CN106872630B - With the screening and application of the relevant biomarker of severe teen bra - Google Patents
With the screening and application of the relevant biomarker of severe teen bra Download PDFInfo
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Abstract
The invention discloses a kind of and relevant biomarker of severe teen bra screening techniques, and the mass spectral analysis of depth has been carried out first with the sperm protein undoded amino acid of NanoHPLC MS/MS mass spectrometer systems and label-free proteomics method weak smart disease few to multigroup severe;Then mass spectrometric data is scanned for using non-limiting aminoacid protein modification analysis method, using multivariate Gaussian mixed distribution clustering, largely identifies undoded amino acid in sperm protein group as far as possible;Finally by the comparison of undoded amino acid in normal and patient's sperm protein group, it obtains lacking azoospermia relevant albumen undoded amino acid site with severe, to lack the molecular marker of azoospermia as severe, lacks azoospermia for severe and provide new diagnosing and treating target spot.
Description
Technical field
The present invention relates to medicine and molecular diagnostic techniques field, and in particular to a kind of and relevant life of severe teen bra
The screening and application of object marker.
Background technology
The infertile healthy reproduction problem having become in worldwide, wherein since infertility caused by male factor is big
It is general to account for 50% or so, and be in the trend risen in recent years.The main reason for causing male sterility is oligoasthenospermia disease.According to the world
Health Organization criterion provides, if a grades of sperm counts<25%, (a+b) grade sperm count<50%, and sperm motility rate is less than 60%
Words, so that it may be diagnosed as asthénospermie.Oligospermatism refers to the sperm number in sperm has fecundity male less than normal
A kind of illness is just oligospermatism when the sperm of male is less than 2,000 ten thousand at every milliliter.
The research about sperm protein matter group has much at present.Saraswat etc. is using the method for UPLC-MS in Human Sperm
667 albumen have been quantified in son, and have analyzed the differential protein in 20 Healthy Peoples and azoospermia patient's sperm
(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).).Last updated human sperm's protein group totally 6198 albumen
(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 etc. utilize high-resolution
Mass spectrum identifies 4675 albumen (Wang G, Guo Y, Zhou T et al.In-depth proteomic in human sperm
analysis of the human sperm reveals complex protein compositions.Journal of
proteomics,79,114-122(2013).).Tyrosine phosphorylation is for processes such as the movement of sperm, capacitation, super sharp movements
Important function.Chying-Chyuan Chan etc. carry out proteomics by the sperm to 20 groups of normal persons and azoospermia patient
Analysis finds that peroxophosphoric acid (Chan CC, Shui HA, Wu CH et have occurred in 12 kinds of albumen including TUBGCP2
al.Motility and Protein Phosphorylation in Healthy and Asthenozoospermic
Sperm.Journal of proteome research,8(11),5382-5386(2009))。
Undoded amino acid includes posttranslational modification and amino acid mutation, is the important side of modulin function and structure
Formula, therefore abnormal under morbid state or quantity is changed into great undoded amino acid as the biomarker of disease,
And then the process for diagnosing the illness is of great significance.But at present also not about undoded amino acid as few weak with severe
The report of the relevant biomarker of sperm disease.
Invention content
For the above-mentioned prior art, it is an object of the invention to provide a kind of and relevant biomarkers of severe teen bra
Screening and application.The invention firstly uses NanoHPLC-MS/MS mass spectrometer systems and label-free proteomics method pair
The sperm protein undoded amino acid of the few weak smart disease of multigroup severe has carried out the mass spectral analysis of depth;Then non-limiting ammonia is utilized
Base acid protein modification analysis method scans for mass spectrometric data, using multivariate Gaussian mixed distribution clustering, to the greatest extent
Undoded amino acid in sperm protein group may largely be identified;Finally by non-coding in normal and patient's sperm protein group
The comparison of amino acid obtains lacking azoospermia relevant albumen undoded amino acid site with severe, to few as severe
The molecular marker of azoospermia.
To achieve the above object, the present invention adopts the following technical scheme that:
The first aspect of the present invention provides a kind of and relevant biomarker of severe teen bra screening side
Method includes the following steps:
(1) spermatoblast holoprotein is extracted;
(2) spermatoblast holoprotein is detached using gel electrophoresis, cuts glue enzymolysis, desalination is carried out to the peptide fragment after enzymolysis,
Sample is prepared;
(3) by the sample of step (2) using receiving flow liquid phase chromatographic isolation, through receive the sample after flow liquid phase chromatographic isolation again into
Row Mass Spectrometer Method acquires mass spectrometric data;
(4) mass spectrometric data is scanned for using non-limiting aminoacid protein modification analysis method, using multivariable
Gaussian Mixture distributed clustering analysis largely identifies undoded amino acid in sperm protein group as far as possible;Finally by normal
The comparison of undoded amino acid, obtains lacking the relevant egg of azoospermia with severe in individual and the few weak individual sperm protein group of essence of severe
White undoded amino acid site, as with the relevant biomarker of severe teen bra.
In step (1), the method that uses of extraction spermatoblast holoprotein for:Sperm sample is washed using DPBS, is added
RIPA lysate 1~2min of ultrasound, are placed in and are incubated 30min cracking on ice, and centrifugation takes supernatant.
Preferably, it is centrifuged under conditions of 4 DEG C, centrifugal rotational speed 14,000g, centrifugation time 20min.
In step (2), it is preferred that detached to albumen using 10% polyacrylamide gel electrophoresis (SDS-PAGE).
In step (2), it is preferred that carry out desalination to the peptide fragment after enzymolysis using ziptip.
In step (3), the chromatographic condition of flow liquid phase chromatographic isolation received is:Mobile phase A:Water containing 0.1% formic acid, flowing
Phase B:Acetonitrile containing 0.1% formic acid;Flow liquid phase mass spectrometry system of receiving is Orbitrap Elite (Thermo
Scientific)
Elution requirement is:0-100min, 95-68% mobile phase A, 5-32% Mobile phase Bs;100-120min, 68-20% flow
Dynamic phase A, 32-80% Mobile phase B;120-150min, 20% mobile phase A, 80% Mobile phase B;
Flow velocity is 300nL/min.
In step (3), the condition of Mass Spectrometer Method is:The full scan of 350-1800m/z, 60,000 (m/z of resolution ratio
200).When second order spectrum scans, soak time 10ms, isolation width is 2m/z;Fragmentation pattern is that induction is collisionally dissociated
(collision-induced dissociation, CID), normalization collision energy are set as 35%, and dynamic efflux time is
90s。
In step (4), the parameter that mass spectrometric data scans for is set as:Protease is trypsase, and leakage enzyme site is set
It is set to 2, parent ion mass deviation is 10ppm, and the mass deviation of fragment ion is 0.6Da, and the blind upper limit of searching is set as 1000, blind to search down
Limit is set as -200, and albumen FDR is 0.01;
Select peptide fragment score>200 and FDR<The unknown modifications that 0.01 peptide fragment is searched as Wildcard SearchTM
Data form the one-dimensional data matrix (- 200Da-400Da) of mass change, then by data according to the variation range of 1Da, 0.5Da
For boundary, it is divided into 601 data windows.
In step (4), the method for multivariate Gaussian mixed distribution clustering is:For each data window, with R languages
The mclust program bags called the turn do Gaussian Mixture distributed clustering analysis, take optimal value according to BIC, then merge to each peak
Then analysis uses each peak of Gauss Distribution Fitting, determines peak value;Peptide fragment number of sites included in each peak after cluster
According to, be distributed according to site amino acids, select distribution more than 5% data as one kind undoded amino acid.
In step (4), the undoded amino acid of the few weak essence individual of normal individual and severe is detected into the T of frequency according to it and is examined
Test (p<And ratio (ratio 0.05)>2) it is screened, to obtain difference undoded amino acid.
Above-mentioned screening technique is for obtaining biomarker, and is not to obtain the diagnosing and treating result of disease as mesh
's;The biomarker obtained through above-mentioned screening technique can be used for the theoretical research of severe teen bra or opening for novel drugs
Hair.
The second aspect of the present invention, provide screened according to above-mentioned screening technique it is relevant with severe teen bra
Biomarker, the biomarker include but not limited to:
The serine of 8+79.96685 mass shifts of generation of AKAP3 protein 20s (is labeled as S+79.96685;According to quality
Deviant determines that phosphorylation modification has occurred in the serine of the position);
The asparagine (being labeled as N-113.05347) of 6-113.05347 mass shifts of generation of AKAP4 protein 18s;
The asparagine (being labeled as N-114.04278) of 6-114.04278 mass shifts of generation of AKAP4 protein 18s;
The glutamine (being labeled as Q-17.02660) of 617-17.02660 mass shifts of generation of AKAP4 albumen;
The lysine (being labeled as K+211.09682) of 733+211.09682 mass shifts of generation of AKAP4 albumen;
The lysine of 1+42.01108 mass shift of generation of ATP5A1 protein 53s (is labeled as K+42.01108;According to matter
Deviant is measured, determines that acetylation modification has occurred in the lysine of the position);
The lysine of 87+42.01108 mass shifts of generation of COX4I1 albumen (is labeled as K+42.01108;According to quality
Deviant determines that acetylation modification has occurred in the lysine of the position);
The threonine of 64+79.96685 mass shifts of generation of GAPDHS albumen (is labeled as T+79.96685;According to quality
Deviant determines that phosphorylation modification has occurred in the threonine of the position);
(it is labeled as S+79.96685 with the serine of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s;Root
According to quality offset value, determine that phosphorylation modification has occurred in the serine of the position).
The third aspect of the present invention provides the serine conduct of 8+79.96685 mass shifts of generation of AKAP3 protein 20s
Purposes of the biomarker in preparing severe and lacking the diagnostic reagent of weak essence.
Preferably, it is few weak to be also used as severe for the serine of 8+79.96685 mass shifts of generation of AKAP3 protein 20s
The target of essence treatment, for severe lacks the treatment of weak essence.
Further, the present invention also provides the serine conducts of 8+79.96685 mass shifts of generation of AKAP3 protein 20s
Purposes of the biomarker in preparing severe and lacking the medicine of weak essence.
The present invention also provides a kind of kits for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (serines of 8+79.96685 mass shifts of generation of AKAP3 protein 20s).
AKAP3 protein 20s 8 can be made by containing the present invention also provides a kind of drug treated severe and lack weak essence, in the drug
Position serine carries out the component of phosphorylation modification.
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, step is:Detect sample to be tested AKAP3 protein 20s 8
The frequency of+79.96685 mass shifts occurs for position serine, if the detection frequency is less than 1.5, is judged to less weak smart patient.
The fourth aspect of the present invention provides the asparagine of 6-113.05347 mass shifts of generation of AKAP4 protein 18s
As purposes of the biomarker in preparing severe and lacking the diagnostic reagent of weak essence.
The present invention also provides a kind of kits for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (asparagines of 6-113.05347 mass shifts of generation of AKAP4 protein 18s).
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, step is:Detect sample to be tested AKAP4 protein 18s 6
The frequency of -113.05347 mass shifts occurs for position asparagine, when the detection frequency is less than 0.5, is judged to less weak smart patient.
The fifth aspect of the present invention provides the asparagine of 6-114.04278 mass shifts of generation of AKAP4 protein 18s
As purposes of the biomarker in preparing severe and lacking the diagnostic reagent of weak essence.
The present invention also provides a kind of kits for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (asparagines of 6-114.04278 mass shifts of generation of AKAP4 protein 18s).
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, step is:Detect sample to be tested AKAP4 protein 18s 6
The frequency of -114.04278 mass shifts occurs for position asparagine, when the detection frequency is less than 0.5, is judged to less weak smart patient.
The sixth aspect of the present invention, the glutamine for providing 617-17.02660 mass shifts of generation of AKAP4 albumen are made
For purposes of the biomarker in preparing severe and lacking the diagnostic reagent of weak essence.
The present invention also provides a kind of kits for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (glutamine of 617-17.02660 mass shifts of generation of AKAP4 albumen).
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, step is:Detect sample to be tested AKAP4 albumen 617
The frequency of -17.02660 mass shifts occurs for position glutamine, when the detection frequency is less than 0.5, is judged to less weak smart patient.
The seventh aspect of the present invention provides the lysine conduct of 733+211.09682 mass shifts of generation of AKAP4 albumen
Purposes of the biomarker in preparing severe and lacking the diagnostic reagent of weak essence.
The present invention also provides a kind of kits for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (lysines of 733+211.09682 mass shifts of generation of AKAP4 albumen).
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, step is:Detect sample to be tested AKAP4 albumen 733
The frequency of+211.09682 mass shifts occurs for position lysine, when the detection frequency is less than 3.5, is judged to less weak smart patient.
The eighth aspect of the present invention provides the lysine conduct of 1+42.01108 mass shift of generation of ATP5A1 protein 53s
Purposes of the biomarker in preparing severe and lacking the diagnostic reagent of weak essence.
Preferably, it is few weak to be also used as severe for the lysine of 1+42.01108 mass shift of generation of ATP5A1 protein 53s
The target of essence treatment, for severe lacks the treatment of weak essence.
Further, the present invention also provides the lysine conducts of 1+42.01108 mass shift of generation of ATP5A1 protein 53s
Purposes of the biomarker in preparing severe and lacking the medicine of weak essence.
The present invention also provides a kind of kits for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (lysines of 1+42.01108 mass shift of generation of ATP5A1 protein 53s).
ATP5A1 albumen can be made by containing the present invention also provides a kind of drug treated severe and lack weak essence, in the drug
531 lysines carry out the component of acetylation modification.
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, step is:Detect sample to be tested ATP5A1 protein 53s 1
The frequency of+42.01108 mass shifts occurs for position lysine, if the detection frequency is less than 0.5, is judged to less weak smart patient.
The ninth aspect of the present invention provides the lysine conduct of 87+42.01108 mass shifts of generation of COX4I1 albumen
Purposes of the biomarker in preparing severe and lacking the diagnostic reagent of weak essence.
Preferably, it is few weak to be also used as severe for the lysine of 87+42.01108 mass shifts of generation of COX4I1 albumen
The target of essence treatment, for severe lacks the treatment of weak essence.
Further, the present invention also provides the lysine conducts of 87+42.01108 mass shifts of generation of COX4I1 albumen
Purposes of the biomarker in preparing severe and lacking the medicine of weak essence.
The present invention also provides a kind of kits for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (lysines of 87+42.01108 mass shifts of generation of COX4I1 albumen).
COX4I1 albumen 87 can be made by containing the present invention also provides a kind of drug treated severe and lack weak essence, in the drug
Position lysine carries out the component of acetylation modification.
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, step is:Detect sample to be tested COX4I1 albumen 87
The frequency of+42.01108 mass shifts occurs for position lysine, if the detection frequency is less than 0.5, is judged to less weak smart patient.
The tenth aspect of the present invention provides the threonine conduct of 64+79.96685 mass shifts of generation of GAPDHS albumen
Purposes of the biomarker in preparing severe and lacking the diagnostic reagent of weak essence.
Preferably, it is few weak to be also used as severe for the threonine of 64+79.96685 mass shifts of generation of GAPDHS albumen
The target of essence treatment, for severe lacks the treatment of weak essence.
Further, the present invention also provides the threonine conducts of 64+79.96685 mass shifts of generation of GAPDHS albumen
Purposes of the biomarker in preparing severe and lacking the medicine of weak essence.
The present invention also provides a kind of kits for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (threonines of 64+79.96685 mass shifts of generation of GAPDHS albumen).
GAPDHS albumen 64 can be made by containing the present invention also provides a kind of drug treated severe and lack weak essence, in the drug
Position threonine carries out the component of phosphorylation modification.
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, step is:Detect sample to be tested GAPDHS albumen 64
The frequency of+79.96685 mass shifts occurs for position threonine, if the detection frequency is less than 3.5, is judged to less weak smart patient.
The eleventh aspect of the present invention provides the serine of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s
As purposes of the biomarker in preparing severe and lacking the diagnostic reagent of weak essence.
Preferably, it is few to be also used as severe for the serine of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s
The target of weak essence treatment, for severe lacks the treatment of weak essence.
Further, the present invention also provides the serine works of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s
For purposes of the biomarker in preparing severe and lacking the medicine of weak essence.
The present invention also provides a kind of kits for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (serines of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s).
KIAA1683 albumen can be made by containing the present invention also provides a kind of drug treated severe and lack weak essence, in the drug
692 serines carry out the component of phosphorylation modification.
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, step is:Detect sample to be tested KIAA1683 albumen
The frequency of+79.96685 mass shifts occurs for 692 serines, if the detection frequency is less than 0.5, is judged to less weak smart patient.
Beneficial effects of the present invention:
(1) present invention establishes a kind of and relevant biomarker of severe teen bra screening technique for the first time, leads to
It crosses and analyzing processing is carried out to the mass spectrometric data of great amount of samples sperm protein, largely identify non-volume in sperm protein group as far as possible
Code amino acid;Finally by the comparison of undoded amino acid in normal and patient's sperm protein group, obtain lacking azoospermia with severe
Relevant albumen undoded amino acid site, to lack the molecular marker of azoospermia as severe.
(2) biomarker that the present invention further obtains above-mentioned screening technique is studied, and discovery can pass through
The inspection frequency of above-mentioned biomarker lacks azoospermia to diagnose severe, and lacking azoospermia for severe provides new diagnosing and treating
Target spot.
Description of the drawings
The accompanying drawings which form a part of this application are used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation do not constitute the improper restriction to the application for explaining the application.
Fig. 1:The ROC curve of phosphorylation modification S+79.96685 detection frequencies on 8 serines of AKAP3 protein 20s.
Fig. 2:Phosphorylation modification S+79.96685 inspections in healthy and few weak smart sample on 8 serines of AKAP3 protein 20s
Measured frequency compares.
Fig. 3:The ROC curve of 6 undoded amino acid N-113.05347 detection frequencies of AKAP4 protein 18s.
Fig. 4:6 undoded amino acid N-113.05347 of AKAP4 protein 18s of healthy and few weak smart sample detect frequency ratio
Compared with.
Fig. 5:The ROC curve of 6 undoded amino acid N-114.04278 detection frequencies of AKAP4 protein 18s.
Fig. 6:6 undoded amino acid N-114.04278 of AKAP4 protein 18s of healthy and few weak smart sample detect frequency ratio
Compared with.
Fig. 7:The ROC curve of 617 undoded amino acid Q-17.02660 detection frequencies of AKAP4 albumen.
Fig. 8:617 undoded amino acid Q-17.02660 of AKAP4 albumen of healthy and few weak smart sample detect frequency ratio
Compared with.
Fig. 9:The ROC curve of 733 undoded amino acid K+211.09682 detection frequencies of AKAP4 albumen.
Figure 10:The undoded amino acid K+211.09682 detections frequency of healthy and few weak smart sample compares.
Figure 11:The ROC curve of 1 lysine acetylation modification K+42.01108 detection frequency of ATP5A1 protein 53s.
Figure 12:1 lysine acetylation modification K+42.01108 detection of ATP5A1 protein 53s of healthy and few weak smart sample
Frequency compares.
Figure 13:The ROC curve of 87 lysine acetylation modification K+42.01108 detection frequencies of COX4I1 albumen.
Figure 14:87 lysine acetylation modification K+42.01108 detection frequencies of COX4I1 albumen of healthy and few weak smart sample
Rate compares.
Figure 15:The ROC curve of phosphorylation modification T+79.96685 detection frequencies on 64 threonines of GAPDHS albumen.
Figure 16:Phosphorylation modification T+79.96685 inspections in healthy and few weak smart sample on 64 threonines of GAPDHS albumen
Measured frequency compares.
Figure 17:The ROC curve of 2 serine phosphorylation modification S+79.96685 detection frequencies of KIAA1683 protein 69s.
Figure 18:2 serine phosphorylation modification S+79.96685 inspections of KIAA1683 protein 69s in healthy and few weak smart sample
Measured frequency compares.
Specific implementation mode
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific implementation mode, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or combination thereof.
As background technology is introduced, also weak essence is not lacked as with severe about undoded amino acid in the prior art
The report of the sub- relevant biomarker of disease.Based on this, the present invention proposes a kind of and relevant biology of severe teen bra
The screening technique of marker and application.
In a kind of embodiment of the application, it is proposed that a kind of and the relevant biomarker of severe teen bra
Screening technique includes the following steps:
(1) spermatoblast holoprotein is extracted;
(2) spermatoblast holoprotein is detached using gel electrophoresis, cuts glue enzymolysis, desalination is carried out to the peptide fragment after enzymolysis,
Sample is prepared;
(3) by the sample of step (2) using receiving flow liquid phase chromatographic isolation, through receive the sample after flow liquid phase chromatographic isolation again into
Row Mass Spectrometer Method acquires mass spectrometric data;
(4) mass spectrometric data is scanned for using non-limiting aminoacid protein modification analysis method, using multivariable
Gaussian Mixture distributed clustering analysis largely identifies undoded amino acid in sperm protein group as far as possible;Finally by normal
The comparison of undoded amino acid, obtains lacking the relevant egg of azoospermia with severe in individual and the few weak individual sperm protein group of essence of severe
White undoded amino acid site, as with the relevant biomarker of severe teen bra.
Ready availability due to sperm sample, its transcription and translation stays cool after spermioteleosis, this also for we
It studies teen bra on protein level to provide a convenient, the application is first with NanoHPLC-MS/MS mass spectrometer systems and non-
The sperm protein undoded amino acid of label quantitative proteomics method weak smart disease few to multigroup severe has carried out depth
Mass spectral analysis;Then mass spectrometric data is scanned for using non-limiting aminoacid protein modification analysis method, using changeable
Gaussian Mixture distributed clustering analysis is measured, largely identifies undoded amino acid in sperm protein group as far as possible.It finally will be normal
The T of frequency is detected according to it examine (p with the undoded amino acid of illness group<And ratio (ratio 0.05)>2) it is screened, from
And obtain difference undoded amino acid.Then difference undoded amino acid ROC curve is made using SPSS softwares, and calculates its song
Area (AUC) under line, and then judge its diagnostic value.
Using the above-mentioned screening technique of the application, series and the relevant biomarker of severe teen bra have been obtained,
It is specific as follows:
The serine of 8+79.96685 mass shifts of generation of AKAP3 protein 20s (is labeled as S+79.96685;According to quality
Deviant determines that phosphorylation modification has occurred in the serine of the position);
The asparagine (being labeled as N-113.05347) of 6-113.05347 mass shifts of generation of AKAP4 protein 18s;
The asparagine (being labeled as N-114.04278) of 6-114.04278 mass shifts of generation of AKAP4 protein 18s;
The glutamine (being labeled as Q-17.02660) of 617-17.02660 mass shifts of generation of AKAP4 albumen;
The lysine (being labeled as K+211.09682) of 733+211.09682 mass shifts of generation of AKAP4 albumen;
The lysine of 1+42.01108 mass shift of generation of ATP5A1 protein 53s (is labeled as K+42.01108;According to matter
Deviant is measured, determines that acetylation modification has occurred in the lysine of the position);
The lysine of 87+42.01108 mass shifts of generation of COX4I1 albumen (is labeled as K+42.01108;According to quality
Deviant determines that acetylation modification has occurred in the lysine of the position);
The threonine of 64+79.96685 mass shifts of generation of GAPDHS albumen (is labeled as T+79.96685;According to quality
Deviant determines that phosphorylation modification has occurred in the threonine of the position);
(it is labeled as S+79.96685 with the serine of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s;Root
According to quality offset value, determine that phosphorylation modification has occurred in the serine of the position).
In the another embodiment of the application, it is proposed that a kind of kit for the few weak essence diagnosis of severe, it is described
Kit includes the reagent of the above-mentioned biomarker of specific detection.
By being detected to above-mentioned biomarker, the diagnosis for lacking azoospermia to severe may be implemented.
In the another embodiment of the application, it is proposed that a kind of drug treated severe and lack weak essence, in the drug
Containing 28 serines of AKAP3 protein 20s, 64 threonines of GAPDHS albumen or KIAA1683 protein 69s serines can be made
The component of phosphorylation modification is carried out, or containing 87 bad ammonia of 1 lysine of ATP5A1 protein 53s or COX4I1 albumen can be made
Acid carries out the component of acetylation modification.
It has been investigated that 8 serines of the AKAP3 protein 20s of phosphorylation modification, 64 threonines of GAPDHS albumen and
The downward of 2 serines of KIAA1683 protein 69s conspicuousness in the few weak smart sample of severe, the ATP5A1 albumen of acetylation modification
87 lysines of 531 lysines or COX4I1 albumen also lack the downward of conspicuousness in weak smart sample in severe.It is possible thereby to close
Reason is expected, using above-mentioned biomarker as target, by carrying out phosphorus to the amino acid at the few weak smart patient target of multiplicity
Acidification or acetylation modification, can play the therapeutic effect for lacking weak essence to severe.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Test material used in the embodiment of the present invention is the test material of this field routine, can pass through commercial channel
It is commercially available.
Embodiment 1:With the screening of the relevant biomarker of severe teen bra
Specific screening technique is as follows:
One, sample process and experimental analysis
1. the extraction of spermatoblast holoprotein:The few weak essence of the severe of equivalent and eupyrene sperm sample wash three with DPBS respectively
It is secondary, equivalent RIPA lysate 1~2min of ultrasound are added, is placed in and is incubated 30min cracking on ice, 4 DEG C of centrifugation 14,000g × 20min
Take supernatant.Albumen concentration is measured using Bradford methods.
2. proteolysis:The about few weak essence of severe and each 150 μ g sperm proteins of eupyrene sperm sample are taken, 10% polypropylene is used
Acyl ammonia gel electrophoresis (SDS-PAGE) detaches albumen, is respectively divided into 5 parts and carries out cutting glue enzymolysis.Using ziptip to peptide fragment into
Row desalination.
3. mass spectral analysis:Receive flow liquid phase chromatographic isolation:A phases:Water containing 0.1% formic acid;B phases:Contain 0.1% formic acid
Acetonitrile
Each sample is respectively with 13.5 μ L A phased solns, and sample injection volume is 4 μ L, and flow liquid phase mass spectrometry system of receiving is
Orbitrap Elite(Thermo Scientific).Sample separation before respectively use 4 μ L A balance each other homemade pre-column and divide
Analyse column.The specification of pre-column and analytical column is respectively:Pre-column (5 μm of 4cm × 150 μm I.D., C18 packing material size,), analysis
Column (30cm × 75 μm I.D., C18 filler are filled, 3 μm of grain size,Dr.Maisch GmbH,Germany).After balance
Then sample load sample first under the drive of A phases carries out liquid phase separation in pre-column under different gradients.150min chromatography gradients become
Change as follows:5-32% Mobile phase Bs 100min;32-80% Mobile phase Bs, 20min;80% Mobile phase B, 30min.Flow velocity is protected always
It holds in 300nL/min.It is directly entered ESI ionsprays source by receiving the sample of flow liquid phase separation and enters Orbitrap Elite
Mass Spectrometer Method is carried out in mass spectrograph.
Mass spectrometric data acquires:The full scan of 350-1800m/z, resolution ratio 60,000 (m/z 200).Second order spectrum scans
When, soak time 10ms, isolation width is 2m/z.Fragmentation pattern is that induction is collisionally dissociated (collision-induced
Dissociation, CID), normalization collision energy is set as 35%, and dynamic efflux time is 90s.
Two, MASS SPECTRAL DATA ANALYSIS
Byonic is analyzed:In order to identify the undoded amino acid of sperm protein, we are analyzing 21 pairs just with ByonicTM
Often lack the protamine mass spectrometric data of weak smart patient with severe.Search parameter is as follows:Protease is trypsase, leakage enzyme site setting
It is 2, parent ion mass deviation is 10ppm, and the mass deviation of fragment ion is 0.6Da, and the blind upper limit of searching is set as 1000, blind to search lower limit
It is 0.01 to be set as -200. albumen FDR.
Select peptide fragment score>200 and FDR<The unknown modifications that 0.01 peptide fragment is searched as Wildcard SearchTM
Data form the one-dimensional data matrix (- 200Da-400Da) of mass change, then by data according to the variation range of 1Da, 0.5Da
For boundary, it is divided into 601 data windows.For each data window, it is mixed to be Gauss with the mclust program bags in R language
Distributed clustering analysis is closed, optimal value is taken according to BIC, then analysis is merged to each peak, it is then every with Gauss Distribution Fitting
One peak, determines peak value.Peptide fragment site data, are distributed according to site amino acids included in each peak after cluster, choosing
Data of the distribution more than 5% are selected as a kind of undoded amino acid.
(p is examined by normally the T of frequency is detected according to it with the undoded amino acid of illness group<And ratio (ratio 0.05)
>2) it is screened, to obtain difference undoded amino acid.Then difference undoded amino acid ROC is made using SPSS softwares
Curve, and its area under the curve (AUC) is calculated, and then judge its diagnostic value.
Three, experimental result:
Through MASS SPECTRAL DATA ANALYSIS and by normally compared with the undoded amino acid of illness group, to obtain 9 non-volumes of difference
Code amino acid, can as with the relevant biomarker of severe teen bra, it is specific as follows:
The serine of 8+79.96685 mass shifts of generation of 1.AKAP3 protein 20s (is labeled as S+79.96685;According to matter
Deviant is measured, determines that phosphorylation modification has occurred in the serine of the position)
We have found that phosphorylation modification S+79.96685 has occurred on 8 serines of AKAP3 protein 20s, by it was found that
This phosphorylation modification has lowered 10.7 times in the few weak smart sample significance of severe, p value 7.49E-15<0.05.
It is few to severe weak that frequency is detected for the phosphorylation modification S+79.96685 on evaluation 8 serines of AKAP3 protein 20s
The diagnostic of essence, present invention employs ROC curve analysis, AUC is the area under ROC curve, is most common evaluation ROC bent
The parameter of line feature is important experimental accuracy index.If AUC is below 0.7, then it represents that the accuracy rate of diagnosis is relatively low;AUC
0.7 or more, then it can meet the requirement of clinical diagnosis.
Fig. 1 is the ROC curve of the phosphorylation modification S+79.96685 detection frequencies on 8 serines of AKAP3 protein 20s,
ROC is analysis shows that the AUC of this phosphorylation modification is 0.856>0.7, illustrate that there is preferable diagnosis effect, i.e. AKAP3 albumen
Phosphorylation modification S+79.96685 on 208 serines can lack the diagnosis marker of weak essence as severe.
When it is 1.5 to examine the frequency, sensitivity 73%, specificity 88.9%.When carrying out individual detection, detection frequency
It is secondary when being less than 1.5, it is judged to less weak smart patient's (false positive rate 11.1%).
Phosphorylation modification S+79.96685 detection frequencies in healthy and few weak smart sample on 8 serines of AKAP3 protein 20s
Rate comparison result is shown in Fig. 2, as seen from Figure 2 this undoded amino acid averagely had occurred in Healthy People sample 4.6 times and
It is had occurred in pathology sample 0.4 time (solid line in figure), and its median (dotted line in figure) difference is farther out, illustrates in weak essence less
There is this undoded amino acid in sample largely loses.
In view of the above results, the phosphorylation modification S+79.96685 on 8 serines of AKAP3 protein 20s can be used as few weak
The potential source biomolecule marker of sperm disease, to predict this illness.
The asparagine (being labeled as N-113.05347) of 6-113.05347 mass shifts of generation of 2.AKAP4 protein 18s
By MASS SPECTRAL DATA ANALYSIS, it has been found that 186 asparagines of AKAP4 albumen have -113.05347 matter
Amount offset (N-113.05347), by it was found that this undoded amino acid N-113.05347 is aobvious in the few weak smart sample of severe
Work property has lowered 9.2 times, p value 4.60E-13<0.05.
Fig. 3 is the ROC curve that 6 undoded amino acid N-113.05347 of AKAP4 protein 18s detect frequency, and ROC analyses are aobvious
Show that the AUC of this undoded amino acid N-113.05347 is 0.852>0.7, illustrate that there is preferable diagnosis effect.Examining frequency
It is secondary when being 1.5, sensitivity 65.1%, specificity 93.7%.When carrying out individual detection, when the detection frequency is less than 1.5, quilt
It is judged to less weak smart patient's (false positive rate 6.3%).
Fig. 4 compares undoded amino acid N-113.05347 in healthy and weak smart sample less detection frequency, it can be seen that
This undoded amino acid averagely has occurred 2.9 times in Healthy People sample and 0.3 time has occurred in pathology sample (in figure in fact
Line), and its median (dotted line in figure) difference is farther out, illustrates that this undoded amino acid has larger journey in weak smart sample less
It loses on degree ground.
In view of the above results, this non-coding amino of the asparagine N-113.05347 in 186 sites of AKAP4 albumen
Acid can be as the potential source biomolecule marker of teen bra, to predict this illness.
The asparagine (being labeled as N-114.04278) of 6-114.04278 mass shifts of generation of 3.AKAP4 protein 18s
By MASS SPECTRAL DATA ANALYSIS, it has been found that 186 asparagines of AKAP4 albumen have -114.04278 matter
Amount offset (N-114.04278), by it was found that this undoded amino acid N-114.04278 is aobvious in the few weak smart sample of severe
Work property has lowered 7.3 times, p value 1.11E-11<0.05.
Fig. 5 is the ROC curve that 6 undoded amino acid N-114.04278 of AKAP4 protein 18s detect frequency, and ROC analyses are aobvious
Show that the AUC of this undoded amino acid N-114.04278 is 0.817>0.7, illustrate that there is preferable diagnosis effect.Examining frequency
It is secondary when being 0.5, sensitivity 74.6%, specificity 84.1%.When carrying out individual detection, when the detection frequency is less than 0.5, quilt
It is judged to less weak smart patient's (false positive rate 15.9%).
6 undoded amino acid N-114.04278 detections frequencies of AKAP4 protein 18s of healthy and few weak smart sample, which compare, sees
Fig. 6, as seen from Figure 6 this undoded amino acid averagely had occurred in Healthy People sample 1.6 times and in pathology sample
It has occurred 0.2 time (solid line in figure), and its median (dotted line in figure) difference is farther out, illustrates that this is non-in weak smart sample less
Coded amino acid, which has, largely to be lost.
In view of the above results, this non-coding amino of the asparagine N-114.04278 in 186 sites of AKAP4 albumen
Acid can be as the potential source biomolecule marker of teen bra, to predict this illness.
The glutamine (being labeled as Q-17.02660) of 617-17.02660 mass shifts of generation of 4.AKAP4 albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that 617 glutamine of AKAP4 albumen have -17.02660 quality
It deviates (Q-17.02660), by it was found that this undoded amino acid Q-17.02660 lacks weak smart sample significance in severe
4.8 times are lowered, p value 2.21E-09<0.05.
Fig. 7 is the ROC curve that 617 undoded amino acid Q-17.02660 of AKAP4 albumen detect frequency, and ROC analyses are aobvious
Show that the AUC of this undoded amino acid Q-17.02660 is 0.802>0.7, illustrate that there is preferable diagnosis effect.Examining frequency
It is secondary when being 0.5, sensitivity 77.8%, specificity 79.4%.When carrying out individual detection, when the detection frequency is less than 0.5, quilt
It is judged to less weak smart patient's (false positive rate 20.6%).
617 undoded amino acid Q-17.02660 detections frequencies of AKAP4 albumen of healthy and few weak smart sample, which compare, sees
Fig. 8, as seen from Figure 8 this undoded amino acid averagely had occurred in Healthy People sample 2.7 times and in pathology sample
It has occurred 0.6 time (solid line in figure), and its median (dotted line in figure) difference is farther out, illustrates that this is non-in weak smart sample less
Coded amino acid, which has, largely to be lost.
In view of the above results, this undoded amino acid of the glutamine Q-17.02660 in 617 sites of AKAP4 albumen can
Using the potential source biomolecule marker as teen bra, to predict this illness.
The lysine (being labeled as K+211.09682) of 733+211.09682 mass shifts of generation of 5.AKAP4 albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that 733 lysine of AKAP4 albumen has 211.09682 quality inclined
It moves (K+211.09682), by it was found that this undoded amino acid K+211.09682 lacks weak smart sample significance in severe
4.4 times are lowered, p value 5.79E-11<0.05.
Fig. 9 is the ROC curve that 733 undoded amino acid K+211.09682 of AKAP4 albumen detect frequency, and ROC analyses are aobvious
Show that the AUC of this undoded amino acid K+211.09682 is 0.804>0.7, illustrate that there is preferable diagnosis effect.Examining frequency
It is secondary when being 3.5, sensitivity 74.6%, specificity 71.4%.When carrying out individual detection, when the detection frequency is less than 3.5, quilt
It is judged to less weak smart patient's (false positive rate 28.6%).
The undoded amino acid K+211.09682 detections frequency of healthy and few weak smart sample, which compares, sees Figure 10, can by Figure 10
To find out that this undoded amino acid averagely has occurred 14 times in Healthy People sample and 3 (figures have occurred in pathology sample
Middle solid line), and its median (dotted line in figure) difference farther out, illustrate less it is weak essence sample in this undoded amino acid have compared with
Lose to big degree.
In view of the above results, this undoded amino acid of the lysine K+211.09682 in 733 sites of AKAP4 albumen can
Using the potential source biomolecule marker as teen bra, to predict this illness.
The lysine of 1+42.01108 mass shift of generation of 6.ATP5A1 protein 53s
By MASS SPECTRAL DATA ANALYSIS, it has been found that acetylation modification K+ has occurred on 1 lysine of ATP5A1 protein 53s
42.01108, by it was found that this acetylation modification has lowered 10.5 times in the few weak smart sample significance of severe, p value is
3.48E-16<0.05。
Figure 11 is that 1 lysine acetylation of ATP5A1 protein 53s modifies the ROC curve that K+42.01108 detects frequency, ROC
Analysis shows that the AUC of this acetylation modification is 0.848>0.7, illustrate that there is preferable diagnosis effect.It is 0.5 examining the frequency
When, sensitivity 76.2%, specificity 85.7%.When carrying out individual detection, when the detection frequency is less than 0.5, it is judged to few
Weak essence patient (false positive rate 14.3%).
1 lysine acetylation modification K+42.01108 of ATP5A1 protein 53s of healthy and few weak smart sample detects frequency ratio
Relatively see Figure 12, it can be seen that this undoded amino acid averagely has occurred 1.7 times in Healthy People sample and in pathology sample
It has occurred 0.2 time (solid line in figure), and its median (dotted line in figure) difference is farther out, illustrates that this is non-in weak smart sample less
Coded amino acid, which has, largely to be lost.
In view of the above results, the acetylation modification K+42.01108 on 1 lysine of ATP5A1 protein 53s can be used as few
The potential source biomolecule marker of asthénospermie, to predict this illness.
The lysine (being labeled as K+42.01108) of 87+42.01108 mass shifts of generation of 7.COX4I1 albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that acetylation modification K+ has occurred on 87 lysines of COX4I1 albumen
42.01108, by it was found that this acetylation modification has lowered 11.3 times in the few weak smart sample significance of severe, p value is
5.06E-13<0.05。
Figure 13 is that 87 lysine acetylations of COX4I1 albumen modify the ROC curve that K+42.01108 detects frequency, and ROC divides
Analysis shows that the AUC of this acetylation modification is 0.803>0.7, illustrate that there is preferable diagnosis effect.It is 0.5 examining the frequency
When, sensitivity 66.7%, specificity 95.2%.When carrying out individual detection, when the detection frequency is less than 0.5, it is judged to few
Weak essence patient (false positive rate 4.8%).
Figure 14 is that 87 lysine acetylations of COX4I1 albumen of healthy and few weak smart sample modify K+42.01108 detections
Frequency compares, this undoded amino acid averagely has occurred 1.1 times in Healthy People sample and in pathology as seen from Figure 14
It has occurred 0.1 time (solid line in figure), and its median (dotted line in figure) difference is farther out, illustrates in weak smart sample less in sample
This undoded amino acid, which has, largely to be lost.
In view of the above results, the acetylation modification K+42.01108 on 87 lysines of COX4I1 albumen can be used as few weak
The potential source biomolecule marker of sperm disease, to predict this illness.
The threonine (being labeled as T+79.96685) of 64+79.96685 mass shifts of generation of 8.GAPDHS albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that phosphorylation modification T+ has occurred on 64 threonines of GAPDHS albumen
79.96685, by it was found that this phosphorylation modification has lowered 4.1 times in the few weak smart sample significance of severe, p value is
1.82E-14<0.05。
Figure 15 is the ROC curve of the phosphorylation modification T+79.96685 detection frequencies on 64 threonines of GAPDHS albumen,
ROC is analysis shows that the AUC of this phosphorylation modification is 0.868>0.7, illustrate that there is preferable diagnosis effect.It is examining the frequency
When 3.5, sensitivity 71.4%, specificity 92.1%.When carrying out individual detection, when the detection frequency is less than 3.5, it is judged to
Few weak smart patient's (false positive rate 7.9%).
Figure 16 is the phosphorylation modification T+79.96685 on 64 threonines of GAPDHS albumen in healthy and few weak smart sample
Detection frequency compare, as seen from Figure 16 this undoded amino acid averagely had occurred in Healthy People sample 5.8 times and
It has occurred 1.4 times (solid lines in figure), and its median (dotted line in figure) difference is farther out, illustrates in weak smart sample less in pathology sample
There is this undoded amino acid in this largely loses.
In view of the above results, the phosphorylation modification T+79.96685 on 64 threonines of GAPDHS albumen can be used as few weak
The potential source biomolecule marker of sperm disease, to predict this illness.
The serine (being labeled as S+79.96685) of 2+79.96685 mass shifts of generation of 9.KIAA1683 protein 69s
By MASS SPECTRAL DATA ANALYSIS, it has been found that phosphorylation modification S+ has occurred on 2 serines of KIAA1683 protein 69s
79.96685, by it was found that this phosphorylation modification has lowered 22 times in the few weak smart sample significance of severe, p value is
2.73E-10<0.05。
Figure 17 is that 2 serine phosphorylations of KIAA1683 protein 69s modify the ROC curve that S+79.96685 detects frequency,
ROC is analysis shows that the AUC of this phosphorylation modification is 0.808>0.7, illustrate that there is preferable diagnosis effect.It is examining the frequency
When 0.5, sensitivity 65.1%, specificity 95.2%.When carrying out individual detection, when the detection frequency is less than 0.5, it is judged to
Few weak smart patient's (false positive rate 4.8%).
Figure 18 is that 2 serine phosphorylations of KIAA1683 protein 69s modify S+79.96685 inspections in healthy and few weak smart sample
Measured frequency compares, this undoded amino acid averagely has occurred 2.1 times in Healthy People sample and in disease as seen from Figure 18
It has occurred 0.1 time (solid line in figure), and its median (dotted line in figure) difference is farther out, illustrates in weak smart sample less in reason sample
In this undoded amino acid have and largely lose.
In view of the above results, the phosphorylation modification S+79.96685 on 2 serines of KIAA1683 protein 69s can conduct
The potential source biomolecule marker of teen bra, to predict this illness.
Embodiment 2:Clinical detection is verified
It is verified, is distinguished as research object using the few weak smart sample of severe of 4 healthy samples, 8 clinical definites
Detect 6 generations of serine, AKAP4 protein 18s-of 8+79.96685 mass shifts of generation of AKAP3 protein 20s of above-mentioned sample
The aspartoyl of 6-114.04278 mass shifts of generation of asparagine, AKAP4 protein 18s of 113.05347 mass shifts
733 generations+211.09682 of glutamine, AKAP4 albumen of 617 amine, AKAP4 albumen -17.02660 mass shifts of generation
87, lysine, the COX4I1 albumen of 1+42.01108 mass shift of generation of lysine, ATP5A1 protein 53s of mass shift
Occur 64, lysine, GAPDHS albumen+79.96685 mass shifts of generation of+42.01108 mass shifts threonines and
The respective detection frequency of serine of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s, and by each in embodiment 1
Criterion when biomarker carries out individual detection diagnoses sample to be tested.
The result shows that:When individually being diagnosed with above-mentioned each biomarker respectively, diagnostic result is consistent with known results.
Illustrate that the present invention screens 9 obtained biomarkers and can lack the diagnosis marker of weak essence respectively as severe.
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
Claims (8)
1. a kind of and relevant biomarker of severe teen bra screening technique, which is characterized in that include the following steps:
(1) spermatoblast holoprotein is extracted;
(2) spermatoblast holoprotein is detached using gel electrophoresis, cuts glue enzymolysis, desalination is carried out to the peptide fragment after enzymolysis, is prepared
Obtain sample;
(3) by the sample of step (2) using receiving flow liquid phase chromatographic isolation, matter is carried out again through receiving the sample after flow liquid phase chromatographic isolation
Spectrum detection;
The chromatographic condition of flow liquid phase chromatographic isolation received is:Mobile phase A:Water containing 0.1% formic acid, Mobile phase B:Contain 0.1%
The acetonitrile of formic acid;It is 4 μ L that each sample uses 13.5 μ L A phased solns, sample injection volume respectively, receives flow liquid phase mass spectrometry system
For the Orbitrap Elite of Thermo Scientific research and development;
It is balanced each other homemade pre-column and analytical column with 4 μ L A respectively before sample separation;The specification of pre-column and analytical column is respectively:
Size is 5 μm of 4cm × 150 μm I.D.C18 packing material sizes,Pre-column, size be 30cm × 75 μm I.D., C18 fillers
Filling, 3 μm of grain size,Analytical column, pre- Zhuhe analytical column is purchased from Dr.Maisch GmbH, Germany companies;Balance
Later then sample load sample first under the drive of A phases carries out liquid phase separation in pre-column under different gradients;
150min chromatography gradeds are as follows:5-32% Mobile phase Bs 100min;32-80% Mobile phase Bs, 20min;80% flowing
Phase B, 30min;Flow velocity is 300nL/min;It is directly entered ESI ionsprays source by receiving the sample of flow liquid phase separation and enters
Mass Spectrometer Method, gathered data are carried out in Orbitrap Elite mass spectrographs;
(4) mass spectrometric data is scanned for using non-limiting aminoacid protein modification analysis method, using multivariate Gaussian
Mixed distribution clustering largely identifies undoded amino acid in sperm protein group as far as possible;Finally by normal individual
With the comparison of undoded amino acid in the few weak individual sperm protein group of essence of severe, obtain lacking the relevant albumen of azoospermia with severe non-
Coded amino acid site as diagnoses and/or treats the biomarker that severe lacks weak sperm disease.
2. screening technique as described in claim 1, which is characterized in that in step (1), extraction spermatoblast holoprotein uses
Method is:Sperm sample is washed using DPBS, RIPA lysate 1~2min of ultrasound are added, incubation 30min on ice is placed in and splits
Solution, centrifugation, takes supernatant.
3. screening technique as claimed in claim 2, which is characterized in that it is centrifuged under conditions of 4 DEG C, centrifugal rotational speed 14,
000g, centrifugation time 20min.
4. screening technique as described in claim 1, which is characterized in that in step (2), using 10% polyacrylamide gel electricity
Swimming detaches albumen.
5. screening technique as described in claim 1, which is characterized in that in step (2), using ziptip to the peptide fragment after enzymolysis
Carry out desalination.
6. screening technique as described in claim 1, which is characterized in that in step (3), the condition of Mass Spectrometer Method is:350-
The full scan of 1800m/z, resolution ratio 60,000, charge-mass ratio 200;When second order spectrum scans, soak time 10ms, isolation
Width is 2m/z;Fragmentation pattern is that induction is collisionally dissociated, and normalization collision energy is set as 35%, and dynamic efflux time is 90s.
7. screening technique as described in claim 1, which is characterized in that in step (4), parameter that mass spectrometric data is scanned for
It is set as:Protease is trypsase, and leakage enzyme site is set as 2, and parent ion mass deviation is 10ppm, the quality of fragment ion
Deviation is 0.6Da, and the blind upper limit of searching is set as 1000, and blind lower limit of searching is set as -200, and albumen FDR is 0.01;
Select peptide fragment score>200 and FDR<0.01 peptide fragment is as Wildcard SearchTMThe unknown modifications data searched,
The one-dimensional data matrix of the ranging from -200Da-400Da of mass change is formed, then by data according to the variation range of 1Da,
0.5Da is boundary, is divided into 601 data windows.
8. screening technique as described in claim 1, which is characterized in that in step (4), multivariate Gaussian mixed distribution cluster point
The method of analysis is:For each data window, Gaussian Mixture distributed clustering analysis is done with the mclust program bags in R language,
Optimal value is taken according to BIC, then analysis is merged to each peak, each peak of Gauss Distribution Fitting is then used, determines peak value;
Peptide fragment site data, are distributed according to site amino acids included in each peak after cluster, number of the selection distribution more than 5%
According to as a kind of undoded amino acid.
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CN110514838B (en) * | 2018-05-21 | 2020-10-23 | 山东大学 | Application of 184-bit N +22.968 of AKAP4 protein in preparation of diagnostic reagent for severe oligospermia |
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CN110514837B (en) * | 2018-05-21 | 2020-10-23 | 山东大学 | Application of 203-position S +79.967 of AKAP3 protein in preparation of diagnostic reagent for severe oligospermia |
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WO2012061578A2 (en) * | 2010-11-03 | 2012-05-10 | The University Of North Carolina At Chapel Hill | Sperm motility analyzer and related methods |
CN103361336A (en) * | 2012-03-31 | 2013-10-23 | 上海南方模式生物研究中心 | Animal model with male reproductive disorders, as well as preparation method and application thereof |
CN106996981B (en) * | 2017-03-29 | 2018-11-02 | 山东大学 | Purposes of 6 N-114.04278 of AKAP4 protein 18s in preparing the few weak smart diagnostic reagent of severe |
CN106932597B (en) * | 2017-03-29 | 2018-07-20 | 山东大学 | Purposes of the lysine of 1 generation mass shift of ATP5A1 protein 53s in preparing the few weak smart diagnostic reagent of severe |
CN107037172B (en) * | 2017-03-29 | 2018-03-06 | 山东大学 | Purposes of the lysine of 87 generation mass shifts of COX4I1 albumen in the few weak smart diagnostic reagent of severe is prepared |
CN106996980B (en) * | 2017-03-29 | 2018-11-02 | 山东大学 | Purposes of the lysine of 733 generation mass shifts of AKAP4 albumen in preparing the few weak smart diagnostic reagent of severe |
CN106872630B (en) * | 2017-03-29 | 2018-07-24 | 山东大学 | With the screening and application of the relevant biomarker of severe teen bra |
CN106996979B (en) * | 2017-03-29 | 2018-11-02 | 山东大学 | Purposes of 6 N-113.05347 of AKAP4 protein 18s in preparing the few weak smart diagnostic reagent of severe |
CN107015005B (en) * | 2017-03-29 | 2018-07-24 | 山东大学 | Purposes of the threonine of 64 generation mass shifts of GAPDHS albumen in preparing the few weak smart diagnostic reagent of severe |
CN107024553B (en) * | 2017-03-29 | 2018-03-06 | 山东大学 | Purposes of the serine of 8 generation mass shifts of AKAP3 protein 20s in the few weak smart diagnostic reagent of severe is prepared |
CN106990177B (en) * | 2017-03-29 | 2019-03-22 | 山东大学 | Purposes of the glutamine of 617, AKAP4 albumen generation mass shifts in the few weak smart diagnostic reagent of preparation severe |
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2017
- 2017-03-29 CN CN201710196769.9A patent/CN106872630B/en active Active
- 2017-10-19 WO PCT/CN2017/106915 patent/WO2018176808A1/en active Application Filing
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