Application of co-immunoprecipitation coupled LC-MS / MS for identification of sperm immunogenic membrane antigens

Objective: Antisperm antibodies play a main role in immunological infertility, as they impair sperm function by binding to the sperm membrane. In this study, we conducted screening experiments tried to screen sperm membrane proteins interacting with antisperm antibodies by the method of co-immunoprecipitation and liquid chromatogram mass/mass (LC-MS/MS). Methods: The serum samples from infertile persons were previously screened as positive for ASA in ELISA and reproduced with mixed antiglobulin reaction. The swim-up method was used to separate mature and motile sperm. Hypoosmotic swelling homogenization and sonication were sequentially conducted to extract sperm membranes. The purified human sperm membrane proteins were then mixed with serum from disease group (positive for antisperm antibodies) and control (not containing antisperm antibodies) serum samples. The binding proteins of antisperm antibodies were enriched using co-immunoprecipitation assay. The immunoprecipitates were separated on SDS-polyactylamide gel, then the binding proteins were cut from the gel and analyzed by LC-MS/MS after the enzymolysis. Results: The serum samples from infertile persons (39 females and 17 males) were previously screened as positive for ASA in ELISA and conformed to MAR. The healthy controls (17 females and 14 males) were ASA-negative in ELISA and already possessed healthy offspring. 107 proteins that interacted with antisperm antibodies were obtained from the study via LC-MS/MS. These proteins could be divided into three groups: 13 antigens detected by control serum samples only, 14 antigens recognized by both infertile patients and control sera, 80 antigens specific for patients with antisperm antibodies. 15 novel sperm membrane proteins are CatSper1, CatSper3, CatSper4, SPAG9, Apolipoprotein A-I, Dynein heavy chain 14, axonemal, Cylicin-2 (CYLC2), Izumo sperm-egg fusion protein 4, Thioredoxin domain-containing protein 2, IQ domain-containing protein H (IQCH), IQ domain-containing protein F1 (IQCF1), Spermatogenesis-associated protein 5 (SPATA5), Sperm acrosome membrane-associated protein 1, E3 ubiquitin-protein ligase RNF 114. Conclusions: Fifteen novel sperm membrane proteins discovered with co-immunoprecipitation coupled LC-MS/MS analysis could be referred as male immunoinfertility-related antigens. These proteins may be valuable as an indicator in the clinical diagnosis and monitoring treatment of infertility.