Fig. 5. Identification of tyrosine-phosphorylated proteins on the surface of spermatozoa. (A) Cauda epididymal spermatozoa were capacitated for 90 minutes in BWW–Ca²⁺+Sr²⁺+ptx+cAMP. Surface proteins were affinity-purified and resolved by SDS-PAGE. Tyrosine-phosphorylated surface proteins were immunoblotted with anti-phosphotyrosine. Three major bands (P1, P2 and P3) were identified and corresponding bands sequenced from a Coomassie-stained gel by tandem mass spectrometry. (B-D) Alignment of peptide sequences from (B) P1 with mouse hexokinase type 1 (hk1, accession number P19376), (C) P2 with mouse endoplasmin (erp99, accession number P08113) and (D) P3 with mouse heat shock protein 60 (hsp60, accession number NP_034607). Sequenced peptides are underlined. Potential tyrosine phosphorylation sites predicted by NetPhos 2.0 (Blom, 1999) appear in bold italics.