TEX101 is a testis-specific protein expressed exclusively in male germ cells and is a validated biomarker of male infertility. Studies in mice suggest that TEX101 is a cell-surface chaperone which regulates, through protein-protein interactions, the maturation of proteins involved in spermatozoa transit and oocyte binding. Male TEX101-null mice are sterile. Here, we identified by co-immunoprecipitation-mass spectrometry the interactome of human TEX101 in testicular tissues and spermatozoa. The testis-specific cell-surface dipeptidase 3 (DPEP3) emerged as the top hit. We further validated the TEX101-DPEP3 complex by using hybrid immunoassays. Combinations of antibodies recognizing different epitopes of TEX101 and DPEP3 facilitated development of a simple immunoassay to screen for disruptors of TEX101-DPEP3 complex. As a proof-of-a-concept, we demonstrated that anti-TEX101 clone 34ED604 (antibody T4) disrupted the native TEX101-DPEP3 complex. Disrupting antibodies may be used to study the human TEX101-DPEP3 complex, and to develop modulators for male fertility.

To verify TEX101 interactome, we developed and applied Tier 2 targeted mass spectrometry analysis. Two multiplexed SRM assays coupled to co-IP were used to monitor the candidate proteins in testicular tissue and spermatozoa, respectively. Our MS and MS/MS identification data (including potential post-translational modifications) was used to select proteotypic peptides. Peptides with 7-20 aa and without oxidation, deamidation or potential missed cleavages were selected. Selected peptides were also confirmed with SRM Atlas database (www.srmatlas.org). To facilitate accurate relative quantification, synthetic heavy isotope-labeled peptides were obtained for all proteins. Survey unscheduled SRM assays with all possible y- and b-ion fragments were prepared for light and heavy peptides and monitored in testicular tissue or spermatozoa lysates on TSQ QuantivaTM. Intensity and interferences were assessed for each transition, and the three most intense transitions were selected for each heavy and light forms. Two separate multiplex SRM assays were finally developed for candidates identified in testicular tissues (20 heavy and light peptides for 9 candidates, and TEX101) and spermatozoa (20 heavy and light peptides for 9 candidates, and TEX101). All peptides were scheduled within 2-min intervals during a 30-min gradient. TEX101-interacting proteins were verified in pools of independent testicular tissue and spermatozoa lysates (one biological replicate for each type of specimen). Three full process replicates were performed independently (from co-IP to trypsin digestion) for each specimen. Each process replicate was analysed in duplicate, and raw files were analyzed with Skyline software (v3.6.0.10493). The relative abundance of each endogenous peptide and corresponding protein was calculated according to the heavy-to-light ratio and the amount of the heavy peptides spiked in each sample. Non-specific mouse IgG antibody was used as a negative control. Co-IP of TEX101 complexes in testicular tissues and spermatozoa was performed, as described above. Prior to trypsin digestion, 500 fmoles of heavy isotope-labeled TEX101 and DPEP3 proteotypic peptides (AGTETAILATK*-JPTtag and SWSEEELQGVLR*-JPTtag) were added to all samples. Eight heavy isotope-labeled peptides for TEX101 interactome in testicular tissue, and eight heavy peptides for TEX101 interactome in spermatozoa, were pooled and diluted to a final concentration of 100 fmol/µL. Five µL of the heavy peptide pool were spiked to each sample after digestion. Initial testicular tissue and spermatozoa lysates (10 µg) were digested, to calculate the recovery of each protein after co-IP. Digests were desalted, and peptides were separated with a 30-min gradient and quantified by TSQ QuantivaTM mass spectrometer.

Testicular tissues with active spermatogenesis (confirmed by histological examination) were obtained with informed consent by orchiectomy from men with scrotal pain or testicular masses. Upon removal, testicular tissues were subjected to snap-freezing, and stored in liquid nitrogen. Semen samples were collected from healthy fertile pre-vasectomy patients, they were allowed to liquefy at RT for 1 hour, and then aliquoted and centrifuged 3 times at 13,000 g for 15 min at RT. The SP and sperm cells were separated and stored at -80°C. Sample collection was approved by the institutional review boards.