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Protamine 1 (P1) and protamine 2 family (P2) are extremely basic sperm-specific proteins, packing 85-95% of the paternal DNA. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3 and HP4) arise from the proteolysis of the precursor (pre-P2). Due to the particular protamine physical-chemical properties, their identification by standardized bottom-up mass spectrometry (MS) strategies is not straightforward. Therefore, the aim of this study was to identify the sperm protamine proteoforms profile, including their post-translational modifications, in normozoospermic individuals using two complementary strategies, a top-down MS approach and a proteinase K-digestion based bottom-up MS approach. By top-down MS, described and novel truncated P1 and pre-P2 proteoforms were identified. Intact P1, pre-P2, and P2 mature proteoforms and their phosphorylation pattern were also detected. Additionally, a +61 Da modification in different proteoforms was observed. By the bottom-up MS approach, phosphorylated residues for pre-P2, as well as the new P2 isoform 2, which is not annotated at UniProtKB database, were revealed. Implementing these strategies in comparative studies of different infertile phenotypes, together with the evaluation of P1/P2 and pre-P2/P2 MS-derived ratios, would permit determining specific alterations in the protamine proteoforms and elucidate the role of phosphorylation/dephosphorylation dynamics in male fertility.
This article was published in the following journal.
Name: Journal of proteome research
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