SUMMARYTEX101 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 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.Non-standard abbreviationsTEX101Testis-expressed sequence 101 proteinDPEP3Dipeptidase 3AC-MSAffinity capture-mass spectrometryco-IP-MSCoimmunoprecipitation-mass spectrometryGPIGlycosylphosphatidylinositolLFQLabel-free quantificationmAbMonoclonal antibodyNHSN-hydroxysuccinimidePRMParallel reaction monitoringPTMPosttranslational modificationSPSeminal plasmaSRMSelected reaction monitoringFDRfalse detection rate
A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry