CN106996980A - Purposes of the lysine of 733 generation mass shifts of AKAP4 albumen in the few weak smart diagnostic reagent of severe is prepared - Google Patents
Purposes of the lysine of 733 generation mass shifts of AKAP4 albumen in the few weak smart diagnostic reagent of severe is prepared Download PDFInfo
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Abstract
Purposes the invention discloses the lysine of 733+211.09682 mass shifts of generation of AKAP4 albumen as biomarker in the few weak smart diagnostic reagent of severe is prepared.Present invention discover that：By the inspection frequencys of 733, AKAP4 albumen+211.09682 mass shifts of generation new diagnosis and therapy target can be provided for the few azoospermia of severe for the few azoospermia of diagnosis severe.
Description
Technical field
The present invention relates to medical science and molecular diagnostic techniques field, and in particular to it is inclined that quality occurs for a kind of 733, AKAP4 albumen
Purposes of the lysine of shifting as biomarker in the few weak smart diagnostic reagent of severe is prepared.
Background technology
It is infertile to turn into the healthy reproduction problem in 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 risen.The main cause for causing male sterility is oligoasthenospermia disease.According to the world
Health Organization criterion is provided, if a grades of sperm counts<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's less than normal
A kind of illness, is just oligospermatism when the sperm of male is being less than 2,000 ten thousand for every milliliter.
The research on sperm protein matter group has much at present.Saraswat etc. is using UPLC-MS method 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).).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 of albumen including TUBGCP2 there occurs peroxophosphoric acid (Chan CC, Shui HA, Wu CH et
al.Motility and Protein Phosphorylation in Healthy and Asthenozoospermic
Sperm.Journal of proteome research,8(11),5382-5386(2009))。
Undoded amino acid includes posttranslational modification and amino acid mutation, is the important side of modulin function and structure
Formula, thus using the great undoded amino acid of abnormal under morbid state or number change as disease biomarker,
And then it is significant for the process diagnosed the illness.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 and 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 clustering, to the greatest extent
Substantial amounts of it may identify undoded amino acid in sperm protein group；Finally by non-coding in normal and patient's sperm protein group
The comparison of amino acid, obtains the albumen undoded amino acid site related to the few azoospermia of severe, so that few as severe
The molecular marker of azoospermia.
To achieve the above object, the present invention is adopted the following technical scheme that：
There is provided a kind of screening side of the biomarker related to severe teen bra for the first aspect of the present invention
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 comparison of undoded amino acid, obtains the egg related to the few azoospermia of severe in individual and the few weak individual sperm protein group of essence of severe
White undoded amino acid site, is the biomarker related to severe teen bra.
In step (1), extract the method that uses of spermatoblast holoprotein for：Sperm sample is washed using DPBS, added
RIPA lysate 1~2min of ultrasound, are placed in and are incubated 30min cracking on ice, and centrifugation takes supernatant.
It is preferred that, 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：350-1800m/z full scan, 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 is set
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；
Select peptide fragment fraction>200 and FDR<0.01 peptide fragment is used as Wildcard SearchTMThe unknown modifications searched
Data, constitute the one-dimensional data matrix (- 200Da-400Da) of mass change, then by excursion of the data according to 1Da, 0.5Da
For boundary, 601 data windows are divided into.
In step (4), the method for multivariate Gaussian mixed distribution clustering is：For each data window, R languages are used
The mclust program bags called 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 being distributed according to site amino acids, selection distribution is used as a class undoded amino acid more than 5% data.
In step (4), the undoded amino acid of the few weak essence individual of normal individual and severe is detected that the T of frequency is examined according to it
Test (p<0.05) with ratio (ratio>2) screened, so as to obtain difference undoded amino acid.
Above-mentioned screening technique is to be used to obtain biomarker, and is not diagnosis to obtain disease and treatment results 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 there is provided according to above-mentioned screening technique screen obtain it is related to severe teen bra
Biomarker, the biomarker includes 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)；
The asparagine (being labeled as N-113.05347) of 6-113.05347 mass shifts of generation of AKAP4 protein 18s；
The asparagine (being labeled as N-114.04278) of 6-114.04278 mass shifts of generation of AKAP4 protein 18s；
The glutamine (being labeled as Q-17.02660) of 617-17.02660 mass shifts of generation of AKAP4 albumen；
The lysine (being labeled as K+211.09682) of 733+211.09682 mass shifts of generation of AKAP4 albumen；
The lysine of 1+42.01108 mass shift of generation of ATP5A1 protein 53s (is labeled as K+42.01108；According to matter
Deviant is measured, determines that 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)；
(S+79.96685 is labeled as with the serine of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s；Root
According to quality offset value, determine that the serine of the position there occurs phosphorylation modification).
There is provided the serine conduct of 8+79.96685 mass shifts of generation of AKAP3 protein 20s for the third aspect of the present invention
Purposes of the biomarker in the few weak smart diagnostic reagent of severe is prepared.
It is preferred that, it is few weak that 8 serines for occurring+79.96685 mass shifts of AKAP3 protein 20s are also used as severe
The target of essence treatment, so that for the few weak smart treatment of severe.
Further, the present invention also provides the serine conduct of 8+79.96685 mass shifts of generation of AKAP3 protein 20s
Purposes of the biomarker in the few weak smart medicine of severe is prepared.
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 (serines of 8+79.96685 mass shifts of generation of AKAP3 protein 20s).
The present invention also provides a kind of treat in the few weak smart medicine of severe, the medicine containing can make AKAP3 protein 20s 8
Position serine carries out the component of phosphorylation modification.
The present invention also provides a kind of severe few weak smart diagnostic method, and step is：Detect sample to be tested AKAP3 protein 20s 8
The frequency of+79.96685 mass shifts occurs for position serine, if the detection frequency is less than 1.5, is judged to less weak smart patient.
There is provided the asparagine of 6-113.05347 mass shifts of generation of AKAP4 protein 18s for the fourth aspect of the present invention
It is used as purposes of the biomarker in the few weak smart diagnostic reagent of severe is prepared.
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 (asparagines of 6-113.05347 mass shifts of generation of AKAP4 protein 18s).
The present invention also provides a kind of severe few weak smart diagnostic method, and step is：Detect sample to be tested AKAP4 protein 18s 6
The frequency of -113.05347 mass shifts occurs for position asparagine, when the detection frequency is less than 0.5, is judged to less weak smart patient.
There is provided the asparagine of 6-114.04278 mass shifts of generation of AKAP4 protein 18s for the fifth aspect of the present invention
It is used as purposes of the biomarker in the few weak smart diagnostic reagent of severe is prepared.
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 (asparagines of 6-114.04278 mass shifts of generation of AKAP4 protein 18s).
The present invention also provides a kind of severe few weak smart diagnostic method, and step is：Detect sample to be tested AKAP4 protein 18s 6
The frequency of -114.04278 mass shifts occurs for position asparagine, when the detection frequency is less than 0.5, is judged to less weak smart patient.
There is provided the glutamine work of 617-17.02660 mass shifts of generation of AKAP4 albumen for the sixth aspect of the present invention
For purposes of the biomarker in the few weak smart diagnostic reagent of severe is prepared.
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 (glutamine of 617-17.02660 mass shifts of generation of AKAP4 albumen).
The present invention also provides a kind of severe few weak smart diagnostic method, and step is：Detect sample to be tested AKAP4 albumen 617
The frequency of -17.02660 mass shifts occurs for position glutamine, when the detection frequency is less than 0.5, is judged to less weak smart patient.
There is provided the lysine conduct of 733+211.09682 mass shifts of generation of AKAP4 albumen for the seventh aspect of the present invention
Purposes of the biomarker in the few weak smart diagnostic reagent of severe is prepared.
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 (lysines of 733+211.09682 mass shifts of generation of AKAP4 albumen).
The present invention also provides a kind of severe few weak smart diagnostic method, and step is：Detect sample to be tested AKAP4 albumen 733
The frequency of+211.09682 mass shifts occurs for position lysine, when the detection frequency is less than 3.5, is judged to less weak smart patient.
There is provided the lysine conduct of 1+42.01108 mass shift of generation of ATP5A1 protein 53s for the eighth aspect of the present invention
Purposes of the biomarker in the few weak smart diagnostic reagent of severe is prepared.
It is preferred that, it is few weak that 1 lysine for occurring+42.01108 mass shifts of ATP5A1 protein 53s is also used as severe
The target of essence treatment, so that for the few weak smart treatment of severe.
Further, the present invention also provides the lysine conduct of 1+42.01108 mass shift of generation of ATP5A1 protein 53s
Purposes of the biomarker in the few weak smart medicine of severe is prepared.
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 (lysines of 1+42.01108 mass shift of generation of ATP5A1 protein 53s).
The present invention also provides a kind of treat in the few weak smart medicine of severe, the medicine containing can make ATP5A1 albumen
531 lysines carry out the component of acetylation modification.
The present invention also provides a kind of severe few weak smart diagnostic method, and step is：Detect sample to be tested ATP5A1 protein 53s 1
The frequency of+42.01108 mass shifts occurs for position lysine, if the detection frequency is less than 0.5, is judged to less weak smart patient.
There is provided the lysine conduct of 87+42.01108 mass shifts of generation of COX4I1 albumen for the ninth aspect of the present invention
Purposes of the biomarker in the few weak smart diagnostic reagent of severe is prepared.
It is preferred that, it is few weak that 87 lysines for occurring+42.01108 mass shifts of COX4I1 albumen are also used as severe
The target of essence treatment, so that for the few weak smart treatment of severe.
Further, the present invention also provides the lysine conduct of 87+42.01108 mass shifts of generation of COX4I1 albumen
Purposes of the biomarker in the few weak smart medicine of severe is prepared.
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 (lysines of 87+42.01108 mass shifts of generation of COX4I1 albumen).
The present invention also provides a kind of treat in the few weak smart medicine of severe, the medicine containing can make COX4I1 albumen 87
Position lysine carries out the component of acetylation modification.
The present invention also provides a kind of severe few weak smart diagnostic method, and step is：Detect sample to be tested COX4I1 albumen 87
The frequency of+42.01108 mass shifts occurs for position lysine, if the detection frequency is less than 0.5, is judged to less weak smart patient.
There is provided the threonine conduct of 64+79.96685 mass shifts of generation of GAPDHS albumen for the tenth aspect of the present invention
Purposes of the biomarker in the few weak smart diagnostic reagent of severe is prepared.
It is preferred that, it is few weak that 64 threonines for occurring+79.96685 mass shifts of GAPDHS albumen are also used as severe
The target of essence treatment, so that for the few weak smart treatment of severe.
Further, the present invention also provides the threonine conduct of 64+79.96685 mass shifts of generation of GAPDHS albumen
Purposes of the biomarker in the few weak smart medicine of severe is prepared.
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 (threonines of 64+79.96685 mass shifts of generation of GAPDHS albumen).
The present invention also provides a kind of treat in the few weak smart medicine of severe, the medicine containing can make GAPDHS albumen 64
Position threonine carries out the component of phosphorylation modification.
The present invention also provides a kind of severe few weak smart diagnostic method, and step is：Detect sample to be tested GAPDHS albumen 64
The frequency of+79.96685 mass shifts occurs for position threonine, if the detection frequency is less than 3.5, is judged to less weak smart patient.
There is provided the serine of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s for the eleventh aspect of the present invention
It is used as purposes of the biomarker in the few weak smart diagnostic reagent of severe is prepared.
It is preferred that, it is few that 2 serines for occurring+79.96685 mass shifts of KIAA1683 protein 69s are also used as severe
The target of weak essence treatment, so that for the few weak smart treatment of severe.
Further, the present invention also provides the serine work of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s
For purposes of the biomarker in the few weak smart medicine of severe is prepared.
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 (serines of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s).
The present invention also provides a kind of treat in the few weak smart medicine of severe, the medicine containing can make KIAA1683 albumen
692 serines carry out the component of phosphorylation modification.
The present invention also provides a kind of severe few weak smart diagnostic method, and step is：Detect sample to be tested KIAA1683 albumen
The frequency of+79.96685 mass shifts occurs for 692 serines, if the detection frequency is less than 0.5, is judged to less weak smart patient.
Beneficial effects of the present invention：
(1) present invention establishes a kind of screening technique of the biomarker related to severe teen bra first, leads to
Cross and the mass spectrometric data of great amount of samples sperm protein is analyzed and processed, it is as substantial amounts of as possible to identify non-volume in sperm protein group
Code amino acid；Finally by the comparison 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 that as the molecular marker of 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 lacks azoospermia to diagnose severe, and new diagnosis and treatment is provided for the few azoospermia of severe
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 illustrate be used for explain the application, do 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.
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 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
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation, device, component and/or combinations thereof.
As background technology is introduced, undoded amino acid is not related to also in the prior art as with severe and lacks weak essence
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 comparison of undoded amino acid, obtains the egg related to the few azoospermia of severe in individual and the few weak individual sperm protein group of essence of severe
White undoded amino acid site, is the biomarker 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
Gaussian Mixture distributed clustering analysis is measured, it is as substantial amounts of as possible to identify undoded amino acid in sperm protein group.Finally will be normal
T inspections (the p of frequency is detected according to it with the undoded amino acid of illness group<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 has been obtained,
It is specific as follows：
The serine of 8+79.96685 mass shifts of generation of AKAP3 protein 20s (is labeled as S+79.96685；According to quality
Deviant, determines that the serine of the position there occurs phosphorylation modification)；
The asparagine (being labeled as N-113.05347) of 6-113.05347 mass shifts of generation of AKAP4 protein 18s；
The asparagine (being labeled as N-114.04278) of 6-114.04278 mass shifts of generation of AKAP4 protein 18s；
The glutamine (being labeled as Q-17.02660) of 617-17.02660 mass shifts of generation of AKAP4 albumen；
The lysine (being labeled as K+211.09682) of 733+211.09682 mass shifts of generation of AKAP4 albumen；
The lysine of 1+42.01108 mass shift of generation of ATP5A1 protein 53s (is labeled as K+42.01108；According to matter
Deviant is measured, determines that 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)；
(S+79.96685 is labeled as with the serine of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s；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.
By being detected to 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 to treat in the few weak smart medicine of severe, the medicine
Containing 8 serines of AKAP3 protein 20s, 2 serines of 64 threonines of GAPDHS albumen or KIAA1683 protein 69s can be made
The component of phosphorylation modification is carried out, or containing 87 bad ammonia of 1 lysine of ATP5A1 protein 53s or COX4I1 albumen can be made
Acid carries out the component of acetylation modification.
It has been investigated that, 8 serines of AKAP3 protein 20s of phosphorylation modification, 64 threonines of GAPDHS albumen and
The downward of 2 serines of KIAA1683 protein 69s conspicuousness in the few weak smart sample of severe, the ATP5A1 albumen of acetylation modification
87 lysines of 531 lysines or COX4I1 albumen also lack the downward of conspicuousness in weak smart sample in severe.It is possible thereby to close
Reason is expected, using above-mentioned biomarker as target, by carrying out phosphorus to the amino acid at the few weak smart patient target of multiplicity
Acidifying or acetylation modification, can be played to the few weak smart therapeutic action of severe.
In order that the technical scheme of the application can clearly be understood by obtaining those skilled in the art, 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 pass through 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, is placed in and is incubated 30min cracking on ice, 4 DEG C of centrifugation 14,000g × 20min
Take supernatant.Protein concentration is determined using Bradford methods.
2. proteolysis：The about few weak essence of severe and each 150 μ g sperm proteins of eupyrene sperm sample are taken, 10% polypropylene is used
Acyl ammonia gel electrophoresis (SDS-PAGE) is separated to albumen, is respectively divided into 5 parts and is carried 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
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 balanced each other respectively with 4 μ L A homemade pre-column and divide
Analyse 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.It is directly entered ESI ionsprays source by receiving the sample of flow liquid phase separation and enters Orbitrap Elite
Mass Spectrometer Method is carried out in mass spectrograph.
Mass spectrometric data is gathered：350-1800m/z full scan, 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 that induction is collisionally dissociated (collision-induced
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 use ByonicTM21 pairs of analysis is 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 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 to 1000, and blind lower limit of searching is set
It is 0.01 for -200. albumen FDR.
Select peptide fragment fraction>200 and FDR<0.01 peptide fragment is used as Wildcard SearchTMThe unknown modifications searched
Data, constitute the one-dimensional data matrix (- 200Da-400Da) of mass change, then by excursion of the data according to 1Da, 0.5Da
For boundary, 601 data windows are divided into.For each data window, Gauss is of the mclust program bags in R language and is mixed
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 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 detected to the T inspections (p of frequency according to it<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：
Compared through MASS SPECTRAL DATA ANALYSIS and by the normal undoded amino acid with ill group, so as to obtain the non-volume of 9 differences
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
Deviant is measured, determines that the serine of the position there occurs phosphorylation modification)
We have found that there occurs phosphorylation modification S+79.96685 on 8 serines of AKAP3 protein 20s, by it was found that
This phosphorylation modification has lowered 10.7 times in the few weak smart sample significance of severe, 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 8 serines of AKAP3 protein 20s
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 used as the few weak smart diagnosis marker of severe.
When it is 1.5 to examine the frequency, sensitivity is 73%, and specificity is 88.9%.When carrying out individual detection, detection frequency
It is secondary when being less than 1.5, it is judged to less weak smart patient (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 averagely there occurs in Healthy People sample 4.6 times and
It 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
The undoded amino acid of this in sample, which has, largely to be lost.
In view of the phosphorylation modification S+79.96685 on the above results, 8 serines of AKAP3 protein 20s can be as few weak
The potential source biomolecule mark of sperm disease, so as to be predicted to this illness.
The asparagine (being labeled as N-113.05347) of 6-113.05347 mass shifts of generation of 2.AKAP4 protein 18s
By MASS SPECTRAL DATA ANALYSIS, it has been found that the asparagine of 186 of AKAP4 albumen has -113.05347 matter
Amount skew (N-113.05347), by it was found that this undoded amino acid N-113.05347 shows 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 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.Examining frequency
Secondary when being 1.5, sensitivity is 65.1%, and specificity is 93.7%.When carrying out individual detection, when the detection frequency is less than 1.5, quilt
It is judged to less weak smart patient (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, this non-coding amino of the asparagine N-113.05347 in 186 sites of AKAP4 albumen
Acid can as teen bra potential source biomolecule mark, so as to be predicted to this illness.
The asparagine (being labeled as N-114.04278) of 6-114.04278 mass shifts of generation of 3.AKAP4 protein 18s
By MASS SPECTRAL DATA ANALYSIS, it has been found that the asparagine of 186 of AKAP4 albumen has -114.04278 matter
Amount skew (N-114.04278), by it was found that this undoded amino acid N-114.04278 shows 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 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.Examining frequency
Secondary when being 0.5, sensitivity is 74.6%, and specificity is 84.1%.When carrying out individual detection, when the detection frequency is less than 0.5, quilt
It is judged to less weak smart patient (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, which compare, sees
Fig. 6, as seen from Figure 6 this undoded amino acid averagely there occurs in Healthy People sample 1.6 times and in pathology sample
It 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, 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 as teen bra potential source biomolecule mark, so as to be predicted to this illness.
The glutamine (being labeled as Q-17.02660) of 617-17.02660 mass shifts of generation of 4.AKAP4 albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that the glutamine of 617 of AKAP4 albumen has -17.02660 quality
Offset (Q-17.02660), by it was found that this undoded amino acid Q-17.02660 is in the few weak smart sample significance of 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 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.Examining frequency
Secondary when being 0.5, sensitivity is 77.8%, and specificity is 79.4%.When carrying out individual detection, when the detection frequency is less than 0.5, quilt
It is judged to less weak smart patient (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, which compare, sees
Fig. 8, as seen from Figure 8 this undoded amino acid averagely there occurs in Healthy People sample 2.7 times and in pathology sample
It 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, 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 mark as teen bra, so as to be predicted to this illness.
The lysine (being labeled as K+211.09682) of 733+211.09682 mass shifts of generation of 5.AKAP4 albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that the lysine of 733 of AKAP4 albumen has 211.09682 quality inclined
Move (K+211.09682), by it was found that this undoded amino acid K+211.09682 is in the few weak smart sample significance of 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 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.Examining frequency
Secondary when being 3.5, sensitivity is 74.6%, and specificity is 71.4%.When carrying out individual detection, when the detection frequency is less than 3.5, quilt
It is judged to less weak smart patient (false positive rate is 28.6%).
The undoded amino acid K+211.09682 detections frequency of healthy and few weak smart sample, which compares, sees Figure 10, can by Figure 10
3 (figures are there occurs in pathology sample to find out this undoded amino acid averagely to there occurs in Healthy People sample 14 times
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 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 mark as teen bra, so as to be predicted to this illness.
The lysine of 1+42.01108 mass shift of generation of 6.ATP5A1 protein 53s
By MASS SPECTRAL DATA ANALYSIS, it has been found that there occurs acetylation modification K+ on 1 lysine of ATP5A1 protein 53s
42.01108, by it was found that this acetylation modification has lowered 10.5 times in the few weak smart sample significance of severe, p value is
3.48E-16<0.05。
Figure 11 is 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 examining the frequency
When, sensitivity is 76.2%, and specificity is 85.7%.When carrying out individual detection, 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 averagely there occurs 1.7 times in Healthy People sample and in pathology sample
It 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, which has, largely to be lost.
In view of the acetylation modification K+42.01108 on the above results, 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.
The lysine (being labeled as K+42.01108) of 87+42.01108 mass shifts of generation of 7.COX4I1 albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that there occurs acetylation modification K+ 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 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 examining the frequency
When, sensitivity is 66.7%, and specificity is 95.2%.When carrying out individual detection, 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 detects for 87 lysine acetylation modification K+42.01108 of COX4I1 albumen of healthy and few weak smart sample
Frequency compares, and this undoded amino acid averagely there occurs 1.1 times in Healthy People sample and in pathology as seen from Figure 14
It 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, which has, largely to be lost.
In view of the acetylation modification K+42.01108 on the above results, 87 lysines of COX4I1 albumen can be as few weak
The potential source biomolecule mark of sperm disease, so as to be predicted to this illness.
The threonine (being labeled as T+79.96685) of 64+79.96685 mass shifts of generation of 8.GAPDHS albumen
By MASS SPECTRAL DATA ANALYSIS, it has been found that there occurs phosphorylation modification T+ on 64 threonines of GAPDHS albumen
79.96685, by it was found that this phosphorylation modification has lowered 4.1 times in the few weak smart sample significance of severe, p value is
1.82E-14<0.05。
Figure 15 is the ROC curve of the phosphorylation modification T+79.96685 detection frequencies on 64 threonines of GAPDHS albumen,
ROC analyses show that the AUC of this phosphorylation modification is 0.868>0.7, illustrate that there is preferable diagnosis effect.It is examining the frequency
When 3.5, sensitivity is 71.4%, and specificity is 92.1%.When carrying out individual detection, 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 compare, as seen from Figure 16 this undoded amino acid averagely there occurs in Healthy People sample 5.8 times and
It 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
There being this undoded amino acid in this largely loses.
In view of the phosphorylation modification T+79.96685 on the above results, 64 threonines of GAPDHS albumen can be as few weak
The potential source biomolecule mark of sperm disease, so as to be predicted to this illness.
The serine (being labeled as S+79.96685) of 2+79.96685 mass shifts of generation of 9.KIAA1683 protein 69s
By MASS SPECTRAL DATA ANALYSIS, it has been found that there occurs phosphorylation modification S+ on 2 serines of KIAA1683 protein 69s
79.96685, by it was found that this phosphorylation modification has lowered 22 times in the few weak smart sample significance of severe, p value is
2.73E-10<0.05。
Figure 17 is 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 examining the frequency
When 0.5, sensitivity is 65.1%, and specificity is 95.2%.When carrying out individual detection, 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, and this undoded amino acid averagely there occurs 2.1 times in Healthy People sample and in disease as seen from Figure 18
It 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 phosphorylation modification S+79.96685 on the above results, 2 serines of KIAA1683 protein 69s can conduct
The potential source biomolecule mark of teen bra, so as to be predicted to this illness.
Embodiment 2：Clinical detection is verified
Verified, distinguished as research object using the few weak smart sample of severe of 4 healthy samples, 8 clinical definites
Serine, the AKAP4 protein 18s 6 for detecting+79.96685 mass shifts of generation of AKAP3 protein 20s 8 of above-mentioned sample occur-
The aspartoyl of 6-114.04278 mass shifts of generation of asparagine, AKAP4 protein 18s of 113.05347 mass shifts
Amine, 733, glutamine, the AKAP4 albumen of 617-17.02660 mass shifts of generation of AKAP4 albumen occur+211.09682
87, lysine, the COX4I1 albumen of 1+42.01108 mass shift of generation of lysine, ATP5A1 protein 53s of mass shift
Occur the threonine of 64, lysine, GAPDHS albumen+79.96685 mass shifts of generation of+42.01108 mass shifts and
The respective detection frequency of serine of 2+79.96685 mass shifts of generation of KIAA1683 protein 69s, and by each in embodiment 1
Criterion when biomarker carries out individual detection is diagnosed to sample to be tested.
As a result show：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 few weak smart diagnosis marker 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 substitution, improvement etc., should be included within the protection domain of the application.
Claims (2)
- The lysine of 733+211.09682 mass shifts of generation of 1.AKAP4 albumen is few in preparation severe as biomarker Purposes in weak smart diagnostic reagent.
- 2. a kind of kit for the few weak essence diagnosis of severe, it is characterised in that the kit includes specific detection power Profit requires the reagent of the biomarker described in 1.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018176808A1 (en) * | 2017-03-29 | 2018-10-04 | 山东大学 | Screening and use of biomarker related to severe oligoasthenospermia |
| 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 |
| CN116879563A (en) * | 2023-07-20 | 2023-10-13 | 南昌大学 | Application of 317 th lysine succinylated modified lactate dehydrogenase C as weak sperm disease development marker or target |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060199204A1 (en) * | 2001-10-05 | 2006-09-07 | U.S. Epa | Genetic testing for male factor infertility |
| US20100151498A1 (en) * | 2007-04-13 | 2010-06-17 | Teruaki Iwamoto | Method of Diagnosing Asthenozoospermia |
| CN102323398A (en) * | 2011-05-19 | 2012-01-18 | 国家人口计生委科学技术研究所 | Solution for sperm detection |
| CN202994620U (en) * | 2012-11-30 | 2013-06-12 | 上海市计划生育科学研究所 | Semi-quantification sperm density detection kit |
| CN104007103A (en) * | 2013-02-21 | 2014-08-27 | 湖州海创生物科技有限公司 | Sperm detection method and kit thereof |
| US20140302522A1 (en) * | 2011-11-02 | 2014-10-09 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Method for predicting the presence of reproductive cells in testis |
| CN106148524A (en) * | 2016-07-11 | 2016-11-23 | 吉林大学 | Research human body azoospermia and the method that quickly detects of severe oligospermia and the test kit applying its method |
-
2017
- 2017-03-29 CN CN201710197650.3A patent/CN106996980B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060199204A1 (en) * | 2001-10-05 | 2006-09-07 | U.S. Epa | Genetic testing for male factor infertility |
| US20100151498A1 (en) * | 2007-04-13 | 2010-06-17 | Teruaki Iwamoto | Method of Diagnosing Asthenozoospermia |
| CN102323398A (en) * | 2011-05-19 | 2012-01-18 | 国家人口计生委科学技术研究所 | Solution for sperm detection |
| US20140302522A1 (en) * | 2011-11-02 | 2014-10-09 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Method for predicting the presence of reproductive cells in testis |
| CN202994620U (en) * | 2012-11-30 | 2013-06-12 | 上海市计划生育科学研究所 | Semi-quantification sperm density detection kit |
| CN104007103A (en) * | 2013-02-21 | 2014-08-27 | 湖州海创生物科技有限公司 | Sperm detection method and kit thereof |
| CN106148524A (en) * | 2016-07-11 | 2016-11-23 | 吉林大学 | Research human body azoospermia and the method that quickly detects of severe oligospermia and the test kit applying its method |
Non-Patent Citations (4)
| Title |
|---|
| CHYING-CHYUAN CHAN ET AL.: "Motility and Protein Phosphorylation in Healthy and Asthenozoospermic Sperm", 《JOURNAL OF PROTEOME RESEARCH》 * |
| PRIYANKA P. PARTE ET AL.: "Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC–MSE) reveals altered proteomic signatures in asthenozoospermia", 《JOURNAL OF PROTEOMICS》 * |
| 孙国海: "人精子赖氨酸乙酰化蛋白质组学研究", 《南京医科大学硕士学位论文》 * |
| 石理华等: "精子蛋白表达下调在成人弱精症中的意义分析", 《临床泌尿外科杂志》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018176808A1 (en) * | 2017-03-29 | 2018-10-04 | 山东大学 | Screening and use of biomarker related to severe oligoasthenospermia |
| 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 |
| CN116879563A (en) * | 2023-07-20 | 2023-10-13 | 南昌大学 | Application of 317 th lysine succinylated modified lactate dehydrogenase C as weak sperm disease development marker or target |
| CN116879563B (en) * | 2023-07-20 | 2024-05-14 | 南昌大学 | Application of 317 th lysine succinylated modified lactate dehydrogenase C as weak sperm disease development marker or target |
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