Seminal proteoforms from bulls with contrasting semen freezability: a story deciphered by top-down mass spectrometry

Top-down proteomics was employed to construct a proteoform atlas of sperm and SP from bulls with low semen freezability (LF) and high semen freezability (HF). Sperm and seminal proteins were fractionated by tandem size exclusion chromatography (<30 kDa) and analyzed by reversed-phase liquid chromatography-tandem mass spectrometry. This approach enabled identification of 299 SP (from 46 families) and 267 sperm proteoforms (from 139 families). Seventy proteoforms belonging to beta-defensin 10, c-type natriuretic peptide (NPPC), caltrin, seminal ribonuclease, osteopontin and binder of sperm protein (BSP) 3 families were unique to HF bulls' SP. LF seminal proteins had 77 unique proteoforms, including caltrin, NPPC, osteopontin, BSP3, serpin family A member 5 and β-NGF families. Proteoform families of SP in HF and LF bulls were related to Ca2+ uptake, capacitation, acrosome reaction, sperm protection, fertilization and proteolytic processes. Thirty-three proteoforms of NPPC, caltrin and cylicin-2 families were upregulated in HF sperm. Twenty-two proteoforms of caltrin, cylicin-2, adenosine triphosphate (ATP) synthases and malate dehydrogenase families were among those upregulated in LF sperm. Truncated and acetylated histone H2A and non-truncated and acetylated c-Myc-binding protein were prevalent in LF sperm. Cylicin-2 proteoforms were observed in HF and LF sperm, and truncated glyceraldehyde-3-phosphate dehydrogenases were observed only in HF sperm. In silico analyses indicated the enrichment of mitochondrial metabolic pathways in HF sperm, including fatty acid metabolism and TCA cycle. Our study provides an unprecedented description of the bovine SP and sperm proteoforms. Post-translational processing appears to define the bioproperties of semen proteins and their associations with sperm cryoresistance.