Identification of phosphorylated small ORF-encoded peptides in Hep3B cells by LC/MS/MS

Small ORF-encoded peptides (SEPs) are a class of low molecular weight proteins and peptides comprising <100 amino acids with important functions in various life activities. Although the sequence length is short, SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. In this work, we enriched phosphopeptides with IMAC and TiO₂ materials and analyzed the phosphorylated SEPs in Hep3B cells. A total of 24 phosphorylated SEPs were identified, and 11 SEPs were coded by ncRNA. For the sequence analysis, we found that the general characteristics of phosphorylated SEPs are roughly the same as canonical proteins. Besides, two phosphorylation SEPs have the Stathmin family signature 2 motif, which can regulate the microtubule cytoskeleton. Some SEPs have domains or signal peptides, indicating their specific functions and subcellular locations. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of some SEPs. However, only one-fifth of the predicted phosphorylation sites were identified by LC/MS/MS, indicating that many SEP PTMs are hidden in the dark, waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation. SIGNIFICANCE: Small ORF-encoded peptides (SEPs) are important in various life activities. Although the sequence length is short (<100AA), SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. We enriched phosphopeptides and analyzed the phosphorylated SEPs in Hep3B cells. That is the first time to explore the PTM of SPEs systematically. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of SEPs. More SEP PTMs are hidden in the dark and waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation.