Beyond single modification: Reanalysis of the acetylproteome of human sperm reveals widespread multiple modifications

• We evaluated the results of reanalysis with three variable modifications.
• Ubiquitinated peptides could be co-enriched with acetylated peptides.
• Phosphorylated peptides are randomly eluted with acetylated peptides.
• About 500 proteins contain more than one type of PTM in human sperm.

Sperm is an ideal model for studying post-translational modifications since its transcriptional and translational activities are nearly silent. Thus, sperm functions are mainly regulated at the protein level, especially by means of post-translational modifications. Published proteomic datasets may contain valuable undiscovered information. In this study, we reanalyzed the raw data from previous acetylproteome study on human capacitated sperm to include two additional modifications: phosphorylation and ubiquitination. We successfully identified approximately 500 proteins with multiple types of modifications. Compared with recently developed serial enrichment strategy for multiple modifications, reanalysis of single modification enriched data provides a direct and efficient alternative approach. These results greatly expand our knowledge of protein modifications in human sperm.

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