BONCAT-based Profiling of Nascent Small and Alternative Open Reading Frame-encoded Proteins

Xiongwen Cao; Yanran Chen; Alexandra Khitun; Sarah A Slavoff

doi:10.21769/BioProtoc.4585

BONCAT-based Profiling of Nascent Small and Alternative Open Reading Frame-encoded Proteins

Bio Protoc. 2023 Jan 5;13(1):e4585. doi: 10.21769/BioProtoc.4585.

Authors

Xiongwen Cao^{1

2}, Yanran Chen^{1

2}, Alexandra Khitun^{1

2}, Sarah A Slavoff^{1

2

3}

Affiliations

¹ Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.
² Institute of Biomolecular Design and Discovery, Yale University, West Haven, Connecticut 06516, United States.
³ Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, United States.

Abstract

RIBO-seq and proteogenomics have revealed that mammalian genomes harbor thousands of unannotated small and alternative open reading frames (smORFs, <100 amino acids, and alt-ORFs, >100 amino acids, respectively). Several dozen mammalian smORF-encoded proteins (SEPs) and alt-ORF-encoded proteins (alt-proteins) have been shown to play important biological roles, while the overwhelming majority of smORFs and alt-ORFs remain uncharacterized, particularly at the molecular level. Functional proteomics has the potential to reveal key properties of unannotated SEPs and alt-proteins in high throughput, and an approach to identify SEPs and alt-proteins undergoing regulated synthesis should be of broad utility. Here, we introduce a chemoproteomic pipeline based on bio-orthogonal non-canonical amino acid tagging (BONCAT) (Dieterich et al., 2006) to profile nascent SEPs and alt-proteins in human cells. This approach is able to identify cellular stress-induced and cell-cycle regulated SEPs and alt-proteins in cells. Graphical abstract Schematic overview of BONCAT-based chemoproteomic profiling of nascent, unannotated small and alternative open reading frame-encoded proteins (SEPs and alt-proteins).

Keywords: Alternative protein (alt-protein); Chemoproteomics; Micropeptide; Microprotein; Protein translation; Proteomics; Small open reading frame (smORF); Unnatural amino acid.