Systematic Evaluation of Protein Reduction and Alkylation reveals Massive Unspecific Side Effects by Iodine-containing Reagents

In this study, we systematically evaluated reduction and alkylation of proteins using three reducing agents (DTT, TCEP, and BME) in combination with four alkylation agents (IAA, IAC, AA, and CAA). We tested these conditions using strong anion exchange (SAX) fractionated in-solution digests and in-gel digested fractions of samples enriched for cytosolic proteins of HeLa cells. We analyzed the data using Proteome Discoverer 2 in combination with the MASCOT search engine with different combinations of variable and fixed modifications as well as error tolerant and mass tolerant searches. Furthermore, we compared IAA and AA alkylated samples by dimethyl labeling and performed multi stage activation triggered by the neutral loss of alkylated methionine side chains. This led to the identification of prominent offside alkylation due to iodine containing reagents at tyrosine, serine, threonine, histidine, lysine, methionine, aspartic and glutamic acid as well as the peptide N-terminus with varying abundances based on the reduction and alkylation reagents used.