Evaluation and minimization of over-alkylation in proteomic sample preparation

Reduction and alkylation are essential steps in shotgun proteomic sample preparation, but over-alkylation can occur on peptide N-terminus and amino acid residues other than cysteine, which adversely affects protein identification and quantification. To date, different sample preparation protocols are used in different laboratories, but a systematic comparison of the published protocols has not been done yet in the aspect of over-alkylation. Here, we comprehensively evaluated various protocols using different denaturants, different digestion buffers and different concentrations of reduction and alkylation reagents. The suggested protocols from the manufacturers of RapiGest and ProteaseMAX induce the highest degree of over-alkylation compared with others. Carbamidomethylation at lysine increases over 4 times while using triethylammonium bicarbonate buffer compared with that using others, and it should be minimized for improving the labeling efficiency of stable isotope labeling reagents that label lysine residues. The use of digestion buffers at pH 6 reduces over-alkylation, but greatly introduces some other artificial modifications. Excessive iodoacetamide was proved to be the major cause of over-alkylation during trypsin digestion. Therefore, over-alkylation can be minimized by using low concentration of dithiothreitol and iodoacetamide for reduction and alkylation or quenching the excessive iodoacetamide before digestion.