • the parameters are important test accuracy indicators. If the AUC is below 0.7, the accuracy of the diagnosis is low; if the AUC is above 0.7, the clinical diagnosis can be met.
  • Figure 1 shows the ROC curve of the detection frequency of phosphorylation of S+79.96685 on serine at position 208 of AKAP3 protein.
  • ROC analysis showed that the AUC of this phosphorylated modification was 0.856>0.7, indicating a good diagnostic effect, ie 208 of AKAP3 protein.
  • Phosphorylation on serine S+79.96685 can be used as a diagnostic marker for severe oligos.
  • Fig. 2 The comparison of the detection frequency of phosphorylation of S+79.96685 on serine at position 208 of AKAP3 protein in healthy and oligospermic samples is shown in Fig. 2. It can be seen from Fig. 2 that this non-coding amino acid occurred 4.6 times on average in healthy human samples. 0.4 occurrences in the pathological sample (solid line in the figure), and the median (dashed line in the figure) is far apart, indicating that this non-coding amino acid is largely lost in the sample.
  • phosphorylation of S+79.96685 on serine at position 208 of AKAP3 protein can be used as a potential biomarker for oligozoospermia, thereby predicting this condition.
  • AKAP4 protein 186 occurs with a -113.05347 mass shift of asparagine (labeled N-113.05347)
  • Figure 3 is the ROC curve of the detection frequency of 186 non-coding amino acid N-113.05347 of AKAP4 protein.
  • the ROC analysis showed that the AUC of this non-coding amino acid N-113.05347 was 0.852>0.7, indicating a good diagnostic effect.
  • the frequency of the test is 1.5, the sensitivity is 65.1% and the specificity is 93.7%.
  • the frequency of detection is less than 1.5, it is judged to be a weak and weak patient (false positive rate is 6.3%).
  • Figure 4 compares the frequency of detection of non-coding amino acids N-113.05347 in healthy and weak samples. It can be seen that this non-coding amino acid occurred 2.9 times on average in healthy human samples and 0.3 times in pathological samples (Fig. 4) The medium solid line), and the median (dashed line in the figure) is far apart, indicating that this non-coding amino acid is largely lost in the sample.
  • the non-coding amino acid of asparagine N-113.05347 at position 186 of the AKAP4 protein can be used as a potential biomarker for oligozoospermia, thereby predicting this condition.
  • AKAP4 protein occurs at position 186 - 114.04278 mass shift of asparagine (labeled N-114.04278)
  • Figure 5 is the ROC curve of the detection frequency of 186 non-coding amino acid N-114.04278 of AKAP4 protein. ROC analysis showed that the AUC of this non-coding amino acid N-114.04278 was 0.817>0.7, indicating a good diagnostic effect.
  • the frequency of the test is 0.5
  • the sensitivity is 74.6%
  • the specificity is 84.1%.
  • the individual test when the frequency of detection is less than 0.5, it is judged to be a weak and weak patient (false positive rate is 15.9%).
  • Fig. 6 The comparison of the detection frequency of 186 non-coding amino acid N-114.04278 of AKAP4 protein in healthy and oligospermic samples is shown in Fig. 6. It can be seen from Fig. 6 that this non-coding amino acid averaged 1.6 times in healthy human samples in pathological samples. Occurred 0.2 times (solid line in the figure), and the median (dashed line in the figure) is far apart, indicating that this non-coding amino acid is largely lost in the sample.
  • the non-coding amino acid of asparagine N-114.04278 at position 186 of the AKAP4 protein can be used as a potential biomarker for oligozoospermia, thereby predicting this condition.
  • AKAP4 protein 617 occurs with a -17.02660 mass shift of glutamine (labeled Q-17.02660)
  • Figure 7 is the ROC curve of the detection frequency of the 617 non-coding amino acid Q-17.02660 of AKAP4 protein.
  • the ROC analysis showed that the AUC of this non-coding amino acid Q-17.02660 was 0.802>0.7, indicating a good diagnostic effect.
  • the frequency of the test is 0.5
  • the sensitivity is 77.8% and the specificity is 79.4%.
  • the individual test is performed, when the frequency of detection is less than 0.5, it is judged to be a weak and weak patient (false positive rate is 20.6%).
  • Fig. 8 The comparison of the detection frequency of 617 non-coding amino acid Q-17.02660 of AKAP4 protein in healthy and oligospermic samples is shown in Fig. 8. It can be seen from Fig. 8 that this non-coding amino acid occurred on average 2.7 times in healthy human samples in pathological samples. It occurred 0.6 times (solid line in the figure), and the median (dashed line in the figure) was far from each other, indicating that this non-coding amino acid was largely lost in the sample.
  • the 617 site of AKAP4 protein glutamine Q-17.02660 a non-coding amino acid, can be used as a potential biomarker for oligozoospermia, thereby predicting this condition.
  • AKAP4 protein 733 occurs +211.09682 mass shift of lysine (labeled K+211.09682)
  • Figure 9 is the ROC curve of the detection frequency of 733 non-coding amino acid K+211.09682 of AKAP4 protein. ROC analysis showed that the AUC of this non-coding amino acid K+211.09682 was 0.804>0.7, indicating a good diagnostic effect.
  • the frequency of the test is 3.5, the sensitivity is 74.6% and the specificity is 71.4%.
  • the individual test was performed, when the frequency of detection was less than 3.5, it was judged to be a weak and weak patient (false positive rate was 28.6%).
  • the non-coding amino acid of lysine K+211.09682 at position 733 of AKAP4 protein can be used as a potential biomarker for oligozoospermia, thereby predicting this condition.
  • ATP5A1 protein 531 occurs +42.01108 mass shift of lysine
  • Figure 11 is the ROC curve of the detection frequency of ATP5A1 protein lysine acetylation modification K+42.01108, ROC Analysis showed that the AUC of this acetylation modification was 0.848>0.7, indicating that it has a good diagnostic effect.
  • the frequency of the test is 0.5
  • the sensitivity is 76.2%
  • the specificity is 85.7%.
  • the individual test when the frequency of detection was less than 0.5, it was judged to be a weak and weak patient (false positive rate was 14.3%).
  • the acetylation modification K+42.01108 of lysine at position 531 of ATP5A1 protein can be used as a potential biomarker for oligozoospermia, thereby predicting this condition.
  • Figure 13 is the ROC curve of the detection frequency of lysine acetylation modification K+42.01108 of COX4I1 protein.
  • the ROC analysis showed that the AUC of this acetylation modification was 0.803>0.7, indicating that it has a good diagnostic effect.
  • the frequency of the test is 0.5
  • the sensitivity is 66.7%
  • the specificity is 95.2%.
  • the individual test when the frequency of detection is less than 0.5, it is judged to be a weak and weak patient (false positive rate is 4.8%).
  • Figure 14 is a comparison of the detection frequency of the 87-position lysine acetylation modification K+42.01108 of the COX4I1 protein in healthy and weak-weak samples. It can be seen from Figure 14 that this non-coding amino acid occurred an average of 1.1 times in healthy human samples. The pathological sample occurred 0.1 times (solid line in the figure), and the median (dashed line in the figure) was far apart, indicating that this non-coding amino acid was largely lost in the sample.
  • the acetylation modification K+42.01108 on the 87th lysine of COX4I1 protein can be used as a potential biomarker for oligozoospermia, thereby predicting this condition.
  • GAPDHS protein at position 64 occurs with a loss of +79.96685 mass of threonine (labeled T+79.96685)
  • Figure 15 is the ROC curve of the detection frequency of phosphorylation modification T+79.96685 on the threonine of GAPDHS protein.
  • the ROC analysis showed that the AUC of this phosphorylation modification was 0.868>0.7, indicating a good diagnostic effect.
  • the frequency of the test is 3.5
  • the sensitivity is 71.4%
  • the specificity is 92.1%.
  • the individual test when the frequency of detection was less than 3.5, it was judged to be a weak and weak patient (false positive rate was 7.9%).
  • Figure 16 is a comparison of the detection frequency of the phosphorylated T+79.96685 on the threonine of the GAPDHS protein in healthy and low-weak samples. It can be seen from Figure 16 that this non-coding amino acid occurred 5.8 times on average in healthy human samples. In the pathological sample, 1.4 times (solid line in the figure), and the median (dashed line in the figure) is far apart, indicating that this non-coding amino acid is largely lost in the sample.
  • the phosphorylation modification T+79.96685 on the 64th position of GAPDHS protein can be used as a potential biomarker for oligozoospermia, thereby predicting this condition.
  • Figure 17 shows the ROC curve of the detection frequency of serine phosphorylation modification S+79.96685 of KIAA1683 protein.
  • the ROC analysis showed that the AUC of this phosphorylation modification was 0.808>0.7, indicating a good diagnostic effect.
  • the frequency of the test is 0.5
  • the sensitivity is 65.1% and the specificity is 95.2%.
  • the individual test is performed, when the frequency of detection is less than 0.5, it is judged to be a weak and weak patient (false positive rate is 4.8%).
  • Figure 18 shows the comparison of the detection frequency of serine phosphorylation modification S+79.96685 of KIAA1683 protein in healthy and weak samples. It can be seen from Figure 18 that this non-coding amino acid occurred an average of 2.1 times in healthy human samples in pathological samples. There were 0.1 occurrences (solid line in the figure), and the median (dashed line in the figure) was far apart, indicating that this non-coding amino acid was largely lost in the sample.
  • the phosphorylation modification S+79.96685 on serine at position 692 of KIAA1683 protein can be used as a potential biomarker for oligozoospermia, thereby predicting this condition.
  • the results showed that when the individual biomarkers were separately diagnosed, the diagnostic results were consistent with the known results. Description
  • the nine biomarkers screened by the present invention can each be used as a diagnostic marker for severe oligospermia.

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Abstract

提供一种与重度少弱精子症相关的生物标志物的筛选方法。首先利用NanoHPLC-MS/MS质谱系统和非标记定量蛋白质组学方法对多组重度少弱精疾病的精子蛋白非编码氨基酸进行质谱分析；然后利用非限定氨基酸蛋白质修饰分析方法对质谱数据进行搜索，再经过多变量高斯混合分布聚类分析，尽可能大量的鉴定出精子蛋白组中非编码氨基酸；最后通过正常和病人精子蛋白组中非编码氨基酸的比较，得到与重度少弱精症相关的蛋白非编码氨基酸位点，从而将其作为重度少弱精症的分子标志物，为重度少弱精症提供了新的诊断和治疗靶点。

Description

与重度少弱精子症相关的生物标志物的筛选与应用 技术领域
本发明涉及医学和分子诊断技术领域，具体涉及一种与重度少弱精子症相关的生物标志物的筛选与应用。
背景技术
不孕不育已成为全世界范围内的生殖健康问题，其中由于男性因素造成的不育大概占50％左右，且近年来呈上升的趋势。造成男性不育的主要原因是少精弱精症。根据世界卫生组织标准规定，如果a级精子数<25％，(a+b)级精子数<50％，且精子活率低于60％的话，就可诊断为弱精子症。少精子症是指精液中的精子数目低于正常具有生育能力男性的一种病症，当男性的精子在每毫升低于2千万时，就为少精子症。
目前关于精子蛋白质组的研究有很多。Saraswat等利用UPLC-MS的方法在人类精子中定量了667个蛋白，并且分析出了20个健康人和弱精症患者精子中的差异蛋白(Saraswat M,Joenvaara S,Jain T et al.Human Spermatozoa Quantitative Proteomic Signature Classifies Normo-and Asthenozoospermia.Molecular&cellular proteomics:MCP,16(1),57-72(2017).)。最新更新的人类精子蛋白质组共6198个蛋白(Amaral A,Castillo J,Ramalho-Santos J,Oliva R.The combined human sperm proteome:cellular pathways and implications for basic and clinical science.Human reproduction update,20(1),40-62(2014).)。Gaigai Wang等利用高分辨质谱在人类精子中鉴定出4675个蛋白(Wang G,Guo Y,Zhou T et al.In-depth proteomic analysis of the human sperm reveals complex protein compositions.Journal of proteomics,79,114-122(2013).)。络氨酸磷酸化对于精子的运动、获能、超激运动等过程重要作用。Chying-Chyuan Chan等通过对20组正常人和弱精症患者的精子进行蛋白质组学分析发现有12种包括TUBGCP2在内的蛋白发生了过磷酸化(Chan CC,Shui HA,Wu CH et al.Motility and Protein Phosphorylation in Healthy and Asthenozoospermic Sperm.Journal of proteome research,8(11),5382-5386(2009))。
非编码氨基酸包括翻译后修饰和氨基酸突变，是调控蛋白功能和结构的重要方式，因此将疾病状态下异常的或者数量变化极大的非编码氨基酸作为疾病的生物标志物，进而用于诊断疾病的进程具有重要意义。但目前还未有关于非编码氨基酸作为与重度少弱精子疾病相关的生物标志物的报道。
发明内容
针对上述现有技术，本发明的目的提供一种与重度少弱精子症相关的生物标志物的筛选与应用。本发明首先利用NanoHPLC-MS/MS质谱系统和非标记定量蛋白质组学方法对多组重度少弱精疾病的精子蛋白非编码氨基酸进行了深度的质谱分析；然后利用非限定氨基酸蛋白质修饰分析方法对质谱数据进行搜索，再经过多变量高斯混合分布聚类分析，尽可能大量的鉴定出精子蛋白组中非编码氨基酸；最后通过正常和病人精子蛋白组中非编码氨基酸的比较，得到与重度少弱精症相关的蛋白非编码氨基酸位点，从而将其作为重度少弱精症的分子标志物。
为实现上述目的，本发明采用如下技术方案：
本发明的第一方面，提供了一种与重度少弱精子症相关的生物标志物的筛选方法，包括如下步骤：
(1)提取精子细胞全蛋白；
(2)将精子细胞全蛋白采用凝胶电泳分离，切胶酶解，对酶解后的肽段进行脱盐，制备得到样品；
(3)将步骤(2)的样品采用纳流液相色谱分离，经纳流液相色谱分离后的样品再进行质谱检测，采集质谱数据；
(4)利用非限定氨基酸蛋白质修饰分析方法对质谱数据进行搜索，再经过多变量高斯混合分布聚类分析，尽可能大量的鉴定出精子蛋白组中非编码氨基酸；最后通过正常个体和重度少弱精个体精子蛋白组中非编码氨基酸的比较，得到与重度少弱精症相关的蛋白非编码氨基酸位点，即为与重度少弱精子症相关的生物标志物。
步骤(1)中，提取精子细胞全蛋白采用的方法为：将精子样本采用DPBS洗涤，加入RIPA裂解液超声1～2min，置于冰上孵育30min裂解，离心，取上清。
优选的，在4℃的条件下离心，离心转速为14,000g，离心时间为20min。
步骤(2)中，优选的，采用10％聚丙烯酰氨凝胶电泳(SDS-PAGE)对蛋白进行分离。
步骤(2)中，优选的，采用ziptip对酶解后的肽段进行脱盐。
步骤(3)中，纳流液相色谱分离的色谱条件为：流动相A：含有0.1％甲酸的水，流动相B：含有0.1％甲酸的乙腈；纳流液相质谱分析系统为Orbitrap Elite(Thermo Scientific)
洗脱条件为：0-100min，95-68％流动相A，5-32％流动相B；100-120min，68-20％流动相A，32-80％流动相B；120-150min，20％流动相A，80％流动相B；
流速为300nL/min。
步骤(3)中，质谱检测的条件为：350-1800m/z的全扫描，分辨率为60,000(m/z 200)。 二级图谱扫描时，活化时间为10ms，隔离宽度为2m/z；碎裂方式为诱导碰撞解离(collision-induced dissociation,CID)，归一化碰撞能量设定为35％，动态排出时间为90s。
步骤(4)中，对质谱数据进行搜索的参数设置为：蛋白酶为胰蛋白酶，漏切位点设置为2，母离子质量偏差为10ppm，碎片离子的质量偏差为0.6Da，盲搜上限设为1000，盲搜下限设为-200，蛋白FDR为0.01；
选择肽段分数>200和FDR<0.01的肽段作为Wildcard SearchTM搜索到的未知修饰数据，组成质量变化的一维数据矩阵(-200Da-400Da)，再将数据按照1Da的变化范围，0.5Da为界限，分割成601个数据窗口。
步骤(4)中，多变量高斯混合分布聚类分析的方法为：针对每一个数据窗口，用R语言中的mclust程序包做高斯混合分布聚类分析，根据BIC取最优值，再对每一个峰进行合并分析，然后用高斯分布拟合每一个峰，确定峰值；聚类之后的每个峰中所包含的肽段位点数据，根据位点氨基酸分布，选择分布大于5％的数据作为一类非编码氨基酸。
步骤(4)中，将正常个体和重度少弱精个体的非编码氨基酸按照其检测频率的T检验(p<0.05)和比值(ratio>2)进行筛选，从而得到差异非编码氨基酸。
上述筛选方法是用于获得生物标志物，且不是以获得疾病的诊断和治疗结果为目的；经上述筛选方法获得的生物标志物可用于重度少弱精子症的理论研究或者新药物的开发。
本发明的第二方面，提供根据上述筛选方法筛选得到的与重度少弱精子症相关的生物标志物，所述生物标志物包括但不限于：
AKAP3蛋白208位发生+79.96685质量偏移的丝氨酸(标记为S+79.96685；根据质量偏移值，确定该位置的丝氨酸发生了磷酸化修饰)；
AKAP4蛋白186位发生-113.05347质量偏移的天门冬酰胺(标记为N-113.05347)；
AKAP4蛋白186位发生-114.04278质量偏移的天门冬酰胺(标记为N-114.04278)；
AKAP4蛋白617位发生-17.02660质量偏移的谷氨酰胺(标记为Q-17.02660)；
AKAP4蛋白733位发生+211.09682质量偏移的赖氨酸(标记为K+211.09682)；
ATP5A1蛋白531位发生+42.01108质量偏移的赖氨酸(标记为K+42.01108；根据质量偏移值，确定该位置的赖氨酸发生了乙酰化修饰)；
COX4I1蛋白87位发生+42.01108质量偏移的赖氨酸(标记为K+42.01108；根据质量偏移值，确定该位置的赖氨酸发生了乙酰化修饰)；
GAPDHS蛋白64位发生+79.96685质量偏移的苏氨酸(标记为T+79.96685；根据质量偏移值，确定该位置的苏氨酸发生了磷酸化修饰)；
和KIAA1683蛋白692位发生+79.96685质量偏移的丝氨酸(标记为S+79.96685；根据质量偏移值，确定该位置的丝氨酸发生了磷酸化修饰)。
本发明的第三方面，提供AKAP3蛋白208位发生+79.96685质量偏移的丝氨酸作为生物标志物在制备重度少弱精的诊断试剂中的用途。
优选的，AKAP3蛋白208位发生+79.96685质量偏移的丝氨酸还可以作为重度少弱精治疗的靶标，从而用于重度少弱精的治疗。
进一步的，本发明还提供AKAP3蛋白208位发生+79.96685质量偏移的丝氨酸作为生物标志物在制备重度少弱精的治疗药物中的用途。
本发明还提供一种用于重度少弱精诊断的试剂盒，所述试剂盒中包括特异性检测上述生物标志物(AKAP3蛋白208位发生+79.96685质量偏移的丝氨酸)的试剂。
本发明还提供一种治疗重度少弱精的药物，所述药物中含有能够使AKAP3蛋白208位丝氨酸进行磷酸化修饰的组分。
本发明还提供一种重度少弱精的诊断方法，步骤为：检测待测样本AKAP3蛋白208位丝氨酸发生+79.96685质量偏移的频次，若检测频次小于1.5时，被判为少弱精患者。
本发明的第四方面，提供AKAP4蛋白186位发生-113.05347质量偏移的天门冬酰胺作为生物标志物在制备重度少弱精的诊断试剂中的用途。
本发明还提供一种用于重度少弱精诊断的试剂盒，所述试剂盒中包括特异性检测上述生物标志物(AKAP4蛋白186位发生-113.05347质量偏移的天门冬酰胺)的试剂。
本发明还提供一种重度少弱精的诊断方法，步骤为：检测待测样本AKAP4蛋白186位天门冬酰胺发生-113.05347质量偏移的频次，检测频次小于0.5时，被判为少弱精患者。
本发明的第五方面，提供AKAP4蛋白186位发生-114.04278质量偏移的天门冬酰胺作为生物标志物在制备重度少弱精的诊断试剂中的用途。
本发明还提供一种用于重度少弱精诊断的试剂盒，所述试剂盒中包括特异性检测上述生物标志物(AKAP4蛋白186位发生-114.04278质量偏移的天门冬酰胺)的试剂。
本发明还提供一种重度少弱精的诊断方法，步骤为：检测待测样本AKAP4蛋白186位天门冬酰胺发生-114.04278质量偏移的频次，检测频次小于0.5时，被判为少弱精患者。
本发明的第六方面，提供AKAP4蛋白617位发生-17.02660质量偏移的谷氨酰胺作为生物标志物在制备重度少弱精的诊断试剂中的用途。
本发明还提供一种用于重度少弱精诊断的试剂盒，所述试剂盒中包括特异性检测上述生物标志物(AKAP4蛋白617位发生-17.02660质量偏移的谷氨酰胺)的试剂。
本发明还提供一种重度少弱精的诊断方法，步骤为：检测待测样本AKAP4蛋白617位谷氨酰胺发生-17.02660质量偏移的频次，检测频次小于0.5时，被判为少弱精患者。
本发明的第七方面，提供AKAP4蛋白733位发生+211.09682质量偏移的赖氨酸作为生物标志物在制备重度少弱精的诊断试剂中的用途。
本发明还提供一种用于重度少弱精诊断的试剂盒，所述试剂盒中包括特异性检测上述生物标志物(AKAP4蛋白733位发生+211.09682质量偏移的赖氨酸)的试剂。
本发明还提供一种重度少弱精的诊断方法，步骤为：检测待测样本AKAP4蛋白733位赖氨酸发生+211.09682质量偏移的频次，检测频次小于3.5时，被判为少弱精患者。
本发明的第八方面，提供ATP5A1蛋白531位发生+42.01108质量偏移的赖氨酸作为生物标志物在制备重度少弱精的诊断试剂中的用途。
优选的，ATP5A1蛋白531位发生+42.01108质量偏移的赖氨酸还可以作为重度少弱精治疗的靶标，从而用于重度少弱精的治疗。
进一步的，本发明还提供ATP5A1蛋白531位发生+42.01108质量偏移的赖氨酸作为生物标志物在制备重度少弱精的治疗药物中的用途。
本发明还提供一种用于重度少弱精诊断的试剂盒，所述试剂盒中包括特异性检测上述生物标志物(ATP5A1蛋白531位发生+42.01108质量偏移的赖氨酸)的试剂。
本发明还提供一种治疗重度少弱精的药物，所述药物中含有能够使ATP5A1蛋白531位赖氨酸进行乙酰化修饰的组分。
本发明还提供一种重度少弱精的诊断方法，步骤为：检测待测样本ATP5A1蛋白531位赖氨酸发生+42.01108质量偏移的频次，若检测频次小于0.5时，被判为少弱精患者。
本发明的第九方面，提供COX4I1蛋白87位发生+42.01108质量偏移的赖氨酸作为生物标志物在制备重度少弱精的诊断试剂中的用途。
优选的，COX4I1蛋白87位发生+42.01108质量偏移的赖氨酸还可以作为重度少弱精治疗的靶标，从而用于重度少弱精的治疗。
进一步的，本发明还提供COX4I1蛋白87位发生+42.01108质量偏移的赖氨酸作为生物标志物在制备重度少弱精的治疗药物中的用途。
本发明还提供一种用于重度少弱精诊断的试剂盒，所述试剂盒中包括特异性检测上述生物标志物(COX4I1蛋白87位发生+42.01108质量偏移的赖氨酸)的试剂。
本发明还提供一种治疗重度少弱精的药物，所述药物中含有能够使COX4I1蛋白87位赖氨酸进行乙酰化修饰的组分。
本发明还提供一种重度少弱精的诊断方法，步骤为：检测待测样本COX4I1蛋白87位赖氨酸发生+42.01108质量偏移的频次，若检测频次小于0.5时，被判为少弱精患者。
本发明的第十方面，提供GAPDHS蛋白64位发生+79.96685质量偏移的苏氨酸作为生物标志物在制备重度少弱精的诊断试剂中的用途。
优选的，GAPDHS蛋白64位发生+79.96685质量偏移的苏氨酸还可以作为重度少弱精治疗的靶标，从而用于重度少弱精的治疗。
进一步的，本发明还提供GAPDHS蛋白64位发生+79.96685质量偏移的苏氨酸作为生物标志物在制备重度少弱精的治疗药物中的用途。
本发明还提供一种用于重度少弱精诊断的试剂盒，所述试剂盒中包括特异性检测上述生物标志物(GAPDHS蛋白64位发生+79.96685质量偏移的苏氨酸)的试剂。
本发明还提供一种治疗重度少弱精的药物，所述药物中含有能够使GAPDHS蛋白64位苏氨酸进行磷酸化修饰的组分。
本发明还提供一种重度少弱精的诊断方法，步骤为：检测待测样本GAPDHS蛋白64位苏氨酸发生+79.96685质量偏移的频次，若检测频次小于3.5时，被判为少弱精患者。
本发明的第十一方面，提供KIAA1683蛋白692位发生+79.96685质量偏移的丝氨酸作为生物标志物在制备重度少弱精的诊断试剂中的用途。
优选的，KIAA1683蛋白692位发生+79.96685质量偏移的丝氨酸还可以作为重度少弱精治疗的靶标，从而用于重度少弱精的治疗。
进一步的，本发明还提供KIAA1683蛋白692位发生+79.96685质量偏移的丝氨酸作为生物标志物在制备重度少弱精的治疗药物中的用途。
本发明还提供一种用于重度少弱精诊断的试剂盒，所述试剂盒中包括特异性检测上述生物标志物(KIAA1683蛋白692位发生+79.96685质量偏移的丝氨酸)的试剂。
本发明还提供一种治疗重度少弱精的药物，所述药物中含有能够使KIAA1683蛋白692位丝氨酸进行磷酸化修饰的组分。
本发明还提供一种重度少弱精的诊断方法，步骤为：检测待测样本KIAA1683蛋白692位丝氨酸发生+79.96685质量偏移的频次，若检测频次小于0.5时，被判为少弱精患者。
本发明的有益效果：
(1)本发明首次建立了一种与重度少弱精子症相关的生物标志物的筛选方法，通过对大量样本精子蛋白的质谱数据进行分析处理，尽可能大量的鉴定出精子蛋白组中非编码氨基酸；最后通过正常和病人精子蛋白组中非编码氨基酸的比较，得到与重度少弱精症相关的蛋白非 编码氨基酸位点，从而将其作为重度少弱精症的分子标志物。
(2)本发明进一步的对上述筛选方法得到的生物标志物进行研究，发现可以通过上述生物标志物的检验频次来诊断重度少弱精症，为重度少弱精症提供了新的诊断和治疗靶点。
附图说明
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。
图1：AKAP3蛋白208位丝氨酸上的磷酸化修饰S+79.96685检测频率的ROC曲线。
图2：健康和少弱精样本中AKAP3蛋白208位丝氨酸上的磷酸化修饰S+79.96685检测频率比较。
图3：AKAP4蛋白186位非编码氨基酸N-113.05347检测频率的ROC曲线。
图4：健康和少弱精样本的AKAP4蛋白186位非编码氨基酸N-113.05347检测频率比较。
图5：AKAP4蛋白186位非编码氨基酸N-114.04278检测频率的ROC曲线。
图6：健康和少弱精样本的AKAP4蛋白186位非编码氨基酸N-114.04278检测频率比较。
图7：AKAP4蛋白617位非编码氨基酸Q-17.02660检测频率的ROC曲线。
图8：健康和少弱精样本的AKAP4蛋白617位非编码氨基酸Q-17.02660检测频率比较。
图9：AKAP4蛋白733位非编码氨基酸K+211.09682检测频率的ROC曲线。
图10：健康和少弱精样本的非编码氨基酸K+211.09682检测频率比较。
图11：ATP5A1蛋白531位赖氨酸乙酰化修饰K+42.01108检测频率的ROC曲线。
图12：健康和少弱精样本的ATP5A1蛋白531位赖氨酸乙酰化修饰K+42.01108检测频率比较。
图13：COX4I1蛋白87位赖氨酸乙酰化修饰K+42.01108检测频率的ROC曲线。
图14：健康和少弱精样本的COX4I1蛋白87位赖氨酸乙酰化修饰K+42.01108检测频率比较。
图15：GAPDHS蛋白64位苏氨酸上的磷酸化修饰T+79.96685检测频率的ROC曲线。
图16：健康和少弱精样本中GAPDHS蛋白64位苏氨酸上的磷酸化修饰T+79.96685检测频率比较。
图17：KIAA1683蛋白692位丝氨酸磷酸化修饰S+79.96685检测频率的ROC曲线。
图18：健康和少弱精样本中KIAA1683蛋白692位丝氨酸磷酸化修饰S+79.96685检测频率比较。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
正如背景技术所介绍的，现有技术中还未有关于非编码氨基酸作为与重度少弱精子症相关的生物标志物的报道。基于此，本发明提出了一种与重度少弱精子症相关的生物标志物的筛选方法与应用。
在本申请的一种实施方案中，提出了一种与重度少弱精子症相关的生物标志物的筛选方法，包括如下步骤：
(1)提取精子细胞全蛋白；
(2)将精子细胞全蛋白采用凝胶电泳分离，切胶酶解，对酶解后的肽段进行脱盐，制备得到样品；
(3)将步骤(2)的样品采用纳流液相色谱分离，经纳流液相色谱分离后的样品再进行质谱检测，采集质谱数据；
(4)利用非限定氨基酸蛋白质修饰分析方法对质谱数据进行搜索，再经过多变量高斯混合分布聚类分析，尽可能大量的鉴定出精子蛋白组中非编码氨基酸；最后通过正常个体和重度少弱精个体精子蛋白组中非编码氨基酸的比较，得到与重度少弱精症相关的蛋白非编码氨基酸位点，即为与重度少弱精子症相关的生物标志物。
由于精子样本的易得性，精子成熟后其转录和翻译处于停滞状态，这也为我们在蛋白质水平上研究少弱精子症提供了方便，本申请首先利用NanoHPLC-MS/MS质谱系统和非标记定量蛋白质组学方法对多组重度少弱精疾病的精子蛋白非编码氨基酸进行了深度的质谱分析；然后利用非限定氨基酸蛋白质修饰分析方法对质谱数据进行搜索，再经过多变量高斯混合分布聚类分析，尽可能大量的鉴定出精子蛋白组中非编码氨基酸。最后将正常与患病组的非编码氨基酸按照其检测频率的T检验(p<0.05)和比值(ratio>2)进行筛选，从而得到差异非编码氨基酸。然后利用SPSS软件作出差异非编码氨基酸ROC曲线，并计算其曲线下面积(AUC)，进而判断其诊断价值。
采用本申请的上述筛选方法，得到了系列与重度少弱精子症相关的生物标志物，具体如 下：
AKAP3蛋白208位发生+79.96685质量偏移的丝氨酸(标记为S+79.96685；根据质量偏移值，确定该位置的丝氨酸发生了磷酸化修饰)；
AKAP4蛋白186位发生-113.05347质量偏移的天门冬酰胺(标记为N-113.05347)；
AKAP4蛋白186位发生-114.04278质量偏移的天门冬酰胺(标记为N-114.04278)；
AKAP4蛋白617位发生-17.02660质量偏移的谷氨酰胺(标记为Q-17.02660)；
AKAP4蛋白733位发生+211.09682质量偏移的赖氨酸(标记为K+211.09682)；
ATP5A1蛋白531位发生+42.01108质量偏移的赖氨酸(标记为K+42.01108；根据质量偏移值，确定该位置的赖氨酸发生了乙酰化修饰)；
COX4I1蛋白87位发生+42.01108质量偏移的赖氨酸(标记为K+42.01108；根据质量偏移值，确定该位置的赖氨酸发生了乙酰化修饰)；
GAPDHS蛋白64位发生+79.96685质量偏移的苏氨酸(标记为T+79.96685；根据质量偏移值，确定该位置的苏氨酸发生了磷酸化修饰)；
和KIAA1683蛋白692位发生+79.96685质量偏移的丝氨酸(标记为S+79.96685；根据质量偏移值，确定该位置的丝氨酸发生了磷酸化修饰)。
在本申请的另一种实施方案中，提出了一种用于重度少弱精诊断的试剂盒，所述试剂盒中包括特异性检测上述生物标志物的试剂。
通过对上述生物标志物进行检测，可以实现对重度少弱精症的诊断。
在本申请的另一种实施方案中，提出了一种治疗重度少弱精的药物，所述药物中含有能够使AKAP3蛋白208位丝氨酸、GAPDHS蛋白64位苏氨酸或KIAA1683蛋白692位丝氨酸进行磷酸化修饰的组分，或者含有能够使ATP5A1蛋白531位赖氨酸或COX4I1蛋白87位赖氨酸进行乙酰化修饰的组分。
经研究发现，磷酸化修饰的AKAP3蛋白208位丝氨酸、GAPDHS蛋白64位苏氨酸和KIAA1683蛋白692位丝氨酸在重度少弱精样本中显著性的下调，乙酰化修饰的ATP5A1蛋白531位赖氨酸或COX4I1蛋白87位赖氨酸也在重度少弱精样本中显著性的下调。由此可以合理预期，以上述生物标志物作为靶标，通过对多重度少弱精患者该靶标处的氨基酸进行磷酸化或乙酰化修饰，可以起到对重度少弱精的治疗作用。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案，以下将结合具体的实施例详细说明本申请的技术方案。
本发明实施例中所用的试验材料均为本领域常规的试验材料，均可通过商业渠道购买得 到。
实施例1：与重度少弱精子症相关的生物标志物的筛选
具体筛选方法如下：
一、样本处理及实验分析
1.精子细胞全蛋白的提取：等量的重度少弱精和正常精子样本分别用DPBS洗三次，加入等量RIPA裂解液超声1～2min，置于冰上孵育30min裂解，4℃离心14,000g×20min取上清。利用Bradford方法测定蛋白浓度。
2.蛋白酶解：取约重度少弱精和正常精子样本各150μg精子蛋白，使用10％聚丙烯酰氨凝胶电泳(SDS-PAGE)对蛋白进行分离，各分成5份进行切胶酶解。使用ziptip对肽段进行脱盐。
3.质谱分析：纳流液相色谱分离：A相：含有0.1％甲酸的水；B相：含有0.1％甲酸的乙腈
每个样品分别用13.5μL A相溶解,样品进样量为4μL，纳流液相质谱分析系统为Orbitrap Elite(Thermo Scientific)。样品分离之前分别用4μL A相平衡自制的预柱和分析柱。预柱和分析柱的规格分别为：预柱(4cm×150μm I.D.,C18填料粒径5μm,
Figure PCTCN2017106915-appb-000001
)，分析柱(30cm×75μm I.D.,C18填料填充，粒径3μm,
Figure PCTCN2017106915-appb-000002
Dr.Maisch GmbH,Germany)。平衡之后样品在A相的带动下首先载样于预柱，然后在不同梯度下进行液相分离。150min色谱梯度变化如下：5-32％流动相B 100min；32-80％流动相B，20min；80％流动相B，30min。流速始终保持在300nL/min。经过纳流液相分离的样品直接进入ESI离子喷雾源并进入Orbitrap Elite质谱仪中进行质谱检测。
质谱数据采集：350‐1800m/z的全扫描，分辨率为60,000(m/z 200)。二级图谱扫描时，活化时间为10ms，隔离宽度为2m/z。碎裂方式为诱导碰撞解离(collision-induced dissociation,CID)，归一化碰撞能量设定为35％，动态排出时间为90s。
二、质谱数据分析
Byonic分析：为了鉴定出精子蛋白的非编码氨基酸，我们用ByonicTM分析21对正常与重度少弱精患者的精蛋白质谱数据。搜索参数如下：蛋白酶为胰蛋白酶，漏切位点设置为2，母离子质量偏差为10ppm，碎片离子的质量偏差为0.6Da,盲搜上限设为1000，盲搜下限设为-200.蛋白FDR为0.01。
选择肽段分数>200和FDR<0.01的肽段作为Wildcard SearchTM搜索到的未知修饰数据，组成质量变化的一维数据矩阵(-200Da-400Da)，再将数据按照1Da的变化范围，0.5Da为界 限，分割成601个数据窗口。针对每一个数据窗口，用R语言中的mclust程序包做高斯混合分布聚类分析，根据BIC取最优值，再对每一个峰进行合并分析，然后用高斯分布拟合每一个峰，确定峰值。聚类之后的每个峰中所包含的肽段位点数据，根据位点氨基酸分布，选择分布大于5％的数据作为一类非编码氨基酸。
将正常与患病组的非编码氨基酸按照其检测频率的T检验(p<0.05)和比值(ratio>2)进行筛选，从而得到差异非编码氨基酸。然后利用SPSS软件作出差异非编码氨基酸ROC曲线，并计算其曲线下面积(AUC)，进而判断其诊断价值。
三、实验结果：
经质谱数据分析和将正常与患病组的非编码氨基酸比较，从而得到9个差异非编码氨基酸，可以作为与重度少弱精子症相关的生物标志物，具体如下：
1.AKAP3蛋白208位发生+79.96685质量偏移的丝氨酸(标记为S+79.96685；根据质量偏移值，确定该位置的丝氨酸发生了磷酸化修饰)
我们发现AKAP3蛋白208位丝氨酸上发生了磷酸化修饰S+79.96685，经过比较发现这一磷酸化修饰在重度少弱精样本显著性下调了10.7倍，p值为7.49E-15<0.05。
为评价AKAP3蛋白208位丝氨酸上的磷酸化修饰S+79.96685检测频率对重度少弱精的诊断效能，本发明采用了ROC曲线分析，AUC为ROC曲线下的面积，是最常用的评价ROC曲线特征的参数，是重要的试验准确度指标。若AUC在0.7以下，则表示诊断的准确率较低；AUC在0.7以上，则可以满足临床诊断的要求。
图1为AKAP3蛋白208位丝氨酸上的磷酸化修饰S+79.96685检测频率的ROC曲线，ROC分析显示这一磷酸化修饰的AUC为0.856>0.7，说明具有较好的诊断效果，即AKAP3蛋白208位丝氨酸上的磷酸化修饰S+79.96685可以作为重度少弱精的诊断标志物。
在检验频次为1.5时，灵敏度为73％，特异度为88.9％。当进行个体检测时，检测频次小于1.5时，被判为少弱精患者(假阳性率为11.1％)。
健康和少弱精样本中AKAP3蛋白208位丝氨酸上的磷酸化修饰S+79.96685检测频率比较结果见图2，由图2可以看出这一非编码氨基酸在健康人样本中平均发生了4.6次而在病理样本中发生了0.4次(图中实线)，并且其中位数(图中虚线)相差较远，说明在少弱精样本中这一非编码氨基酸有较大程度地丢失。
鉴于上述结果，AKAP3蛋白208位丝氨酸上的磷酸化修饰S+79.96685可以作为少弱精子症的潜在生物标志物，从而对这一病症进行预测。
2.AKAP4蛋白186位发生-113.05347质量偏移的天门冬酰胺(标记为N-113.05347)
经过质谱数据分析，我们发现AKAP4蛋白的186位的天门冬酰胺有-113.05347的质量偏移(N-113.05347)，经过比较发现这一非编码氨基酸N-113.05347在重度少弱精样本显著性下调了9.2倍，p值为4.60E-13<0.05。
图3为AKAP4蛋白186位非编码氨基酸N-113.05347检测频率的ROC曲线，ROC分析显示这一非编码氨基酸N-113.05347的AUC为0.852>0.7，说明具有较好的诊断效果。在检验频次为1.5时，灵敏度为65.1％，特异度为93.7％。当进行个体检测时，检测频次小于1.5时，被判为少弱精患者(假阳性率为6.3％)。
图4比较了非编码氨基酸N-113.05347在健康和少弱精样本的检测频率，可以看出这一非编码氨基酸在健康人样本中平均发生了2.9次而在病理样本中发生了0.3次(图中实线)，并且其中位数(图中虚线)相差较远，说明在少弱精样本中这一非编码氨基酸有较大程度地丢失。
鉴于上述结果，AKAP4蛋白的186位点的天门冬酰胺N-113.05347这一非编码氨基酸可以作为少弱精子症的潜在生物标志物，从而对这一病症进行预测。
3.AKAP4蛋白186位发生-114.04278质量偏移的天门冬酰胺(标记为N-114.04278)
经过质谱数据分析，我们发现AKAP4蛋白的186位的天门冬酰胺有-114.04278的质量偏移(N-114.04278)，经过比较发现这一非编码氨基酸N-114.04278在重度少弱精样本显著性下调了7.3倍，p值为1.11E-11<0.05。
图5为AKAP4蛋白186位非编码氨基酸N-114.04278检测频率的ROC曲线，ROC分析显示这一非编码氨基酸N-114.04278的AUC为0.817>0.7，说明具有较好的诊断效果。在检验频次为0.5时，灵敏度为74.6％，特异度为84.1％。当进行个体检测时，检测频次小于0.5时，被判为少弱精患者(假阳性率为15.9％)。
健康和少弱精样本的AKAP4蛋白186位非编码氨基酸N-114.04278检测频率比较见图6，由图6可以看出这一非编码氨基酸在健康人样本中平均发生了1.6次而在病理样本中发生了0.2次(图中实线)，并且其中位数(图中虚线)相差较远，说明在少弱精样本中这一非编码氨基酸有较大程度地丢失。
鉴于上述结果，AKAP4蛋白的186位点的天门冬酰胺N-114.04278这一非编码氨基酸可以作为少弱精子症的潜在生物标志物，从而对这一病症进行预测。
4.AKAP4蛋白617位发生-17.02660质量偏移的谷氨酰胺(标记为Q-17.02660)
经过质谱数据分析，我们发现AKAP4蛋白的617位的谷氨酰胺有-17.02660的质量偏移(Q-17.02660)，经过比较发现这一非编码氨基酸Q-17.02660在重度少弱精样本显著性下调了 4.8倍，p值为2.21E-09<0.05。
图7为AKAP4蛋白617位非编码氨基酸Q-17.02660检测频率的ROC曲线，ROC分析显示这一非编码氨基酸Q-17.02660的AUC为0.802>0.7，说明具有较好的诊断效果。在检验频次为0.5时，灵敏度为77.8％，特异度为79.4％。当进行个体检测时，检测频次小于0.5时，被判为少弱精患者(假阳性率为20.6％)。
健康和少弱精样本的AKAP4蛋白617位非编码氨基酸Q-17.02660检测频率比较见图8，由图8可以看出这一非编码氨基酸在健康人样本中平均发生了2.7次而在病理样本中发生了0.6次(图中实线)，并且其中位数(图中虚线)相差较远，说明在少弱精样本中这一非编码氨基酸有较大程度地丢失。
鉴于上述结果，AKAP4蛋白的617位点的谷氨酰胺Q-17.02660这一非编码氨基酸可以作为少弱精子症的潜在生物标志物，从而对这一病症进行预测。
5.AKAP4蛋白733位发生+211.09682质量偏移的赖氨酸(标记为K+211.09682)
经过质谱数据分析，我们发现AKAP4蛋白的733位的赖氨酸有211.09682的质量偏移(K+211.09682)，经过比较发现这一非编码氨基酸K+211.09682在重度少弱精样本显著性下调了4.4倍，p值为5.79E-11<0.05。
图9为AKAP4蛋白733位非编码氨基酸K+211.09682检测频率的ROC曲线，ROC分析显示这一非编码氨基酸K+211.09682的AUC为0.804>0.7，说明具有较好的诊断效果。在检验频次为3.5时，灵敏度为74.6％，特异度为71.4％。当进行个体检测时，检测频次小于3.5时，被判为少弱精患者(假阳性率为28.6％)。
健康和少弱精样本的非编码氨基酸K+211.09682检测频率比较见图10，由图10可以看出这一非编码氨基酸在健康人样本中平均发生了14次而在病理样本中发生了3次(图中实线)，并且其中位数(图中虚线)相差较远，说明在少弱精样本中这一非编码氨基酸有较大程度地丢失。
鉴于上述结果，AKAP4蛋白的733位点的赖氨酸K+211.09682这一非编码氨基酸可以作为少弱精子症的潜在生物标志物，从而对这一病症进行预测。
6.ATP5A1蛋白531位发生+42.01108质量偏移的赖氨酸
经过质谱数据分析，我们发现ATP5A1蛋白531位赖氨酸上发生了乙酰化修饰K+42.01108，经过比较发现这一乙酰化修饰在重度少弱精样本显著性下调了10.5倍，p值为3.48E-16<0.05。
图11为ATP5A1蛋白531位赖氨酸乙酰化修饰K+42.01108检测频率的ROC曲线，ROC 分析显示这一乙酰化修饰的AUC为0.848>0.7，说明具有较好的诊断效果。在检验频次为0.5时，灵敏度为76.2％，特异度为85.7％。当进行个体检测时，检测频次小于0.5时，被判为少弱精患者(假阳性率为14.3％)。
健康和少弱精样本的ATP5A1蛋白531位赖氨酸乙酰化修饰K+42.01108检测频率比较见图12，可以看出这一非编码氨基酸在健康人样本中平均发生了1.7次而在病理样本中发生了0.2次(图中实线)，并且其中位数(图中虚线)相差较远，说明在少弱精样本中这一非编码氨基酸有较大程度地丢失。
鉴于上述结果，ATP5A1蛋白531位赖氨酸上的乙酰化修饰K+42.01108可以作为少弱精子症的潜在生物标志物，从而对这一病症进行预测。
7.COX4I1蛋白87位发生+42.01108质量偏移的赖氨酸(标记为K+42.01108)
经过质谱数据分析，我们发现COX4I1蛋白87位赖氨酸上发生了乙酰化修饰K+42.01108，经过比较发现这一乙酰化修饰在重度少弱精样本显著性下调了11.3倍，p值为5.06E-13<0.05。
图13为COX4I1蛋白87位赖氨酸乙酰化修饰K+42.01108检测频率的ROC曲线，ROC分析显示这一乙酰化修饰的AUC为0.803>0.7，说明具有较好的诊断效果。在检验频次为0.5时，灵敏度为66.7％，特异度为95.2％。当进行个体检测时，检测频次小于0.5时，被判为少弱精患者(假阳性率为4.8％)。
图14为健康和少弱精样本的COX4I1蛋白87位赖氨酸乙酰化修饰K+42.01108检测频率比较，由图14可以看出这一非编码氨基酸在健康人样本中平均发生了1.1次而在病理样本中发生了0.1次(图中实线)，并且其中位数(图中虚线)相差较远，说明在少弱精样本中这一非编码氨基酸有较大程度地丢失。
鉴于上述结果，COX4I1蛋白87位赖氨酸上的乙酰化修饰K+42.01108可以作为少弱精子症的潜在生物标志物，从而对这一病症进行预测。
8.GAPDHS蛋白64位发生+79.96685质量偏移的苏氨酸(标记为T+79.96685)
经过质谱数据分析，我们发现GAPDHS蛋白的64位苏氨酸上发生了磷酸化修饰T+79.96685，经过比较发现这一磷酸化修饰在重度少弱精样本显著性下调了4.1倍，p值为1.82E-14<0.05。
图15为GAPDHS蛋白64位苏氨酸上的磷酸化修饰T+79.96685检测频率的ROC曲线，ROC分析显示这一磷酸化修饰的AUC为0.868>0.7，说明具有较好的诊断效果。在检验频次为3.5时，灵敏度为71.4％，特异度为92.1％。当进行个体检测时，检测频次小于3.5时，被判为少弱精患者(假阳性率为7.9％)。
图16为健康和少弱精样本中GAPDHS蛋白64位苏氨酸上的磷酸化修饰T+79.96685检测频率比较，由图16可以看出这一非编码氨基酸在健康人样本中平均发生了5.8次而在病理样本中发生了1.4次(图中实线)，并且其中位数(图中虚线)相差较远，说明在少弱精样本中这一非编码氨基酸有较大程度地丢失。
鉴于上述结果，GAPDHS蛋白64位苏氨酸上的磷酸化修饰T+79.96685可以作为少弱精子症的潜在生物标志物，从而对这一病症进行预测。
9.KIAA1683蛋白692位发生+79.96685质量偏移的丝氨酸(标记为S+79.96685)
经过质谱数据分析，我们发现KIAA1683蛋白692位丝氨酸上发生了磷酸化修饰S+79.96685，经过比较发现这一磷酸化修饰在重度少弱精样本显著性下调了22倍，p值为2.73E-10<0.05。
图17为KIAA1683蛋白692位丝氨酸磷酸化修饰S+79.96685检测频率的ROC曲线，ROC分析显示这一磷酸化修饰的AUC为0.808>0.7，说明具有较好的诊断效果。在检验频次为0.5时，灵敏度为65.1％，特异度为95.2％。当进行个体检测时，检测频次小于0.5时，被判为少弱精患者(假阳性率为4.8％)。
图18为健康和少弱精样本中KIAA1683蛋白692位丝氨酸磷酸化修饰S+79.96685检测频率比较，由图18可以看出这一非编码氨基酸在健康人样本中平均发生了2.1次而在病理样本中发生了0.1次(图中实线)，并且其中位数(图中虚线)相差较远，说明在少弱精样本中这一非编码氨基酸有较大程度地丢失。
鉴于上述结果，KIAA1683蛋白692位丝氨酸上的磷酸化修饰S+79.96685可以作为少弱精子症的潜在生物标志物，从而对这一病症进行预测。
实施例2：临床检测验证
以4例健康样本、8例已临床确诊的重度少弱精样本作为研究对象进行验证，分别检测上述样本的AKAP3蛋白208位发生+79.96685质量偏移的丝氨酸、AKAP4蛋白186位发生-113.05347质量偏移的天门冬酰胺、AKAP4蛋白186位发生-114.04278质量偏移的天门冬酰胺、AKAP4蛋白617位发生-17.02660质量偏移的谷氨酰胺、AKAP4蛋白733位发生+211.09682质量偏移的赖氨酸、ATP5A1蛋白531位发生+42.01108质量偏移的赖氨酸、COX4I1蛋白87位发生+42.01108质量偏移的赖氨酸、GAPDHS蛋白64位发生+79.96685质量偏移的苏氨酸和KIAA1683蛋白692位发生+79.96685质量偏移的丝氨酸各自的检测频次，并按实施例1中各生物标志物进行个体检测时的判断标准对待测样本进行诊断。
结果表明：分别以上述各生物标志物进行单独诊断时，诊断结果与已知结果一致。说明 本发明筛选得到的9个生物标志物可以各自作为重度少弱精的诊断标志物。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。

Claims (10)

  1. 一种与重度少弱精子症相关的生物标志物的筛选方法，其特征在于，包括如下步骤：
    (1)提取精子细胞全蛋白；
    (2)将精子细胞全蛋白采用凝胶电泳分离，切胶酶解，对酶解后的肽段进行脱盐，制备得到样品；
    (3)将步骤(2)的样品采用纳流液相色谱分离，经纳流液相色谱分离后的样品再进行质谱检测，采集质谱数据；
    (4)利用非限定氨基酸蛋白质修饰分析方法对质谱数据进行搜索，再经过多变量高斯混合分布聚类分析，尽可能大量的鉴定出精子蛋白组中非编码氨基酸；最后通过正常个体和重度少弱精个体精子蛋白组中非编码氨基酸的比较，得到与重度少弱精症相关的蛋白非编码氨基酸位点，即为诊断和/或治疗重度少弱精子疾病的生物标志物。
  2. 如权利要求1所述的筛选方法，其特征在于，步骤(1)中，提取精子细胞全蛋白采用的方法为：将精子样本采用DPBS洗涤，加入RIPA裂解液超声1～2min，置于冰上孵育30min裂解，离心，取上清；
  3. 如权利要求2所述的筛选方法，其特征在于，在4℃的条件下离心，离心转速为14,000g，离心时间为20min。
  4. 如权利要求1所述的筛选方法，其特征在于，步骤(2)中，采用10％聚丙烯酰氨凝胶电泳对蛋白进行分离。
  5. 如权利要求1所述的筛选方法，其特征在于，步骤(2)中，采用ziptip对酶解后的肽段进行脱盐。
  6. 如权利要求1所述的筛选方法，其特征在于，步骤(3)中，纳流液相色谱分离的色谱条件为：流动相A：含有0.1％甲酸的水，流动相B：含有0.1％甲酸的乙腈；
    洗脱条件为：0-100min，95-68％流动相A，5-32％流动相B；100-120min，68-20％流动相A，32-80％流动相B；120-150min，20％流动相A，80％流动相B。
  7. 如权利要求1所述的筛选方法，其特征在于，步骤(3)中，质谱检测的条件为：350-1800m/z的全扫描，分辨率为60,000(m/z200)。二级图谱扫描时，活化时间为10ms，隔离宽度为2m/z；碎裂方式为诱导碰撞解离，归一化碰撞能量设定为35％，动态排出时间为90s。
  8. 如权利要求1所述的筛选方法，其特征在于，步骤(4)中，对质谱数据进行搜索的参数设置为：蛋白酶为胰蛋白酶，漏切位点设置为2，母离子质量偏差为10ppm，碎片离子的质量偏差为0.6Da，盲搜上限设为1000，盲搜下限设为-200，蛋白FDR为0.01；
    选择肽段分数>200和FDR<0.01的肽段作为Wildcard SearchTM搜索到的未知修饰数据， 组成质量变化的一维数据矩阵(-200Da-400Da)，再将数据按照1Da的变化范围，0.5Da为界限，分割成601个数据窗口。
  9. 如权利要求1所述的筛选方法，其特征在于，步骤(4)中，多变量高斯混合分布聚类分析的方法为：针对每一个数据窗口，用R语言中的mclust程序包做高斯混合分布聚类分析，根据BIC取最优值，再对每一个峰进行合并分析，然后用高斯分布拟合每一个峰，确定峰值；聚类之后的每个峰中所包含的肽段位点数据，根据位点氨基酸分布，选择分布大于5％的数据作为一类非编码氨基酸。
  10. 权利要求1-9任一项所述的筛选方法筛选得到的与重度少弱精子症相关的生物标志物。
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