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

ABSTRACT: Tandem mass tags (TMT) are among the most popular proteomics quantification techniques due to its ability to simulta-neously quantify multiple samples in a single experiment. The tags can be easily added onto the primary amine groups of peptides and proteins through chemical reactions. Although TMT reagents are widely considered amine-specific rea-gents, they do react to some extent with the hydroxyl groups of serine, threonine, and tyrosine residues under alkaline conditions, which significantly compromises the sensitivity and precision of proteomic analysis. Reducing the molar ex-cess of TMT in labeling reactions would reduce overlabeling for peptides without histidine, but has limited effect on his-tidyl- and hydroxyl-containing peptides. Here, we present a TMT labeling method that overcomes the problem of overla-beling while enabling efficient labeling of amines using only one-fifth of the TMT amount recommended by the manufac-turer. In a deep-scale proteome experiment using a yeast/human two-proteome sample, we systematically compared our method with the standard and TMT-reduced methods, demonstrating that our method significantly increased the num-bers of identified unique peptides and protein groups. More importantly, the quantitative accuracy and precision were also improved with reducing overlabeling, allowing the greater statistical power of our method to detect 42% and 12% more statistically significant yeast proteins compared to the standard and TMT-reduced methods, respectively.

ORGANISM(S): Homo Sapiens

SUBMITTER: Rijing Liao  

PROVIDER: PXD047052 | iProX | Fri Nov 17 00:00:00 GMT 2023

REPOSITORIES: iProX