CN106872630A - The screening of the biomarker related to severe teen bra and application - Google Patents
The screening of the biomarker related to severe teen bra and application Download PDFInfo
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Abstract
The invention discloses a kind of screening technique of the biomarker related to severe teen bra, the mass spectral analysis of depth is carried out to the sperm protein undoded amino acid of the few weak smart disease of multigroup severe first with NanoHPLC MS/MS mass spectrometer systems and label-free proteomics method；Then mass spectrometric data is scanned for using non-limiting aminoacid protein modification analysis method, then by multivariate Gaussian mixed distribution cluster analysis, it is as substantial amounts of as possible to identify undoded amino acid in sperm protein group；Finally by the comparing of undoded amino acid in normal and patient's sperm protein group, obtain the albumen undoded amino acid site related to the few azoospermia of severe, so as to the molecular marker as the few azoospermia of severe, for the few azoospermia of severe provides new diagnosis and therapy target.
Description
Technical field
The present invention relates to medical science and molecular diagnostic techniques field, and in particular to a kind of life related to severe teen bra
The screening of thing mark and application.
Background technology
The infertile healthy reproduction problem turned into worldwide, wherein because the infertility that male factor is caused is big
It is general to account for 50% or so, and in recent years in the trend for rising.The main cause for causing male sterility is oligoasthenospermia disease.According to the world
Health Organization criterion specifies, if a grades of sperm count<25%, (a+b) level sperm count<50%, and sperm motility rate is less than 60%
Words, so that it may be diagnosed as asthénospermie.Oligospermatism refers to that the sperm number in seminal fluid has fecundity male less than normal
A kind of illness, when the sperm of male is oligospermatism when being less than 2,000 ten thousand for every milliliter, just.
The current research on sperm protein matter group has a lot.Saraswat etc. is using the method for UPLC-MS in Human Sperm
667 albumen are quantified in son, and has 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).).Human sperm's protein group of latest update 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).).TYR phosphorylation is for processes such as the motion of sperm, capacitation, super sharp motions
Important function.Chying-Chyuan Chan etc. carry out proteomics by the sperm to 20 groups of normal persons and azoospermia patient
Analysis finds that 12 kinds there occurs peroxophosphoric acid (Chan CC, Shui HA, Wu CH et including the 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, thus using abnormal under the morbid state or great undoded amino acid of number change as disease biomarker,
And then process for diagnosing the illness is significant.But undoded amino acid is not related to also at present as few weak with severe
The report of the related biomarker of sperm disease.
The content of the invention
For above-mentioned prior art, it is an object of the invention to provide a kind of biomarker related to severe teen bra
Screening with application.The present invention is first with 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 is scanned for mass spectrometric data, then by multivariate Gaussian mixed distribution cluster analysis, to the greatest extent
Substantial amounts of may identify undoded amino acid in sperm protein group；Finally by non-coding in normal and patient's sperm protein group
The comparing of amino acid, obtains the albumen undoded amino acid site related to the few azoospermia of severe, so as to few as severe
The molecular marker of azoospermia.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of the first aspect of the present invention, there is provided screening side of the biomarker related to severe teen bra
Method, comprises the following steps：
(1) spermatoblast holoprotein is extracted；
(2) spermatoblast holoprotein is separated using gel electrophoresis, cuts glue enzymolysis, desalination is carried out to the peptide fragment after enzymolysis,
Prepare sample；
(3) by the sample of step (2) using receiving flow liquid phase chromatographic isolation, enter again through receiving the sample after flow liquid phase chromatographic isolation
Row Mass Spectrometer Method, gathers mass spectrometric data；
(4) mass spectrometric data is scanned for using non-limiting aminoacid protein modification analysis method, then by multivariable
Gaussian Mixture distributed clustering analysis, it is as substantial amounts of as possible to identify undoded amino acid in sperm protein group；Finally by normal
The comparing of undoded amino acid, obtains the egg related to the few azoospermia of severe in the few weak essence individuality sperm protein group of individual and severe
White undoded amino acid site, biomarker as related to severe teen bra.
In step (1), extract the method that uses of spermatoblast holoprotein for：Sperm sample is washed using DPBS, is added
RIPA lysate 1~2min of ultrasound, are placed in and be incubated on ice 30min cracking, and centrifugation takes supernatant.
Preferably, it is centrifuged under conditions of 4 DEG C, centrifugal rotational speed is 14,000g, and centrifugation time is 20min.
In step (2), it is preferred that albumen is separated 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, resolution ratio is 60,000 (m/z
200).When second order spectrum is scanned, soak time is 10ms, and isolation width is 2m/z；Fragmentation pattern is collisionally dissociated for induction
(collision-induced dissociation, CID), normalization collision energy is set as 35%, and dynamic efflux time is
90s。
In step (4), the parameter that mass spectrometric data is scanned for is set to：Protease is trypsase, and leakage enzyme site sets
It is set to 2, parent ion mass deviation is 10ppm, and the mass deviation of fragment ion is 0.6Da, the blind upper limit of searching is set to 1000, blind to search down
Limit is set to -200, and albumen FDR is 0.01；
Selection peptide fragment fraction>200 and FDR<The unknown modifications that 0.01 peptide fragment is searched as Wildcard SearchTM
Data, constitute mass change one-dimensional data matrix (- 200Da-400Da), then by data according to 1Da excursion, 0.5Da
It is boundary, is divided into 601 data windows.
In step (4), the method for multivariate Gaussian mixed distribution cluster analysis is：For each data window, R languages are used
The mclust program bags for calling the turn do Gaussian Mixture distributed clustering analysis, take optimal value according to BIC, then each peak is merged
Analysis, then with each peak of Gauss Distribution Fitting, determines peak value；The peptide fragment number of sites included in each peak after cluster
According to, it is distributed according to site amino acids, selection data of the distribution more than 5% are used as a class undoded amino acid.
In step (4), the individual undoded amino acid of the few weak essence of normal individual and severe is examined according to its T for detecting frequency
Test (p<0.05) with ratio (ratio>2) screened, so as to obtain difference undoded amino acid.
Above-mentioned screening technique is, for obtaining biomarker, and not to be diagnosis to obtain disease and treatment results are 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, there is provided according to above-mentioned screening technique screening obtain it is related to severe teen bra
Biomarker, the biomarker is included but is 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 the serine of the position there occurs phosphorylation modification)；
6 asparagines of -113.05347 mass shifts of generation (being labeled as N-113.05347) of AKAP4 protein 18s；
6 asparagines of -114.04278 mass shifts of generation (being labeled as N-114.04278) of AKAP4 protein 18s；
617 glutamine of -17.02660 mass shifts of generation (being labeled as Q-17.02660) of AKAP4 albumen；
733 lysines of+211.09682 mass shifts of generation (being labeled as K+211.09682) 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
Amount deviant, determines that the lysine of the position there occurs acetylation modification)；
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 the lysine of the position there occurs acetylation modification)；
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 the threonine of the position there occurs phosphorylation modification)；
Serine with 2+79.96685 mass shifts of generation of KIAA1683 protein 69s (is labeled as S+79.96685；Root
According to quality offset value, determine that the serine of the position there occurs phosphorylation modification).
The third aspect of the present invention, there is provided 8 serine conducts of+79.96685 mass shifts of generation of AKAP3 protein 20s
Purposes of the biomarker in the diagnostic reagent for preparing the few weak essence of severe.
Preferably, to be also used as severe few weak for the serines of+79.96685 mass shifts of the generation of AKAP3 protein 20s 8
The target of essence treatment, so that for the treatment of the few weak essence of severe.
Further, the present invention also provides AKAP3 protein 20s 8 serine conducts of+79.96685 mass shifts of generation
Purposes of the biomarker in the medicine for preparing the few weak essence of severe.
The present invention also provides a kind of kit for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (8 serines of+79.96685 mass shifts of generation of AKAP3 protein 20s).
The present invention also provides a kind of medicine for treating the few weak essence of severe, and containing in the medicine can make AKAP3 protein 20s 8
Position serine carries out the component of phosphorylation modification.
The present invention also provides a kind of diagnostic method of the few weak essence of severe, and step is：Detection sample to be tested AKAP3 protein 20s 8
There is the frequency of+79.96685 mass shifts in position serine, if the detection frequency is less than 1.5, be judged to less weak smart patient.
The fourth aspect of the present invention, there is provided 6 asparagines of -113.05347 mass shifts of generation of AKAP4 protein 18s
As purposes of the biomarker in the diagnostic reagent for preparing the few weak essence of severe.
The present invention also provides a kind of kit for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (6 asparagines of -113.05347 mass shifts of generation of AKAP4 protein 18s).
The present invention also provides a kind of diagnostic method of the few weak essence of severe, and step is：Detection sample to be tested AKAP4 protein 18s 6
There is the frequency of -113.05347 mass shifts in position asparagine, when the detection frequency is less than 0.5, be judged to less weak smart patient.
The fifth aspect of the present invention, there is provided 6 asparagines of -114.04278 mass shifts of generation of AKAP4 protein 18s
As purposes of the biomarker in the diagnostic reagent for preparing the few weak essence of severe.
The present invention also provides a kind of kit for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (6 asparagines of -114.04278 mass shifts of generation of AKAP4 protein 18s).
The present invention also provides a kind of diagnostic method of the few weak essence of severe, and step is：Detection sample to be tested AKAP4 protein 18s 6
There is the frequency of -114.04278 mass shifts in position asparagine, when the detection frequency is less than 0.5, be judged to less weak smart patient.
The sixth aspect of the present invention, there is provided the glutamine of 617-17.02660 mass shifts of generation of AKAP4 albumen is made
It is purposes of the biomarker in the diagnostic reagent for preparing the few weak essence of severe.
The present invention also provides a kind of kit for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (617 glutamine of -17.02660 mass shifts of generation of AKAP4 albumen).
The present invention also provides a kind of diagnostic method of the few weak essence of severe, and step is：Detection sample to be tested AKAP4 albumen 617
There is the frequency of -17.02660 mass shifts in position glutamine, when the detection frequency is less than 0.5, be judged to less weak smart patient.
The seventh aspect of the present invention, there is provided 733 lysine conducts of+211.09682 mass shifts of generation of AKAP4 albumen
Purposes of the biomarker in the diagnostic reagent for preparing the few weak essence of severe.
The present invention also provides a kind of kit for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (733 lysines of+211.09682 mass shifts of generation of AKAP4 albumen).
The present invention also provides a kind of diagnostic method of the few weak essence of severe, and step is：Detection sample to be tested AKAP4 albumen 733
There is the frequency of+211.09682 mass shifts in position lysine, when the detection frequency is less than 3.5, be judged to less weak smart patient.
The eighth aspect of the present invention, there is provided 1 lysine conduct of+42.01108 mass shifts of generation of ATP5A1 protein 53s
Purposes of the biomarker in the diagnostic reagent for preparing the few weak essence of severe.
Preferably, to be also used as severe few weak for the lysine of+42.01108 mass shifts of the generation of ATP5A1 protein 53s 1
The target of essence treatment, so that for the treatment of the few weak essence of severe.
Further, the present invention also provides ATP5A1 protein 53s 1 lysine conduct of+42.01108 mass shifts of generation
Purposes of the biomarker in the medicine for preparing the few weak essence of severe.
The present invention also provides a kind of kit for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (1 lysine of+42.01108 mass shifts of generation of ATP5A1 protein 53s).
The present invention also provides a kind of medicine for treating the few weak essence of severe, and containing in the medicine can make ATP5A1 albumen
531 lysines carry out the component of acetylation modification.
The present invention also provides a kind of diagnostic method of the few weak essence of severe, and step is：Detection sample to be tested ATP5A1 protein 53s 1
There is the frequency of+42.01108 mass shifts in position lysine, if the detection frequency is less than 0.5, be judged to less weak smart patient.
The ninth aspect of the present invention, there is provided 87 lysine conducts of+42.01108 mass shifts of generation of COX4I1 albumen
Purposes of the biomarker in the diagnostic reagent for preparing the few weak essence of severe.
Preferably, to be also used as severe few weak for the lysines of+42.01108 mass shifts of 87, COX4I1 albumen generation
The target of essence treatment, so that for the treatment of the few weak essence of severe.
Further, the present invention also provides COX4I1 albumen 87 lysine conducts of+42.01108 mass shifts of generation
Purposes of the biomarker in the medicine for preparing the few weak essence of severe.
The present invention also provides a kind of kit for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (87 lysines of+42.01108 mass shifts of generation of COX4I1 albumen).
The present invention also provides a kind of medicine for treating the few weak essence of severe, and containing in the medicine can make COX4I1 albumen 87
Position lysine carries out the component of acetylation modification.
The present invention also provides a kind of diagnostic method of the few weak essence of severe, and step is：Detection sample to be tested COX4I1 albumen 87
There is the frequency of+42.01108 mass shifts in position lysine, if the detection frequency is less than 0.5, be judged to less weak smart patient.
The tenth aspect of the present invention, there is provided 64 threonine conducts of+79.96685 mass shifts of generation of GAPDHS albumen
Purposes of the biomarker in the diagnostic reagent for preparing the few weak essence of severe.
Preferably, to be also used as severe few weak for the threonines of+79.96685 mass shifts of 64, GAPDHS albumen generation
The target of essence treatment, so that for the treatment of the few weak essence of severe.
Further, the present invention also provides GAPDHS albumen 64 threonine conducts of+79.96685 mass shifts of generation
Purposes of the biomarker in the medicine for preparing the few weak essence of severe.
The present invention also provides a kind of kit for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (64 threonines of+79.96685 mass shifts of generation of GAPDHS albumen).
The present invention also provides a kind of medicine for treating the few weak essence of severe, and containing in the medicine can make GAPDHS albumen 64
Position threonine carries out the component of phosphorylation modification.
The present invention also provides a kind of diagnostic method of the few weak essence of severe, and step is：Detection sample to be tested GAPDHS albumen 64
There is the frequency of+79.96685 mass shifts in position threonine, if the detection frequency is less than 3.5, be judged to less weak smart patient.
The eleventh aspect of the present invention, there is provided 2 serines of+79.96685 mass shifts of generation of KIAA1683 protein 69s
As purposes of the biomarker in the diagnostic reagent for preparing the few weak essence of severe.
Preferably, to be also used as severe few for the serines of+79.96685 mass shifts of the generation of KIAA1683 protein 69s 2
The target of weak essence treatment, so that for the treatment of the few weak essence of severe.
Further, the present invention also provides the serine work of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s
It is purposes of the biomarker in the medicine for preparing the few weak essence of severe.
The present invention also provides a kind of kit for the few weak essence diagnosis of severe, and the kit includes specific detection
The reagent of above-mentioned biomarker (2 serines of+79.96685 mass shifts of generation of KIAA1683 protein 69s).
The present invention also provides a kind of medicine for treating the few weak essence of severe, and containing in the medicine can make KIAA1683 albumen
692 serines carry out the component of phosphorylation modification.
The present invention also provides a kind of diagnostic method of the few weak essence of severe, and step is：Detection sample to be tested KIAA1683 albumen
There is the frequency of+79.96685 mass shifts in 692 serines, if the detection frequency is less than 0.5, be judged to less weak smart patient.
Beneficial effects of the present invention：
(1) present invention establishes a kind of screening technique of the biomarker related to severe teen bra first, leads to
Cross and treatment is analyzed to the mass spectrometric data of great amount of samples sperm protein, it is as substantial amounts of as possible to identify non-volume in sperm protein group
Code amino acid；Finally by the comparing of undoded amino acid in normal and patient's sperm protein group, obtain and the few azoospermia of severe
Related albumen undoded amino acid site, so as to the molecular marker as the few azoospermia of severe.
(2) present invention is further studied the biomarker that above-mentioned screening technique is obtained, and discovery can pass through
The inspection frequency of above-mentioned biomarker diagnoses the few azoospermia of severe, for the few azoospermia of severe provides new diagnosis and treatment
Target spot.
Brief description of the drawings
The Figure of description for constituting the part of the application is used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its illustrated for explaining the application, does not constitute the improper restriction to the application.
Fig. 1：Phosphorylation modification S+79.96685 on 8 serines of AKAP3 protein 20s detects the ROC curve of frequency.
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. 3：6 undoded amino acid N-113.05347 of AKAP4 protein 18s detect the ROC curve of frequency.
Fig. 4：6 undoded amino acid N-113.05347 detection frequency ratios of AKAP4 protein 18s of healthy and few weak smart sample
Compared with.
Fig. 5：6 undoded amino acid N-114.04278 of AKAP4 protein 18s detect the ROC curve of frequency.
Fig. 6：6 undoded amino acid N-114.04278 detection frequency ratios of AKAP4 protein 18s of healthy and few weak smart sample
Compared with.
Fig. 7：617 undoded amino acid Q-17.02660 of AKAP4 albumen detect the ROC curve of frequency.
Fig. 8：617 undoded amino acid Q-17.02660 detection frequency ratios of AKAP4 albumen of healthy and few weak smart sample
Compared with.
Fig. 9：733 undoded amino acid K+211.09682 of AKAP4 albumen detect the ROC curve of frequency.
Figure 10：The undoded amino acid K+211.09682 detections frequency of healthy and few weak smart sample compares.
Figure 11：1 lysine acetylation modification K+42.01108 of ATP5A1 protein 53s detects the ROC curve of frequency.
Figure 12：1 lysine acetylation modification K+42.01108 detection of ATP5A1 protein 53s of healthy and few weak smart sample
Frequency compares.
Figure 13：87 lysine acetylation modification K+42.01108 of COX4I1 albumen detect the ROC curve of frequency.
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：Phosphorylation modification T+79.96685 on 64 threonines of GAPDHS albumen detects the ROC curve of frequency.
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 17：2 serine phosphorylation modification S+79.96685 of KIAA1683 protein 69s detect the ROC curve of frequency.
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 embodiment
It is noted that described further below is all exemplary, it is intended to provide further instruction to the application.Unless another
Indicate, all technologies used herein and scientific terminology are with 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 embodiment, 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
Be also intended to include plural form, additionally, it should be understood that, when in this manual use term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation, device, component and/or combinations thereof.
As background technology is introduced, it is not related to undoded amino acid also in the prior art and lacks weak essence as with severe
The report of the related biomarker of sub- disease.Based on this, the present invention proposes a kind of biology related to severe teen bra
The screening technique of mark and application.
In a kind of embodiment of the application, it is proposed that a kind of biomarker related to severe teen bra
Screening technique, comprises the following steps：
(1) spermatoblast holoprotein is extracted；
(2) spermatoblast holoprotein is separated using gel electrophoresis, cuts glue enzymolysis, desalination is carried out to the peptide fragment after enzymolysis,
Prepare sample；
(3) by the sample of step (2) using receiving flow liquid phase chromatographic isolation, enter again through receiving the sample after flow liquid phase chromatographic isolation
Row Mass Spectrometer Method, gathers mass spectrometric data；
(4) mass spectrometric data is scanned for using non-limiting aminoacid protein modification analysis method, then by multivariable
Gaussian Mixture distributed clustering analysis, it is as substantial amounts of as possible to identify undoded amino acid in sperm protein group；Finally by normal
The comparing of undoded amino acid, obtains the egg related to the few azoospermia of severe in the few weak essence individuality sperm protein group of individual and severe
White undoded amino acid site, biomarker as related to severe teen bra.
Ready availability due to sperm sample, its transcription and translation stays cool after spermioteleosis, this also for we
Teen bra is studied on protein level and provides convenient, the application is first with NanoHPLC-MS/MS mass spectrometer systems and non-
Mark quantitative proteomicses method has carried out depth to the sperm protein undoded amino acid of the few weak smart disease of multigroup severe
Mass spectral analysis；Then mass spectrometric data is scanned for using non-limiting aminoacid protein modification analysis method, then by changeable
Amount Gaussian Mixture distributed clustering analysis, it is as substantial amounts of as possible to identify undoded amino acid in sperm protein group.Finally will be normal
Undoded amino acid with ill group checks (p according to its T for detecting frequency<0.05) with ratio (ratio>2) screened, from
And obtain difference undoded amino acid.Then difference undoded amino acid ROC curve is made using SPSS softwares, and it is bent to calculate it
Area (AUC) under line, and then judge its diagnostic value.
Using the above-mentioned screening technique of the application, the series biomarker related to severe teen bra is 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 the serine of the position there occurs phosphorylation modification)；
6 asparagines of -113.05347 mass shifts of generation (being labeled as N-113.05347) of AKAP4 protein 18s；
6 asparagines of -114.04278 mass shifts of generation (being labeled as N-114.04278) of AKAP4 protein 18s；
617 glutamine of -17.02660 mass shifts of generation (being labeled as Q-17.02660) of AKAP4 albumen；
733 lysines of+211.09682 mass shifts of generation (being labeled as K+211.09682) 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
Amount deviant, determines that the lysine of the position there occurs acetylation modification)；
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 the lysine of the position there occurs acetylation modification)；
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 the threonine of the position there occurs phosphorylation modification)；
Serine with 2+79.96685 mass shifts of generation of KIAA1683 protein 69s (is labeled as S+79.96685；Root
According to quality offset value, determine that the serine of the position there occurs phosphorylation modification).
It is described in the another embodiment of the application, it is proposed that a kind of kit for the few weak essence diagnosis of severe
Kit includes the reagent of the above-mentioned biomarker of specific detection.
Detected by above-mentioned biomarker, it is possible to achieve the diagnosis to the few azoospermia of severe.
In the another embodiment of the application, it is proposed that a kind of medicine for treating the few weak essence of severe, in the medicine
Containing 8 serines of AKAP3 protein 20s, 2 serines of 64 threonines of GAPDHS albumen or KIAA1683 protein 69s can be made
Carry out the component of phosphorylation modification, 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 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 in the few weak smart sample of severe conspicuousness downward.It is possible thereby to close
Reason is expected, and using above-mentioned biomarker as target, phosphorus is carried out by the amino acid at the few weak smart patient target of multiplicity
Acidifying or acetylation modification, can play the therapeutic action of weak essence few to severe.
In order that obtaining those skilled in the art can clearly understand the technical scheme of the application, below with reference to tool
The embodiment of body describes the technical scheme of the application in detail.
Test material used is the conventional test material in this area in the embodiment of the present invention, can be by commercial channel
It is commercially available.
Embodiment 1：The screening of the biomarker related to severe teen bra
Specific screening technique is as follows：
First, 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, it is placed in and be incubated on ice 30min cracking, 4 DEG C of centrifugation 14,000g × 20min
Take supernatant.Protein concentration is determined using Bradford methods.
2. proteolysis：The few weak essence of about severe and each 150 μ g sperm proteins of eupyrene sperm sample are taken, 10% polypropylene is used
Acyl ammonia gel electrophoresis (SDS-PAGE) is separated to albumen, and being respectively divided into 5 parts carries out cutting glue enzymolysis.Peptide fragment is entered using ziptip
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
Respectively with 13.5 μ L A phased solns, sample injection volume is 4 μ L to each sample, and flow liquid phase mass spectrometry system of receiving is
Orbitrap Elite(Thermo Scientific).Sample separate before respectively with 4 μ L A balance each other homemade pre-column and point
Analysis post.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
Post (30cm × 75 μm I.D., C18 filler are filled, 3 μm of particle diameter,Dr.Maisch GmbH,Germany).After balance
Load sample, in pre-column, then carries out liquid phase separation to sample under different gradients first under the drive of A phases.150min chromatograms gradient becomes
Change as follows：5-32% Mobile phase Bs 100min；32-80% Mobile phase Bs, 20min；80% Mobile phase B, 30min.Flow velocity is protected all the time
Hold in 300nL/min.ESI ionsprays source and entrance Orbitrap Elite are directly entered by receiving the sample of flow liquid phase separation
Mass Spectrometer Method is carried out in mass spectrograph.
Mass spectrometric data is gathered：The full scan of 350-1800m/z, resolution ratio is 60,000 (m/z 200).Second order spectrum is scanned
When, soak time is 10ms, and isolation width is 2m/z.Fragmentation pattern is collisionally dissociated (collision-induced for induction
Dissociation, CID), normalization collision energy is set as 35%, and dynamic efflux time is 90s.
2nd, MASS SPECTRAL DATA ANALYSIS
Byonic is analyzed：In order to identify the undoded amino acid of sperm protein, we analyze 21 and align with ByonicTM
Often with the protamine mass spectrometric data of the few weak smart patient of severe.Search parameter is as follows：Protease is trypsase, and leakage enzyme site is set
It is 2, parent ion mass deviation is 10ppm, the mass deviation of fragment ion is 0.6Da, the blind upper limit of searching is set to 1000, blind to search lower limit
It is 0.01 to be set to -200. albumen FDR.
Selection peptide fragment fraction>200 and FDR<The unknown modifications that 0.01 peptide fragment is searched as Wildcard SearchTM
Data, constitute mass change one-dimensional data matrix (- 200Da-400Da), then by data according to 1Da excursion, 0.5Da
It is boundary, is divided into 601 data windows.For each data window, it is Gauss with the mclust program bags in R language and mixes
Distributed clustering analysis are 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 included in each peak after cluster, are distributed according to site amino acids, choosing
Data of the distribution more than 5% are selected as a class undoded amino acid.
The normal undoded amino acid with ill group is checked into (p according to its T for detecting frequency<0.05) with ratio (ratio
>2) screened, so as to obtain difference undoded amino acid.Then difference undoded amino acid ROC is made using SPSS softwares
Curve, and its TG-AUC (AUC) is calculated, and then judge its diagnostic value.
3rd, experimental result：
Compare through MASS SPECTRAL DATA ANALYSIS and by the normal undoded amino acid with ill group, so as to obtain 9 non-volumes of difference
Code amino acid, can be specific as follows as the biomarker related to severe teen bra：
The serine of 8+79.96685 mass shifts of generation of 1.AKAP3 protein 20s (is labeled as S+79.96685；According to matter
Amount deviant, determines that the serine of the position there occurs phosphorylation modification)
We have found that phosphorylation modification S+79.96685 is there occurs on 8 serines of AKAP3 protein 20s, by comparing discovery
This phosphorylation modification has lowered 10.7 times in the few weak smart sample significance of severe, and p value is 7.49E-15<0.05.
It is few to severe weak to evaluate the phosphorylation modification S+79.96685 detection frequencies on AKAP3 8 serines of protein 20
The diagnostic of essence, present invention employs ROC curve analysis, AUC is the area under ROC curve, is the most frequently used 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
More than 0.7, then the requirement of clinical diagnosis can be met.
Fig. 1 is the ROC curve of the phosphorylation modification S+79.96685 detection frequencies on 8 serines of AKAP3 protein 20s,
ROC analyses show 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 be as the diagnosis marker of the few weak essence of severe.
When it is 1.5 to check the frequency, sensitivity is 73%, and specificity is 88.9%.When individual detection is carried out, detection frequency
It is secondary when being less than 1.5, it is judged to less weak smart patient (false positive rate is 11.1%).
Phosphorylation modification S+79.96685 detection frequencies in healthy and few weak smart sample on 8 serines of AKAP3 protein 20s
Rate comparative result is shown in Fig. 2, as seen from Figure 2 this undoded amino acid in Healthy People sample average there occurs 4.6 times and
Be there occurs 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
This undoded amino acid has and largely loses in sample.
In view of the above results, the phosphorylation modification S+79.96685 on 8 serines of AKAP3 protein 20s can be as weak less
The potential source biomolecule mark of sperm disease, so as to be predicted to this illness.
6 asparagines of -113.05347 mass shifts of generation (being labeled as N-113.05347) of 2.AKAP4 protein 18s
By MASS SPECTRAL DATA ANALYSIS, it has been found that the asparagine of 186 of AKAP4 albumen has -113.05347 matter
Amount skew (N-113.05347), finds that this undoded amino acid N-113.05347 shows in the few weak smart sample of severe by comparing
Work property has lowered 9.2 times, and p value is 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
The AUC for showing this undoded amino acid N-113.05347 is 0.852>0.7, illustrate that there is preferable diagnosis effect.In inspection frequency
Secondary when being 1.5, sensitivity is 65.1%, and specificity is 93.7%.When individual detection is carried out, when the detection frequency is less than 1.5, quilt
It is judged to less weak smart patient (false positive rate is 6.3%).
Fig. 4 compares detection frequencies of the undoded amino acid N-113.05347 in healthy and few weak smart sample, it can be seen that
This undoded amino acid averagely there occurs 2.9 times in Healthy People sample and 0.3 time there occurs 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
Lose on degree ground.
In view of the above results, the asparagine N-113.05347 in 186 sites of AKAP4 albumen this non-coding amino
Acid can be as the potential source biomolecule mark of teen bra, so as to be predicted to this illness.
6 asparagines of -114.04278 mass shifts of generation (being labeled as N-114.04278) of 3.AKAP4 protein 18s
By MASS SPECTRAL DATA ANALYSIS, it has been found that the asparagine of 186 of AKAP4 albumen has -114.04278 matter
Amount skew (N-114.04278), finds that this undoded amino acid N-114.04278 shows in the few weak smart sample of severe by comparing
Work property has lowered 7.3 times, and p value is 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
The AUC for showing this undoded amino acid N-114.04278 is 0.817>0.7, illustrate that there is preferable diagnosis effect.In inspection frequency
Secondary when being 0.5, sensitivity is 74.6%, and specificity is 84.1%.When individual detection is carried out, when the detection frequency is less than 0.5, quilt
It is judged to less weak smart patient (false positive rate is 15.9%).
6 undoded amino acid N-114.04278 detections frequencies of AKAP4 protein 18s of healthy and few weak smart sample compare sees
Fig. 6, as seen from Figure 6 this undoded amino acid in Healthy People sample it is average there occurs 1.6 times and in pathology sample
There occurs 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 has largely to be lost.
In view of the above results, the asparagine N-114.04278 in 186 sites of AKAP4 albumen this non-coding amino
Acid can be as the potential source biomolecule mark of teen bra, so as to be predicted to this illness.
617 glutamine of -17.02660 mass shifts of generation (being labeled as Q-17.02660) of 4.AKAP4 albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that the glutamine of 617 of AKAP4 albumen has -17.02660 quality
Skew (Q-17.02660), finds this undoded amino acid Q-17.02660 in the few weak smart sample significance of severe by comparing
4.8 times are lowered, p value is 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
The AUC for showing this undoded amino acid Q-17.02660 is 0.802>0.7, illustrate that there is preferable diagnosis effect.In inspection frequency
Secondary when being 0.5, sensitivity is 77.8%, and specificity is 79.4%.When individual detection is carried out, when the detection frequency is less than 0.5, quilt
It is judged to less weak smart patient (false positive rate is 20.6%).
617 undoded amino acid Q-17.02660 detections frequencies of AKAP4 albumen of healthy and few weak smart sample compare sees
Fig. 8, as seen from Figure 8 this undoded amino acid in Healthy People sample it is average there occurs 2.7 times and in pathology sample
There occurs 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 has largely to be lost.
In view of the above results, the glutamine Q-17.02660 in 617 sites of AKAP4 albumen this undoded amino acid can
As the potential source biomolecule mark of teen bra, so as to be predicted to this illness.
733 lysines of+211.09682 mass shifts of generation (being labeled as K+211.09682) of 5.AKAP4 albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that the lysine of 733 of AKAP4 albumen has 211.09682 quality inclined
Move (K+211.09682), find this undoded amino acid K+211.09682 in the few weak smart sample significance of severe by comparing
4.4 times are lowered, p value is 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
The AUC for showing this undoded amino acid K+211.09682 is 0.804>0.7, illustrate that there is preferable diagnosis effect.In inspection frequency
Secondary when being 3.5, sensitivity is 74.6%, and specificity is 71.4%.When individual detection is carried out, when the detection frequency is less than 3.5, quilt
It is judged to less weak smart patient (false positive rate is 28.6%).
The undoded amino acid K+211.09682 detections frequency of healthy and few weak smart sample compares sees Figure 10, can by Figure 10
3 (figures are there occurs in pathology sample to find out this undoded amino acid averagely to be there occurs 14 times in Healthy People sample
Middle solid line), and its median (dotted line in figure) difference is farther out, illustrate in weak smart sample less this undoded amino acid have compared with
Lose on big degree ground.
In view of the above results, the lysine K+211.09682 in 733 sites of AKAP4 albumen this undoded amino acid can
As the potential source biomolecule mark of teen bra, so as to be predicted to this illness.
1 lysine of+42.01108 mass shifts of generation of 6.ATP5A1 protein 53s
By MASS SPECTRAL DATA ANALYSIS, it has been found that there occurs acetylation modification K+ on 1 lysine of ATP5A1 protein 53s
42.01108, find that this acetylation modification has lowered 10.5 times in the few weak smart sample significance of severe by comparing, p value is
3.48E-16<0.05。
Figure 11 is the ROC curve that 1 lysine acetylation modification K+42.01108 of ATP5A1 protein 53s 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 in the inspection frequency
When, sensitivity is 76.2%, and specificity is 85.7%.When individual detection is carried out, when the detection frequency is less than 0.5, it is judged to few
Weak smart patient (false positive rate is 14.3%).
1 lysine acetylation modification K+42.01108 detection frequency ratio of ATP5A1 protein 53s of healthy and few weak smart sample
Relatively see Figure 12, it can be seen that this undoded amino acid in Healthy People sample it is average there occurs 1.7 times and in pathology sample
There occurs 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 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 as few
The potential source biomolecule mark of asthénospermie, so as to be predicted to this illness.
87 lysines of+42.01108 mass shifts of generation (being labeled as K+42.01108) of 7.COX4I1 albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that there occurs acetylation modification K+ on 87 lysines of COX4I1 albumen
42.01108, find that this acetylation modification has lowered 11.3 times in the few weak smart sample significance of severe by comparing, p value is
5.06E-13<0.05。
Figure 13 is the ROC curve that 87 lysine acetylation modification K+42.01108 of COX4I1 albumen detect frequency, ROC points
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 in the inspection frequency
When, sensitivity is 66.7%, and specificity is 95.2%.When individual detection is carried out, when the detection frequency is less than 0.5, it is judged to few
Weak smart patient (false positive rate is 4.8%).
Figure 14 is 87 lysine acetylation modification K+42.01108 detections of COX4I1 albumen of healthy and few weak smart sample
Frequency compares, as seen from Figure 14 this undoded amino acid in Healthy People sample it is average there occurs 1.1 times and in pathology
Be there occurs in sample 0.1 time (solid line in figure), and its median (dotted line in figure) difference is farther out, illustrates in weak smart sample less
This undoded amino acid 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 as weak less
The potential source biomolecule mark of sperm disease, so as to be predicted to this illness.
64 threonines of+79.96685 mass shifts of generation (being labeled as T+79.96685) of 8.GAPDHS albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that there occurs phosphorylation modification T+ on 64 threonines of GAPDHS albumen
79.96685, find that this phosphorylation modification has lowered 4.1 times in the few weak smart sample significance of severe by comparing, 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 analyses show that the AUC of this phosphorylation modification is 0.868>0.7, illustrate that there is preferable diagnosis effect.It is in the inspection frequency
When 3.5, sensitivity is 71.4%, and specificity is 92.1%.When individual detection is carried out, when the detection frequency is less than 3.5, it is judged to
Few weak smart patient (false positive rate is 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 compares, as seen from Figure 16 this undoded amino acid in Healthy People sample average there occurs 5.8 times and
Be there occurs in pathology sample 1.4 times (solid line in figure), and its median (dotted line in figure) difference is farther out, illustrates in weak smart sample less
This undoded amino acid has and largely loses in this.
In view of the above results, the phosphorylation modification T+79.96685 on 64 threonines of GAPDHS albumen can be as weak less
The potential source biomolecule mark of sperm disease, so as to be predicted to this illness.
2 serines of+79.96685 mass shifts of generation (being labeled as S+79.96685) of 9.KIAA1683 protein 69s
By MASS SPECTRAL DATA ANALYSIS, it has been found that there occurs phosphorylation modification S+ on 2 serines of KIAA1683 protein 69s
79.96685, find that this phosphorylation modification has lowered 22 times in the few weak smart sample significance of severe by comparing, p value is
2.73E-10<0.05。
Figure 17 is the ROC curve that 2 serine phosphorylation modification S+79.96685 of KIAA1683 protein 69s detect frequency,
ROC analyses show that the AUC of this phosphorylation modification is 0.808>0.7, illustrate that there is preferable diagnosis effect.It is in the inspection frequency
When 0.5, sensitivity is 65.1%, and specificity is 95.2%.When individual detection is carried out, when the detection frequency is less than 0.5, it is judged to
Few weak smart patient (false positive rate is 4.8%).
Figure 18 is 2 serine phosphorylation modification S+79.96685 inspections of KIAA1683 protein 69s in healthy and few weak smart sample
Measured frequency compares, as seen from Figure 18 this undoded amino acid in Healthy People sample it is average there occurs 2.1 times and in disease
Be there occurs 0.1 time (solid line in figure) in reason sample, and its median (dotted line in figure) difference is farther out, illustrates in weak smart sample less
In this undoded amino acid have and largely lose.
In view of the above results, phosphorylation modification S+79.96685 on 2 serines of KIAA1683 protein 69s can be as
The potential source biomolecule mark of teen bra, so as to be predicted to this illness.
Embodiment 2：Clinical detection is verified
Verified as research object using the few weak smart sample of severe of 4 healthy samples, 8 clinical definites, respectively
Detect the serines of+79.96685 mass shifts of generation of AKAP3 protein 20s 8 of above-mentioned sample, AKAP4 protein 18s 6 occur-
The asparagine of 113.05347 mass shifts, 6 aspartoyls of -114.04278 mass shifts of generation of AKAP4 protein 18s
Amine, 617 glutamine of -17.02660 mass shifts of generation of AKAP4 albumen, 733, AKAP4 albumen occur+211.09682
The lysine of mass shift, ATP5A1 protein 53s 1 lysine, 87, the COX4I1 albumen of+42.01108 mass shifts of generation
Occur the lysine of+42.01108 mass shifts, the threonines of 64+79.96685 mass shifts of generation of GAPDHS albumen and
2 respective detection frequencys of serine of+79.96685 mass shifts of generation of KIAA1683 protein 69s, and by each in embodiment 1
Criterion when biomarker carries out individual detection is diagnosed to sample to be tested.
Result shows：When individually being diagnosed with above-mentioned each biomarker respectively, diagnostic result is consistent with known results.
Illustrate that 9 biomarkers that present invention screening is obtained can be respectively as the diagnosis marker of the few weak essence of severe.
The preferred embodiment of the application is the foregoing is only, the application is not limited to, for the skill of this area
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair
Change, equivalent, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. a kind of screening technique of the biomarker related to severe teen bra, it is characterised in that comprise the following steps：
(1) spermatoblast holoprotein is extracted；
(2) spermatoblast holoprotein is separated using gel electrophoresis, cuts glue enzymolysis, desalination is carried out to the peptide fragment after enzymolysis, 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, gathers mass spectrometric data；
(4) mass spectrometric data is scanned for using non-limiting aminoacid protein modification analysis method, then by multivariate Gaussian
Mixed distribution cluster analysis, it is as substantial amounts of as possible to identify undoded amino acid in sperm protein group；Finally by normal individual
The comparing of undoded amino acid, obtains the albumen related to the few azoospermia of severe non-in weak essence individuality sperm protein group few with severe
Coded amino acid site, as diagnoses and/or treats the biomarker of the few weak sperm disease of severe.
2. screening technique as claimed in claim 1, it is characterised in that in step (1), extracts what spermatoblast holoprotein was used
Method is：Sperm sample is washed using DPBS, RIPA lysate 1~2min of ultrasound are added, incubation 30min on ice is placed in and is split
Solution, centrifugation, takes supernatant.
3. screening technique as claimed in claim 2, it is characterised in that be centrifuged under conditions of 4 DEG C, centrifugal rotational speed is 14,
000g, centrifugation time is 20min.
4. screening technique as claimed in claim 1, it is characterised in that in step (2), using 10% polyacrylamide gel electricity
Swimming is separated to albumen.
5. screening technique as claimed in claim 1, it is characterised in that in step (2), using ziptip to the peptide fragment after enzymolysis
Carry out desalination.
6. screening technique as claimed in claim 1, it is characterised in that in step (3), receives the chromatostrip of flow liquid phase chromatographic isolation
Part is：Mobile phase A：Water containing 0.1% formic acid, Mobile phase B：Acetonitrile containing 0.1% formic acid；
Elution requirement is：0-100min, 95-68% mobile phase A, 5-32% Mobile phase Bs；100-120min, 68-20% mobile phase
A, 32-80% Mobile phase B；120-150min, 20% mobile phase A, 80% Mobile phase B.
7. screening technique as claimed in claim 1, it is characterised in that in step (3), the condition of Mass Spectrometer Method is：350-
The full scan of 1800m/z, resolution ratio is 60,000 (m/z 200).When second order spectrum is scanned, soak time is 10ms, and isolation is wide
It is 2m/z to spend；Fragmentation pattern is collisionally dissociated for induction, and normalization collision energy is set as 35%, and dynamic efflux time is 90s.
8. screening technique as claimed in claim 1, it is characterised in that in step (4), the parameter scanned for mass spectrometric data
It is set to：Protease is trypsase, and leakage enzyme site is set to 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 to 1000, and blind lower limit of searching is set to -200, and albumen FDR is 0.01；
Selection peptide fragment fraction>200 and FDR<The unknown modifications number that 0.01 peptide fragment is searched as Wildcard SearchTM
According to, constitute one-dimensional data matrix (- 200Da-400Da) of mass change, then by data according to 1Da excursion, 0.5Da is
Boundary, is divided into 601 data windows.
9. screening technique as claimed in claim 1, it is characterised 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 are done with the mclust program bags in R language,
Optimal value is taken according to BIC, then analysis is merged to each peak, then with each peak of Gauss Distribution Fitting, determine peak value；
Peptide fragment site data included in each peak after cluster, are distributed according to site amino acids, selection number of the distribution more than 5%
According to as a class undoded amino acid.
10. the biological mark related to severe teen bra that the screening technique screening described in any one of claim 1-9 is obtained
Will thing.
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| WO2018176808A1 (en) * | 2017-03-29 | 2018-10-04 | 山东大学 | Screening and use of biomarker related to severe oligoasthenospermia |
| CN110187128A (en) * | 2019-06-22 | 2019-08-30 | 江西省妇幼保健院 | It is a kind of for diagnosing the biomarker of asthénospermie |
| CN110514834A (en) * | 2018-05-21 | 2019-11-29 | 山东大学 | Application of 1 K+42.011 of ATP5A1 protein 53 in the few weak smart diagnostic reagent of preparation severe |
| CN110514839A (en) * | 2018-05-21 | 2019-11-29 | 山东大学 | Application of 616 N+12.000 of AKAP4 albumen in the few weak smart diagnostic reagent of preparation severe |
| CN110514837A (en) * | 2018-05-21 | 2019-11-29 | 山东大学 | Application of 3 S+79.967 of AKAP3 protein 20 in the few weak smart diagnostic reagent of preparation severe |
| CN110514838A (en) * | 2018-05-21 | 2019-11-29 | 山东大学 | Purposes of 4 N+22.968 of AKAP4 protein 18 in the few weak smart diagnostic reagent of preparation severe |
| CN112782297A (en) * | 2020-12-24 | 2021-05-11 | 郭继生 | Liver cirrhosis related biomarker and screening method and application thereof |
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| CN110514837A (en) * | 2018-05-21 | 2019-11-29 | 山东大学 | Application of 3 S+79.967 of AKAP3 protein 20 in the few weak smart diagnostic reagent of preparation severe |
| CN110514838A (en) * | 2018-05-21 | 2019-11-29 | 山东大学 | Purposes of 4 N+22.968 of AKAP4 protein 18 in the few weak smart diagnostic reagent of preparation severe |
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| CN106872630B (en) | 2018-07-24 |
| WO2018176808A1 (en) | 2018-10-04 |
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