‘Shotgun’ Proteome Analyses Do Not Require Alkylation of Cysteine

It is a common belief that reduction of disulfide bridges and alkylation of thiols in proteins are indispensable steps in proteomic sample preparation. Since this chemical procedure is often incomplete and prone to side reactions and can results in various artefacts we reexamined its importance. We found that the reduction and alkylation do not increase the depth of analysis and quality of proteomic quantification therefore these steps are unessential in shotgun-type investigations of proteomes. Moreover, we found that compared to a standard procedure using iodoacetamide for thiol-alkylation, sample preparation under conditions protecting thiols from oxidation improves quality of peptides and allows identifying of 10-20% more peptides and proteins. Excluding the thiol-alkylation from proteomic sample preparation, shorten the workflows, and decreases the probability of biases resulting from occurrence of artificially modified peptides.