Systematic Analysis of C‑Termini of Small Open Reading Frame-Encoded Peptides in Human Cancer Cell Lines

The C-termini often regulate protein biological functions through specific structures or modifications. Small open reading frame-encoded peptides (SEPs) make up a novel class of gene expression products that participate in various biological activities. Their C-termini have also been found to affect their function, but the polymorphism of SEPs’ C-termini has not yet been systematically elucidated. Using C-terminal proteomics, we identified 3636 C-terminal peptides from 2168 proteins in three human cancer cell lines, including 3364 peptides from 1901 classical proteins and 272 peptides from 267 SEPs. Approximately 20% of all of the identified C-terminal peptides had been reported in previous studies, originating from mRNA alternative splicing or protease cleavage, while more than 85% of the C-terminal peptides from SEPs were novel. Bioinformatics analysis revealed that most new SEP C-termini are likely produced by protease cleavage by the KLK, MMP, and CAT protease families. Others without accurately predicted hydrolysis sites may originate from alternative splicing or protein trimming. The intact and hydrolysis products of some SEPs were verified by immunoblotting. Some cleavage occurs in the predicted domain, which might affect SEPs’ function. This study enriches the SEP sequence information, provides experimental evidence for SEP in vivo processing, and supports the subsequent functional analysis of SEP.