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Ongoing efforts for complete annotation and functional characterization of smORF\u2010encoded proteins have yielded novel regulators and therapeutic targets. However, because they are excluded from protein databases, initiate at non\u2010AUG 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\u2010based detection of translated smORFs in cultured human cells from protein extraction, digestion, and LC\u2010MS\/MS, to database preparation and data analysis. Following proteomic detection, translation from a unique smORF may be validated via siRNA\u2010based 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. \u00a9 2019 by John Wiley &amp; Sons, Inc.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Protein extraction, size selection, and trypsin digestion<\/jats:p><jats:p><jats:bold>Alternate Protocol 1<\/jats:bold>: In\u2010solution C8 column size selection<\/jats:p><jats:p><jats:bold>Support Protocol 1<\/jats:bold>: Chloroform\/methanol precipitation<\/jats:p><jats:p><jats:bold>Support Protocol 2<\/jats:bold>: Reduction, alkylation, and in\u2010solution protease digestion<\/jats:p><jats:p><jats:bold>Support Protocol 3<\/jats:bold>: Peptide de\u2010salting<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Two\u2010dimensional LC\u2010MS\/MS with ERLIC fractionation<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Transcriptomic database construction<\/jats:p><jats:p><jats:bold>Alternate Protocol 2<\/jats:bold>: Transcriptomics database generation with gffread<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Non\u2010annotated peptide identification from LC\u2010MS\/MS data<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: Validation using isotopically labeled synthetic peptide standards and siRNA<\/jats:p><jats:p><jats:bold>Basic Protocol 6<\/jats:bold>: Transcript validation using transient overexpression<\/jats:p>","DOI":"10.1002\/cpch.77","type":"journal-article","created":{"date-parts":[[2019,11,21]],"date-time":"2019-11-21T14:35:16Z","timestamp":1574346916000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":47,"title":["Proteomic Detection and Validation of Translated Small Open Reading Frames"],"prefix":"10.1002","volume":"11","author":[{"given":"Alexandra","family":"Khitun","sequence":"first","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut"},{"name":"Chemical Biology Institute Yale University  West Haven Connecticut"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah A.","family":"Slavoff","sequence":"additional","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut"},{"name":"Chemical Biology Institute Yale University  West Haven Connecticut"},{"name":"Department of Molecular Biophysics and Biochemistry Yale University  New Haven Connecticut"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2019,11,21]]},"reference":[{"key":"e_1_2_15_2_1_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac070997p"},{"key":"e_1_2_15_2_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrg3520"},{"key":"e_1_2_15_2_3_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/7.6.1513"},{"key":"e_1_2_15_2_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(76)90527-3"},{"key":"e_1_2_15_2_5_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1215110"},{"key":"e_1_2_15_2_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3688"},{"key":"e_1_2_15_2_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature11184"},{"key":"e_1_2_15_2_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.2377"},{"key":"e_1_2_15_2_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm.2017.58"},{"key":"e_1_2_15_2_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jproteome.7b00419"},{"key":"e_1_2_15_2_11_1","first-page":"10.25.1","article-title":"Preparation of proteins and peptides for mass spectrometry analysis in a bottom\u2010up proteomics workflow","volume":"90","author":"Gundry R. 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