Sperm Proteomics and Phosphoproteomics: High-Throughput Methods Tailored to Gain Insight into Sperm Cell Signaling Pathways.

Protein phosphorylation is critical to successful sperm cell maturation and oocyte interaction. However, despite longstanding and largely global use of protein tyrosine phosphorylation as a robust marker of "readiness for fertilization," a deeper understanding of sperm-specific phosphorylation events and phosphoproteins is still lacking. To address this critical knowledge gap, here we present a complete guide to produce and analyze highly comprehensive phosphoproteomes from human spermatozoa. This chapter provides a bespoke guide to in vitro human sperm capacitation, phosphopeptide enrichment, and high-throughput sample preparation for mass spectrometry based on the renowned EasyPhos method. Replete with hints for successful application, we intend this guide to be of equal use in improving our understanding of male infertility, as well as establishing new insight for the development of non-hormonal, sperm-targeted contraceptives.