biostudies-literatureSuttapitugsakul SDivision of ChemistryNational Institute of General Medical SciencesNIGMS NIH HHS2574-2582https://www.ebi.ac.uk/biostudies/studies/S-EPMC5698164biostudies-literatureUnknown13(12)Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, incomplete reactions and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with commonly used reagents, i.e., dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, or tris(3-hydroxypropyl)phosphine, and alkylation with iodoacetamide, acrylamide, N-ethylmaleimide, or 4-vinylpyridine. By using digested peptides from a yeast whole-cell lysate, the number of proteins and peptides identified were very similar using four different reducing reagents. The results from four alkylating reagMolecular bioSystemsEvaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics.PMC5698164R01GM118803R01 GM118803CAREER Award, CHE-1454501Suttapitugsakul SXiao HWu RSmeekens JfalseEvaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics.Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, incomplete reactions and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with commonly used reagents, i.e., dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, or tris(3-hydroxypropyl)phosphine, and alkylation with iodoacetamide, acrylamide, N-ethylmaleimide, or 4-vinylpyridine. By using digested peptides from a yeast whole-cell lysate, the number of proteins and peptides identified were very similar using four different reducing reagents. The results from four alkylating reag2017-01-01T00:00:00Z2017 Nov2025-04-05T10:32:21.304Z2019-03-27T00:08:02ZS-EPMC56981642901937010.1039/c7mb00393e