Proteomic Detection and Validation of Translated Small Open Reading Frames

Small open reading frames (smORFs) encode previously unannotated polypeptides or short proteins that regulate translation in cis (eukaryotes) and/or are independently functional (prokaryotes and eukaryotes). Ongoing efforts for complete annotation and functional characterization of smORF‐encoded proteins have yielded novel regulators and therapeutic targets. However, because they are excluded from protein databases, initiate at non‐AUG start codons, and produce few unique tryptic peptides, unannotated small proteins cannot be detected with standard proteomic methods. Here,, we outline a procedure for mass spectrometry‐based detection of translated smORFs in cultured human cells from protein extraction, digestion, and LC‐MS/MS, to database preparation and data analysis. Following proteomic detection, translation from a unique smORF may be validated via siRNA‐based silencing or overexpression and epitope tagging. This is necessary to unambiguously assign a peptide to a smORF within a specific transcript isoform or genomic locus. Provided that sufficient starting material is available, this workflow can be applied to any cell type/organism and adjusted to study specific (patho)physiological contexts including, but not limited to, development, stress, and disease. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1 : Protein extraction, size selection, and trypsin digestion Alternate Protocol 1 : In‐solution C8 column size selection Support Protocol 1 : Chloroform/methanol precipitation Support Protocol 2 : Reduction, alkylation, and in‐solution protease digestion Support Protocol 3 : Peptide de‐salting Basic Protocol 2 : Two‐dimensional LC‐MS/MS with ERLIC fractionation Basic Protocol 3 : Transcriptomic database construction Alternate Protocol 2 : Transcriptomics database generation with gffread Basic Protocol 4 : Non‐annotated peptide identification from LC‐MS/MS data Basic Protocol 5 : Validation using isotopically labeled synthetic peptide standards and siRNA Basic Protocol 6 : Transcript validation using transient overexpression