Influence of overalkylation in enzymatic digestion on the qualitative and quantitative analysis of proteins

Reduction and alkylation with iodoacetamide (IAA) of the disulfide bridges in proteins are important procedures used in protein digestion. But the alkylation with IAA takes place not only on cysteine residues but also on other amino acid residues. Here we conducted a systematic study of all alkylated peptides under the usual protein digestion conditions. It showed that the potentials for alkylation reaction of different amino acid residues were cysteine > N-terminal amino acid > aspartic acid > glutamic acid > histidine > asparagine > lysine > tyrosine. Furthermore, we found that the alkylation reaction happened either exclusively or cooperatively among different amino acid residues in the same peptide. Based on the qualitative results on overalkylation at several peptides, the targeted multiple reaction monitoring (MRM) technique was used to evaluate the effect of overalkylation on quantitative protein analysis. The results showed that overalkylation has large effect on the qualitative and quantitative analysis of proteins and should be avoided in enzymatic digestion.