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  

Intergration of co-immunoprecipitation technology and mass spectrometric analysis for sperm membrane protein screening

作　　者：梁爽[1] 向代军[2] 王红霞[2] 马骏龙[2] 薛丹丹[2] 刘培培[2] 刘萍[2] 姜文灿 苑晓舟 李新军[2] 葛素君 王成彬[2] 

机构地区：[1]河北北方学院研究生部检验学院2014级临床检验诊断学,张家口075000 [2]解放军总医院临床检验科

出　　处：《中华检验医学杂志》2017年第4期298-302,共5页Chinese Journal of Laboratory Medicine

摘　　要：目的为了揭示免疫性不育的机制，研究通过免疫共沉淀联合液相色谱串联二级质谱技术筛选与抗精子抗体（ASA）结合的精子膜蛋白。方法病例对照研究。选取2015年9至12月在中国人民解放军总医院生殖中心门诊就诊的不明原因不孕症血清标本521份，采用ELISA试剂盒进行ASA阳性血清的筛选，再应用混合抗球蛋白试验对ASA阳性血清进行确认，得到ASA阳性血清标本56份作为疾病组，同时选择在该院临床检验科正常查体的血清ASA阴性的已正常生育过的健康人血清标本31份作为对照组。选取2016年1至4月在中国人民解放军总医院行正常查体男性志愿者精液标本48份，混合后提取精子膜蛋白。进行免疫共沉淀反应，精子膜蛋白分别与ASA阳性血清、ASA阴性血清孵育后，同时加入蛋白A/G（protein A/G）孵育，采用十二烷基硫酸钠－聚丙烯酰氨凝胶电泳对免疫沉淀复合物进行分离，切胶酶解后，进行液相色谱串联二级质谱分析，得到相应的蛋白编码。通过UniProt数据库检索查找这些蛋白的名称、生物学功能、细胞定位等信息。结果筛选出ASA阳性不育患者血清56份（女39例、男17例），ASA阴性正常生育者31例（女17例、男14例）。共鉴定出40种相关蛋白，可分为3组蛋白，分别是：仅与正常组血清相结合的蛋白（11种）；既与正常组血清结合，也与ASA阳性不育组血清结合的蛋白（14种）；仅与ASA阳性不育组血清结合的蛋白（15种）。这15种蛋白分别是精子阳离子通道蛋白1，精子阳离子通道蛋白3，精子阳离子通道蛋白4，精子相关抗原9，载脂蛋白A－I, 轴丝动力蛋白重链14 , 多波段多肽－2，精卵融合蛋白4, 硫氧还蛋白结构域蛋白2 , 含有蛋白H的IQ域，含有蛋白F1的IQ域，精子发生相关蛋白5, 精子发生相关蛋白5样蛋白1，精子顶体膜相关蛋白1 ，E3泛素蛋白连接酶RNF114。结论�ObjectiveTo reveal the mechanisms of immunological infertility, the method of co-immunoprecipitation（CO-IP） and liquid chromatogram mass/mass （LC-MS/MS） was used to screen sperm membrane proteins which interacting with antisperm antibodies （ASA）.MethodsThis 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 48 donors 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 be identified as definition, biological function（s） and subcellular localization with Uniprot database.ResultsThe serum samples from infertile persons （39 females and 17 males） 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, 14 antigens recognised by both infertile patients and control sera, and 15 antigens specific for patients with ASA. These 15 proteins are Sperm Cation channel protein 1, Sperm Cation channel protein 3, Sperm Cation channel protein 4, Sperm associated antigen 9, Apolipoprotein A-I, Dynein heavy c

关 键 词：抗体 精子 膜蛋白质类 免疫沉淀法 色谱法 液相 串联质谱法 

分 类 号：O657.63[理学—分析化学] R446.6[理学—化学] R698.2[医药卫生—诊断学]