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

Tandem
mass tags (TMT) are one of the most widely used
techniques
in proteomics quantification due to their ability to accurately and
precisely analyze up to 18 samples in a multiplexed manner. Moreover,
TMT tags are introduced chemically by covalent coupling of the primary
amines of digested proteins, making them universally applicable for
any kind of sample. However, in addition to amine groups, the hydroxyl
groups of serine, threonine, and tyrosine residues can also be labeled
to some extent during TMT labeling, which compromises the analytical
sensitivity and results in lower peptide identification rates compared
to label-free methods. In this work, we investigated in-depth the
chemical nature of TMT overlabeling and revealed that peptides simultaneously
containing histidine and hydroxyl-containing residues were prone to
overlabeling due to an intramolecular catalysis mediated by the histidyl
imidazolyl group. Based on the understanding of the chemical mechanism,
we developed a novel TMT labeling method under acidic pH that completely
overcomes overlabeling. Compared to the standard labeling method provided
by the TMT vendor, our method achieved comparable labeling efficiency
on target groups but greatly reduced overlabeled peptides, resulting
in the identification of 33.9% more unique peptides and 20.9% more
proteins in proteomic analysis.