A Ubiquitous but Overlooked Side Reaction in Dimethyl Labeling of Peptides

Reductive dimethylation using formaldehyde and NaBH₃CN to label peptides or proteins on their N-termini and lysine residues is one of the most widely used labeling methods in the quantitative proteomics field. In this study, we characterized a ubiquitous side reaction in dimethylation labeling, causing mass increments of 26 Da on the N-termini of peptides. It can occur extensively on most peptides, which significantly compromises data quality in terms of sensitivity, dynamic range, and peptide- and protein-identification rates. Nevertheless, this side reaction was so-far overlooked, largely because the current database search algorithms limited the detection of unknown modifications. In order to illustrate the chemical nature of this side reaction, 1D and 2D nuclear magnetic resonance (NMR) was performed to elucidate the exact structure of the modification formed through this side reaction, revealing that the side reaction produced an N-methyl-4-imidazolidinone moiety between the first two residues of the undesirably labeled peptides. On the basis of the mechanism proposed for the side reaction, we optimized the reaction conditions for dimethyl-labeling. Compared with the current typical labeling method, our approach can dramatically suppress the side reactions at both the standard protein and proteome levels. As a result, with our optimal labeling method, peptide- and protein-identification rates were significantly increased compared with those from the traditional labeling method.