CN106990177B - Purposes of the glutamine of 617, AKAP4 albumen generation mass shifts in the few weak smart diagnostic reagent of preparation severe - Google Patents
Purposes of the glutamine of 617, AKAP4 albumen generation mass shifts in the few weak smart diagnostic reagent of preparation severe Download PDFInfo
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Abstract
The invention discloses 617, AKAP4 albumen to occur purposes of the glutamine of -17.02660 mass shifts as biomarker in the diagnostic reagent that preparation severe lacks weak essence.Present invention discover that: the inspection frequency that -17.02660 mass shifts occur by 617, AKAP4 albumen, which can be used to diagnose severe, lacks azoospermia, lacks azoospermia for severe and provides 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 617 generation quality of AKAP4 albumen are inclined
Purposes of the glutamine of shifting as biomarker in the few weak smart diagnostic reagent of preparation severe.
Background technique
The infertile healthy reproduction problem having become in worldwide, wherein the infertility as caused by male factor is big
50% or so generally is accounted for, and in recent years in the trend risen.The main reason for causing male sterility is oligoasthenospermia disease.According to the world
Health Organization criterion regulation, if a grades of sperm count < 25%, (a+b) grade sperm count < 50%, and sperm motility rate is lower than 60%
Words, so that it may be diagnosed as asthénospermie.Oligospermatism refers to that the sperm number in sperm has fecundity male's lower than normal
A kind of illness, when the sperm of male is when being lower than 2,000 ten thousand for every milliliter, with regard to being oligospermatism.
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. utilizes 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. carries 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 has 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.
Summary of the invention
For the above-mentioned prior art, it is an object of the invention to provide a kind of biomarkers relevant to severe teen bra
Screening technique and application.The invention firstly uses NanoHPLC-MS/MS mass spectrometer systems and label-free proteomics side
The sperm protein undoded amino acid of method weak smart disease few to multiple groups severe has carried out the mass spectral analysis of depth；Then non-limit is utilized
Determine aminoacid protein modification analysis method to scan for mass spectrometric data, using multivariate Gaussian mixed distribution cluster point
Analysis, largely identifies undoded amino acid in sperm protein group as far as possible；Finally by normal and patient's sperm protein group
The comparison of undoded amino acid obtains lacking azoospermia relevant albumen undoded amino acid site to severe, thus as
Severe lacks the molecular marker of azoospermia.
To achieve the above object, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of screening side of biomarker relevant to severe teen bra
Method includes the following steps:
(1) spermatoblast holoprotein is extracted；
(2) spermatoblast holoprotein is separated using gel electrophoresis, cuts glue enzymatic hydrolysis, desalination is carried out to the peptide fragment after enzymatic hydrolysis,
Sample is prepared；
(3) by the sample of step (2) using receiving flow liquid phase chromatographic isolation, received 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 in individual and the few weak individual sperm protein group of essence of severe obtains lacking the relevant egg of azoospermia to severe
White undoded amino acid site, biomarker as relevant to severe teen bra.
In step (1), the method that spermatoblast holoprotein uses is extracted are as follows: sperm sample is washed using DPBS, is added
RIPA lysate 1~2min of ultrasound, is placed in and is incubated for 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 separated using 10% polyacrylamide gel electrophoresis (SDS-PAGE) to albumen.
In step (2), it is preferred that carry out desalination to the peptide fragment after enzymatic hydrolysis using ziptip.
In step (3), the chromatographic condition of flow liquid phase chromatographic isolation is received are as follows: mobile phase A: the water containing 0.1% formic acid, flowing
Phase B: the acetonitrile containing 0.1% formic acid；Flow liquid phase mass spectrometry system of receiving is Orbitrap Elite (Thermo
Scientific)
Elution requirement are as follows: 0-100min, 95-68% mobile phase A, 5-32% Mobile phase B；100-120min, 68-20% stream
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 are as follows: the full scan of 350-1800m/z, 60,000 (m/z of resolution ratio
200).When second order spectrum scans, activation 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), parameter setting that mass spectrometric data is scanned for are as follows: 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 the peptide fragment of peptide fragment score>200 and FDR<0.01 as Wildcard SearchTMThe unknown modifications searched
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 are as follows: each data window is directed to, with R language
The mclust program bag called the turn does Gaussian Mixture distributed clustering analysis, takes 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 greater 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 examined according to its T for detecting frequency
It tests (p<0.05) and ratio (ratio>2) 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 to severe teen bra
Biomarker, the biomarker includes but is not limited to:
The serine of AKAP3 protein 20 8+79.96685 mass shifts of generation (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 AKAP4 protein 18 6-113.05347 mass shifts of generation；
The asparagine (being labeled as N-114.04278) of AKAP4 protein 18 6-114.04278 mass shifts of generation；
The glutamine (being labeled as Q-17.02660) of 617, AKAP4 albumen -17.02660 mass shifts of generation；
The lysine (being labeled as K+211.09682) of 733, AKAP4 albumen+211.09682 mass shifts of generation；
The lysine of ATP5A1 protein 53 1+42.01108 mass shifts of generation (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, COX4I1 albumen+42.01108 mass shifts of generation (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, GAPDHS albumen+79.96685 mass shifts of generation (is labeled as T+79.96685；According to quality
Deviant determines that phosphorylation modification has occurred in the threonine of the position)；
The serine that+79.96685 mass shifts occur with KIAA1683 protein 69 2 (is labeled as S+79.96685；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 AKAP3 protein 20 8+79.96685 mass shifts of generation
Purposes of the biomarker in the diagnostic reagent that preparation severe lacks weak essence.
Preferably, it is few weak to be also used as severe for the serine of AKAP3 protein 20 8+79.96685 mass shifts of generation
The target of essence treatment, for severe lacks the treatment of weak essence.
Further, the present invention also provides the serine conducts that AKAP3 protein 20 8 occur+79.96685 mass shifts
Purposes of the biomarker in the therapeutic agent that preparation severe lacks weak essence.
It include specific detection in the kit the present invention also provides a kind of kit for the few weak essence diagnosis of severe
The reagent of above-mentioned biomarker (serines of AKAP3 protein 20 8+79.96685 mass shifts of generation).
The present invention also provides a kind of drug treated severe and lack weak essence, AKAP3 protein 20 8 can be made by containing 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, steps are as follows: detection sample to be tested AKAP3 protein 20 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 AKAP4 protein 18 6-113.05347 mass shifts of generation
As purposes of the biomarker in the diagnostic reagent that preparation severe lacks weak essence.
It include specific detection in the kit the present invention also provides a kind of kit for the few weak essence diagnosis of severe
The reagent of above-mentioned biomarker (asparagines of AKAP4 protein 18 6-113.05347 mass shifts of generation).
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, steps are as follows: detection sample to be tested AKAP4 protein 18 6
The frequency of -113.05347 mass shifts occurs for position asparagine, when detecting the frequency less than 0.5, is judged to less weak smart patient.
The fifth aspect of the present invention provides the asparagine of AKAP4 protein 18 6-114.04278 mass shifts of generation
As purposes of the biomarker in the diagnostic reagent that preparation severe lacks weak essence.
It include specific detection in the kit the present invention also provides a kind of kit for the few weak essence diagnosis of severe
The reagent of above-mentioned biomarker (asparagines of AKAP4 protein 18 6-114.04278 mass shifts of generation).
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, steps are as follows: detection sample to be tested AKAP4 protein 18 6
The frequency of -114.04278 mass shifts occurs for position asparagine, when detecting the frequency less than 0.5, is judged to less weak smart patient.
The sixth aspect of the present invention, the glutamine for providing 617, AKAP4 albumen -17.02660 mass shifts of generation are made
For purposes of the biomarker in the diagnostic reagent that preparation severe lacks weak essence.
It include specific detection in the kit the present invention also provides a kind of kit for the few weak essence diagnosis of severe
The reagent of above-mentioned biomarker (glutamine of 617, AKAP4 albumen -17.02660 mass shifts of generation).
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, steps are as follows: detection sample to be tested AKAP4 albumen 617
The frequency of -17.02660 mass shifts occurs for position glutamine, when detecting the frequency less than 0.5, is judged to less weak smart patient.
The seventh aspect of the present invention provides the lysine conduct of 733, AKAP4 albumen+211.09682 mass shifts of generation
Purposes of the biomarker in the diagnostic reagent that preparation severe lacks weak essence.
It include specific detection in the kit the present invention also provides a kind of kit for the few weak essence diagnosis of severe
The reagent of above-mentioned biomarker (lysines of 733, AKAP4 albumen+211.09682 mass shifts of generation).
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, steps are as follows: detection sample to be tested AKAP4 albumen 733
The frequency of+211.09682 mass shifts occurs for position lysine, when detecting the frequency less than 3.5, is judged to less weak smart patient.
The eighth aspect of the present invention provides the lysine conduct of ATP5A1 protein 53 1+42.01108 mass shifts of generation
Purposes of the biomarker in the diagnostic reagent that preparation severe lacks weak essence.
Preferably, it is few weak to be also used as severe for the lysine of ATP5A1 protein 53 1+42.01108 mass shifts of generation
The target of essence treatment, for severe lacks the treatment of weak essence.
Further, the present invention also provides the lysine conducts that ATP5A1 protein 53 1 occurs+42.01108 mass shifts
Purposes of the biomarker in the therapeutic agent that preparation severe lacks weak essence.
It include specific detection in the kit the present invention also provides a kind of kit for the few weak essence diagnosis of severe
The reagent of above-mentioned biomarker (lysines of ATP5A1 protein 53 1+42.01108 mass shifts of generation).
The present invention also provides a kind of drug treated severe and lack weak essence, ATP5A1 albumen can be made by containing 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, steps are as follows: detection sample to be tested ATP5A1 protein 53 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, COX4I1 albumen+42.01108 mass shifts of generation
Purposes of the biomarker in the diagnostic reagent that preparation severe lacks weak essence.
Preferably, it is few weak to be also used as severe for the lysine of 87, COX4I1 albumen+42.01108 mass shifts of generation
The target of essence treatment, for severe lacks the treatment of weak essence.
Further, the present invention also provides the lysine conducts that 87, COX4I1 albumen occur+42.01108 mass shifts
Purposes of the biomarker in the therapeutic agent that preparation severe lacks weak essence.
It include specific detection in the kit the present invention also provides a kind of kit for the few weak essence diagnosis of severe
The reagent of above-mentioned biomarker (lysines of 87, COX4I1 albumen+42.01108 mass shifts of generation).
The present invention also provides a kind of drug treated severe and lack weak essence, COX4I1 albumen 87 can be made by containing 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, steps are as follows: detection 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, GAPDHS albumen+79.96685 mass shifts of generation
Purposes of the biomarker in the diagnostic reagent that preparation severe lacks weak essence.
Preferably, it is few weak to be also used as severe for the threonine of 64, GAPDHS albumen+79.96685 mass shifts of generation
The target of essence treatment, for severe lacks the treatment of weak essence.
Further, the present invention also provides the threonine conducts that 64, GAPDHS albumen occur+79.96685 mass shifts
Purposes of the biomarker in the therapeutic agent that preparation severe lacks weak essence.
It include specific detection in the kit the present invention also provides a kind of kit for the few weak essence diagnosis of severe
The reagent of above-mentioned biomarker (threonines of 64, GAPDHS albumen+79.96685 mass shifts of generation).
The present invention also provides a kind of drug treated severe and lack weak essence, GAPDHS albumen 64 can be made by containing 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, steps are as follows: detection 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 KIAA1683 protein 69 2+79.96685 mass shifts of generation
As purposes of the biomarker in the diagnostic reagent that preparation severe lacks weak essence.
Preferably, it is few to be also used as severe for the serine of KIAA1683 protein 69 2+79.96685 mass shifts of generation
The target of weak essence treatment, for severe lacks the treatment of weak essence.
Further, the present invention also provides the serine works that KIAA1683 protein 69 2 occur+79.96685 mass shifts
For purposes of the biomarker in the therapeutic agent that preparation severe lacks weak essence.
It include specific detection in the kit the present invention also provides a kind of kit for the few weak essence diagnosis of severe
The reagent of above-mentioned biomarker (serines of KIAA1683 protein 69 2+79.96685 mass shifts of generation).
The present invention also provides a kind of drug treated severe and lack weak essence, KIAA1683 albumen can be made by containing 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, steps are as follows: detection 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 screening technique of biomarker relevant to severe teen bra for the first time, leads to
It crosses and the mass spectrometric data of great amount of samples sperm protein is analyzed and processed, 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, lacks azoospermia for severe and provides new diagnosing and treating
Target spot.
Detailed description of the invention
The accompanying drawings constituting a part of this application is used to provide further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation are not constituted an undue limitation on the present application for explaining the application.
The ROC curve of phosphorylation modification S+79.96685 detection frequency on Fig. 1: the AKAP3 serine of protein 20 8.
Fig. 2: the phosphorylation modification S+79.96685 inspection in healthy and few weak smart sample on 8 serines of AKAP3 protein 20
Measured frequency compares.
The ROC curve of Fig. 3: the AKAP4 undoded amino acid N-113.05347 of protein 18 6 detection frequency.
Fig. 4: 6 undoded amino acid N-113.05347 of AKAP4 protein 18 of healthy and few weak smart sample detect frequency ratio
Compared with.
The ROC curve of Fig. 5: the AKAP4 undoded amino acid N-114.04278 of protein 18 6 detection frequency.
Fig. 6: 6 undoded amino acid N-114.04278 of AKAP4 protein 18 of healthy and few weak smart sample detect frequency ratio
Compared with.
The ROC curve of Fig. 7: AKAP4 617, albumen undoded amino acid Q-17.02660 detection frequency.
Fig. 8: 617 undoded amino acid Q-17.02660 of AKAP4 albumen of healthy and few weak smart sample detect frequency ratio
Compared with.
The ROC curve of Fig. 9: AKAP4 733, albumen undoded amino acid K+211.09682 detection frequency.
Figure 10: the undoded amino acid K+211.09682 detection frequency of healthy and few weak smart sample compares.
Figure 11: the ATP5A1 lysine acetylation of protein 53 1 modifies the ROC curve of K+42.01108 detection frequency.
Figure 12: 1 lysine acetylation modification K+42.01108 detection of ATP5A1 protein 53 of healthy and few weak smart sample
Frequency compares.
Figure 13: COX4I1 87, albumen lysine acetylation modifies the ROC curve of K+42.01108 detection frequency.
Figure 14: 87 lysine acetylation modification K+42.01108 detection frequencies of COX4I1 albumen of healthy and few weak smart sample
Rate compares.
The ROC curve of phosphorylation modification T+79.96685 detection frequency on Figure 15: GAPDHS 64, albumen threonine.
Figure 16: the phosphorylation modification T+79.96685 inspection in healthy and few weak smart sample on 64 threonines of GAPDHS albumen
Measured frequency compares.
Figure 17: the KIAA1683 serine phosphorylation of protein 69 2 modifies the ROC curve of S+79.96685 detection frequency.
Figure 18: 2 serine phosphorylation modification S+79.96685 inspections of KIAA1683 protein 69 in healthy and few weak smart sample
Measured frequency compares.
Specific embodiment
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 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 singular
Also it is 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 their combination.
As background technique is introduced, also weak essence is not lacked as with severe about undoded amino acid in the prior art
The report of the relevant biomarker of sub- disease.Based on this, the invention proposes a kind of biologies relevant to severe teen bra
The screening technique of marker and application.
In a kind of embodiment of the application, a kind of biomarker relevant to severe teen bra is proposed
Screening technique includes the following steps:
(1) spermatoblast holoprotein is extracted；
(2) spermatoblast holoprotein is separated using gel electrophoresis, cuts glue enzymatic hydrolysis, desalination is carried out to the peptide fragment after enzymatic hydrolysis,
Sample is prepared；
(3) by the sample of step (2) using receiving flow liquid phase chromatographic isolation, received 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 in individual and the few weak individual sperm protein group of essence of severe obtains lacking the relevant egg of azoospermia to severe
White undoded amino acid site, biomarker as relevant to 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 convenience, the application is first with NanoHPLC-MS/MS mass spectrometer system and non-
The sperm protein undoded amino acid of label quantitative proteomics method weak smart disease few to multiple groups 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
(p<0.05) and ratio (ratio>2) is examined to be screened according to its T for detecting frequency with the undoded amino acid of illness group, from
And obtain difference undoded amino acid.Then difference undoded amino acid ROC curve is made using SPSS software, and calculates its song
Area (AUC) under line, and then judge its diagnostic value.
Using the above-mentioned screening technique of the application, series biomarker relevant to severe teen bra has been obtained,
It is specific as follows:
The serine of AKAP3 protein 20 8+79.96685 mass shifts of generation (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 AKAP4 protein 18 6-113.05347 mass shifts of generation；
The asparagine (being labeled as N-114.04278) of AKAP4 protein 18 6-114.04278 mass shifts of generation；
The glutamine (being labeled as Q-17.02660) of 617, AKAP4 albumen -17.02660 mass shifts of generation；
The lysine (being labeled as K+211.09682) of 733, AKAP4 albumen+211.09682 mass shifts of generation；
The lysine of ATP5A1 protein 53 1+42.01108 mass shifts of generation (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, COX4I1 albumen+42.01108 mass shifts of generation (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, GAPDHS albumen+79.96685 mass shifts of generation (is labeled as T+79.96685；According to quality
Deviant determines that phosphorylation modification has occurred in the threonine of the position)；
The serine that+79.96685 mass shifts occur with KIAA1683 protein 69 2 (is labeled as S+79.96685；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, a kind of kit for the few weak essence diagnosis of severe is proposed, it is described
It include the reagent of the above-mentioned biomarker of specific detection in kit.
By detecting to above-mentioned biomarker, the diagnosis for lacking azoospermia to severe may be implemented.
In the another embodiment of the application, a kind of drug treated severe and lack weak essence is proposed, in the drug
Containing 28 serines of AKAP3 protein 20,64 threonines of GAPDHS albumen or KIAA1683 protein 69 serines can be made
The component of phosphorylation modification is carried out, or containing 87 bad ammonia of 1 lysine of ATP5A1 protein 53 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 20 of phosphorylation modification, 64 threonines of GAPDHS albumen and
The downward of 2 serines of KIAA1683 protein 69 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: the screening of biomarker relevant to 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 is added, is placed in and is incubated for 30min cracking, 4 DEG C of centrifugation 14,000g × 20min on ice
Take supernatant.Protein concentration is measured using Bradford method.
2. proteolysis: taking the about few weak essence of severe and each 150 μ g sperm protein of eupyrene sperm sample, use 10% polypropylene
Acyl ammonia gel electrophoresis (SDS-PAGE) separates albumen, is respectively divided into 5 parts and carries out cutting glue enzymatic hydrolysis.Using ziptip to peptide fragment into
Row desalination.
3. mass spectral analysis: receiving flow liquid phase chromatographic isolation: A phase: the water containing 0.1% formic acid；B phase: contain 0.1% formic acid
Acetonitrile
Each sample uses 13.5 μ L A phased solns respectively, 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 with 4 μ L A balance each other homemade pre-column and divide
Analyse column.The specification of pre-column and analytical column be respectively as follows: pre-column (5 μm of 4cm × 150 μm I.D., C18 packing material size,), analysis
Column (filling of 30cm × 75 μm I.D., C18 filler, 3 μm of partial size,Dr.Maisch GmbH,Germany).After balance
Then sample load sample first under the drive of A phase carries out liquid phase separation in pre-column under different gradients.150min chromatography gradient becomes
Change as follows: 5-32% Mobile phase B 100min；32-80% Mobile phase B, 20min；80% Mobile phase B, 30min.Flow velocity is protected always
It holds in 300nL/min.Through receiving stream liquid phase separation sample be directly entered ESI ionspray source and enter Orbitrap Elite
Mass Spectrometer Method is carried out in mass spectrograph.
Mass spectrometric data acquisition: the full scan of 350-1800m/z, resolution ratio 60,000 (m/z 200).Second order spectrum scanning
When, activation 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 analysis: the undoded amino acid in order to identify sperm protein, we use ByonicTM21 pairs of analysis normal
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 as
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, and blind lower limit of searching is set
It is 0.01 for -200. albumen FDR.
Select the peptide fragment of peptide fragment score>200 and FDR<0.01 as Wildcard SearchTMThe unknown modifications searched
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 that Gauss is with the mclust program bag 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 greater than 5% are selected as a kind of undoded amino acid.
Will (p < 0.05) and ratio (ratio normally be examined according to its T for detecting frequency with the undoded amino acid of illness group
> 2) it is screened, to obtain difference undoded amino acid.Then difference undoded amino acid ROC is made using SPSS software
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 be used as biomarker relevant to severe teen bra, specific as follows:
The serine of 1.AKAP3 protein 20 8+79.96685 mass shifts of generation (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 20, by it was found that
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 that frequency is detected for the phosphorylation modification S+79.96685 on evaluation 8 serines of AKAP3 protein 20
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 lower；AUC
0.7 or more, then it can satisfy the requirement of clinical diagnosis.
Fig. 1 is the ROC curve of the phosphorylation modification S+79.96685 detection frequency on 8 serines of AKAP3 protein 20,
ROC illustrate with preferable diagnosis effect, i.e. AKAP3 albumen analysis shows that the AUC of this phosphorylation modification is 0.856 > 0.7
Phosphorylation modification S+79.96685 on 208 serines can be used as the diagnosis marker that severe lacks weak essence.
When examining the frequency is 1.5, sensitivity 73%, specificity 88.9%.When carrying out individual detection, detection frequency
It is secondary less than 1.5 when, be judged to less weak smart patient's (false positive rate 11.1%).
Phosphorylation modification S+79.96685 in healthy and few weak smart sample on 8 serines of AKAP3 protein 20 detects frequency
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 20 can be used as less weak
The potential source biomolecule marker of sperm disease, to predict this illness.
The asparagine (being labeled as N-113.05347) of 2.AKAP4 protein 18 6-113.05347 mass shifts of generation
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, 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 18 detect frequency, and ROC analysis is aobvious
Show that the AUC of this undoded amino acid N-113.05347 is 0.852 > 0.7, illustrates 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 detecting the frequency 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 the detection frequency of healthy and few weak smart sample, 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 used as the potential source biomolecule marker of teen bra, to predict this illness.
The asparagine (being labeled as N-114.04278) of 3.AKAP4 protein 18 6-114.04278 mass shifts of generation
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, 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 18 detect frequency, and ROC analysis is aobvious
Show that the AUC of this undoded amino acid N-114.04278 is 0.817 > 0.7, illustrates 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 detecting the frequency 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 detection frequencies of AKAP4 protein 18 of healthy and few weak smart sample, which compare, sees
Fig. 6, this undoded amino acid averagely has occurred 1.6 times in Healthy People sample and in pathology sample as seen from Figure 6
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
Coding 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 used as the potential source biomolecule marker of teen bra, to predict this illness.
The glutamine (being labeled as Q-17.02660) of 617,4.AKAP4 albumen -17.02660 mass shifts of generation
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 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 analysis is aobvious
Show that the AUC of this undoded amino acid Q-17.02660 is 0.802 > 0.7, illustrates 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 detecting the frequency 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 detection frequencies of AKAP4 albumen of healthy and few weak smart sample, which compare, sees
Fig. 8, this undoded amino acid averagely has occurred 2.7 times in Healthy People sample and in pathology sample as seen from Figure 8
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
Coding 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 733,5.AKAP4 albumen+211.09682 mass shifts of generation
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 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 analysis is aobvious
Show that the AUC of this undoded amino acid K+211.09682 is 0.804 > 0.7, illustrates 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 detecting the frequency 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 detection 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 6.ATP5A1 protein 53 1+42.01108 mass shifts of generation
By MASS SPECTRAL DATA ANALYSIS, it has been found that acetylation modification K+ has occurred on 1 lysine of ATP5A1 protein 53
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 53 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.The frequency is being examined to be 0.5
When, sensitivity 76.2%, specificity 85.7%.When carrying out individual detection, when detecting the frequency 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 53 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
Coding 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 53 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 87,7.COX4I1 albumen+42.01108 mass shifts of generation
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, illustrates there is preferable diagnosis effect.The frequency is being examined to be 0.5
When, sensitivity 66.7%, specificity 95.2%.When carrying out individual detection, when detecting the frequency 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 detection
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 less weak
The potential source biomolecule marker of sperm disease, to predict this illness.
The threonine (being labeled as T+79.96685) of 64,8.GAPDHS albumen+79.96685 mass shifts of generation
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 frequency on 64 threonines of GAPDHS albumen,
ROC illustrate with preferable diagnosis effect analysis shows that the AUC of this phosphorylation modification is 0.868 > 0.7.It is in the inspection frequency
When 3.5, sensitivity 71.4%, specificity 92.1%.When carrying out individual detection, when detecting the frequency 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 in healthy and few weak smart sample on 64 threonines of GAPDHS albumen
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 line 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 less weak
The potential source biomolecule marker of sperm disease, to predict this illness.
The serine (being labeled as S+79.96685) of 9.KIAA1683 protein 69 2+79.96685 mass shifts of generation
By MASS SPECTRAL DATA ANALYSIS, it has been found that phosphorylation modification S+ has occurred on 2 serines of KIAA1683 protein 69
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 69 modify the ROC curve that S+79.96685 detects frequency,
ROC illustrate with preferable diagnosis effect analysis shows that the AUC of this phosphorylation modification is 0.808 > 0.7.It is in the inspection frequency
When 0.5, sensitivity 65.1%, specificity 95.2%.When carrying out individual detection, when detecting the frequency 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 69 modify S+79.96685 inspection 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 69 be can be used as
The potential source biomolecule marker of teen bra, to predict this illness.
Embodiment 2: clinical detection verifying
It is verified, is distinguished as research object using the few weak smart sample of severe of 4 healthy samples, 8 clinical definites
Detect serine, AKAP4 protein 18 6 generations-of AKAP3 protein 20 8+79.96685 mass shifts of generation of above-mentioned sample
The aspartoyl of asparagine, AKAP4 protein 18 6-114.04278 mass shifts of generation of 113.05347 mass shifts
Amine, 617, AKAP4 albumen 733, glutamine, AKAP4 albumen that -17.02660 mass shifts occur occur+211.09682
87, lysine, the COX4I1 albumen of lysine, ATP5A1 protein 53 1+42.01108 mass shifts of generation of mass shift
Occur 64, lysine, GAPDHS albumen of+42.01108 mass shifts+79.96685 mass shifts of generation threonine and
The respective detection frequency of serine of KIAA1683 protein 69 2+79.96685 mass shifts of generation, and by each in embodiment 1
Judgment criteria 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 preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (2)
- The glutamine of 617,1.AKAP4 albumen -17.02660 mass shifts of generation is few in preparation severe as biomarker Purposes in the diagnostic reagent of weak essence.
- 2. a kind of kit for the few weak essence diagnosis of severe, which is characterized in that include that specific detection is weighed in the kit Benefit require 1 described in biomarker reagent.
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