Protein phosphorylation in mammalian sperm during capacitation

Subcellular fractionation revealed that pp97, pp96 and pp64 are head protein whereas pp90 and pp55 are tail proteins. Advanced proteomic analysis (GeLC-MS) identified 37 proteins, including AKAP4, AKAP3, CALI, HSPAlL and HSP70 as candidates for the dephosphorylated proteins. AKAP4 was excluded because it was localised to the tail. Two AKAP3 antibodies showed non-specific binding and a better quality antibody will be needed for further investigations. CALI was excluded because it was localised to the tail fraction. HSP70/72 and HSPA1L were strong candidates for pp64. However, immunoprecipitation of dephosphorylated proteins using phospho (S/T) PKA substrate Ab and HSPA1L Ab was unsuccessful and further work is now required to address this