An Efficient, Amine-Specific, and Cost-Effective Method for TMT 6/11-plex Labeling Improves the Proteome Coverage, Quantitative Accuracy and Precision

Tandem mass tags (TMT) are widely
used in proteomics
to simultaneously
quantify multiple samples in a single experiment. The tags can be
easily added to the primary amines of peptides/proteins through chemical
reactions. In addition to amines, TMT reagents also partially react
with the hydroxyl groups of serine, threonine, and tyrosine residues
under alkaline conditions, which significantly compromises the analytical
sensitivity and precision. Under alkaline conditions, reducing the
TMT molar excess can partially mitigate overlabeling of histidine-free
peptides, but has a limited effect on peptides containing histidine
and hydroxyl groups. Here, we present a method under acidic conditions
to suppress overlabeling while efficiently labeling amines, using
only one-fifth of the TMT amount recommended by the manufacturer.
In a deep-scale analysis of a yeast/human two-proteome sample, we
systematically evaluated our method against the manufacturer’s
method and a previously reported TMT-reduced method. Our method reduced
overlabeled peptides by 9-fold and 6-fold, respectively, resulting
in the substantial enhancement in peptide/protein identification rates.
More importantly, the quantitative accuracy and precision were improved
as overlabeling was reduced, endowing our method with greater statistical
power to detect 42% and 12% more statistically significant yeast proteins
compared to the standard and TMT-reduced methods, respectively. Mass
spectrometric data have been deposited in the ProteomeXchange Consortium
via the iProX partner repository with the data set identifier PXD047052.