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

Xiongwen Cao; Yanran Chen; Alexandra  Khitun; Sarah  Slavoff

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

2023

Xiongwen Cao | Yanran Chen | Alexandra Khitun | Sarah Slavoff

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)

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