Intergration of co-immunoprecipitation technology and mass spectrometric analysis for sperm membrane protein screening
10.3760/cma.j.issn.1009-9158.2017.04.017
- VernacularTitle:免疫共沉淀联合液相色谱串联质谱技术应用于精子膜蛋白筛选
- Author:
Shuang LIANG
;
Daijun XIANG
;
Hongxia WANG
;
Junlong MA
;
Dandan XUE
;
Peipei LIU
;
Ping LIU
;
Wencan JIANG
;
Xiaozhou YUAN
;
Xinjun LI
;
Sujun GE
;
Chengbin WANG
- Keywords:
Antibodies;
Spermatozoa;
Membrane proteins;
Immunoprecipitation;
Chromatography,liquid;
Tandem mass spectrometry
- From:
Chinese Journal of Laboratory Medicine
2017;40(4):298-302
- CountryChina
- Language:Chinese
-
Abstract:
Objective To reveal the mechanisms of immunological infertility,the method of coimmunoprecipitation(CO-IP) and liquid chromatogram mass/mass (LC-MS/MS) was used to screen sperm membrane proteins which interacting with antisperm antibodies (ASA).Methods This study was designed as a case-control.The disease group including 56 serum samples from 521 cryptogenic infertile patients were screened ASA positive by ELISA and conformed with mixed antiglobulin reaction(MAR).The controls were 31 serum samples which ASA is negative and already possessed healthy offspring.All subjects were enrolled from September 2015 to December 2015 in China PLA General Hospital.Spermatozoa samples from 48donors with normal sperm parameters were from January 2016 to April 2016 in China PLA General Hospital.The purified human sperm membrane proteins were then mixed with serum from disease group (positive for ASA) and control group (not containing ASA).The binding proteins of antisperm antibodies were enriched using CO-IP assay.The immunoprecipitates were separated on sodium dodecyl sulfate-polyactylamide gel (SDS-PAGE),then the binding proteins were cut from the gel and analyzed by LC-MS/MS after the enzymolysis.These proteins could bc idcntified as definition,biological function (s) and subccllular localization with Uniprot database.Results The serum samples from infertile persons (39 females and 17males) were screened ASA positive by ELISA and conformed with MAR.The healthy controls (17 females and 14 males) were ASA-negative in ELISA.Forty proteins that interact with ASA were obtained from the study and these could be divided into three groups:11 antigens detected by control serum samples only,14antigens recognised by both infertile patients and control sera,and 15 antigens specific for patients with ASA.These 15 proteins are Sperm Cation channcl protein 1,Sperm Cation channel protein 3,Sperm Cation channel protein 4,Sperm associated antigen 9,Apolipoprotein A-I,Dynein heavy chain 14,Cylicin-2,Izumo sperm-egg fusion protein 4,Thioredoxin domain-containing protein 2,IQ domain-containing protein H,IQ domain-containing protein F1,Spermatogenesis-associated protein 5,Spermatogenesis-associated protein 5-like protein 1,Sperm acrosome membrane-associated protein 1,E3 ubiquitin-protein ligase RNF 114.Conclusion Fifteen proteins discovered with CO-IP technology and LC-MS/MS analysis could be referred as male immunoinfertility-related antigens and they may hold the great importance in revealing the secret of immunological infertility.
