TMT Labeling under Acidic pH Overcomes Detrimental Overlabeling and Improves Peptide Identification Rates

Tandem mass tags (TMT) are one of the most widely usedtechniquesin proteomics quantification due to their ability to accurately andprecisely analyze up to 18 samples in a multiplexed manner. Moreover,TMT tags are introduced chemically by covalent coupling of the primaryamines of digested proteins, making them universally applicable forany kind of sample. However, in addition to amine groups, the hydroxylgroups of serine, threonine, and tyrosine residues can also be labeledto some extent during TMT labeling, which compromises the analyticalsensitivity and results in lower peptide identification rates comparedto label-free methods. In this work, we investigated in-depth thechemical nature of TMT overlabeling and revealed that peptides simultaneouslycontaining histidine and hydroxyl-containing residues were prone tooverlabeling due to an intramolecular catalysis mediated by the histidylimidazolyl group. Based on the understanding of the chemical mechanism,we developed a novel TMT labeling method under acidic pH that completelyovercomes overlabeling. Compared to the standard labeling method providedby the TMT vendor, our method achieved comparable labeling efficiencyon target groups but greatly reduced overlabeled peptides, resultingin the identification of 33.9% more unique peptides and 20.9% moreproteins in proteomic analysis.