Improved Identification and Analysis of Small Open Reading Frame Encoded Polypeptides

Computational,
genomic, and proteomic approaches have been used
to discover nonannotated protein-coding small open reading frames
(smORFs). Some novel smORFs have crucial biological roles in cells
and organisms, which motivates the search for additional smORFs. Proteomic
smORF discovery methods are advantageous because they detect smORF-encoded
polypeptides (SEPs) to validate smORF translation and SEP stability.
Because SEPs are shorter and less abundant than average proteins,
SEP detection using proteomics faces unique challenges. Here, we optimize
several steps in the SEP discovery workflow to improve SEP isolation
and identification. These changes have led to the detection of several
new human SEPs (novel human genes), improved confidence in the SEP
assignments, and enabled quantification of SEPs under different cellular
conditions. These improvements will allow faster detection and characterization
of new SEPs and smORFs.