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.