Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents
Torsten Mueller and Dominic Winter
Molecular & Cellular Proteomics, 16 (2017) 1173-1187
Despite the near-ubiquitous nature of reduction and alkylation in proteomics workflows, there has not been a systematic examination of how it impacts the proteins identified. In this paper the authors evaluated the reduction and alkylation of proteins using three reducing agents (DTT, TCEP, and BME) in combination with four alkylation agents (IAA, IAC, AA, and CAA). They tested these conditions using strong anion exchange fractionated in-solution digests and in-gel digested fractions of samples enriched for cytosolic proteins of HeLa cells.
Among the evaluated alkylation reagents, IAA and IAC resulted in the lowest efficiencies of peptide identification across all conditions. This indicates that the iodine-containing reagents lead to the highest numbers of non-specifically modified peptides.
Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, they identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. There were differences of more than 9-fold in the numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and non-iodine-containing alkylation reagents.
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