Overcoming the detrimental O-acylation in TMTpro labeling improves the proteome depth and quantification precision

With the advent of proline-based reporter isobaric Tandem Mass Tag (TMTpro) reagents, the sample multiplexing capacity of tandem mass tags (TMTs) has been expanded, and up to 18 samples can be quantified in a multiplexed manner. Like classic TMT reagents, TMTpro reagents contain a tertiary amine group, which markedly enhances their reactivity toward hydroxyl groups and results in O-acylation of serine, threonine and tyrosine residues. This overlabeling significantly compromises proteome analysis in terms of depth and precision. In particular, the reactivity of hydroxyl-containing residues can be dramatically enhanced when coexisting with a histidine in the same peptides, leading to a severe systematic bias against the analysis of these peptides. Although some protocols using a reduced molar excess of TMT under alkaline conditions can alleviate overlabeling of histidine-free peptides to some extent, they have a limited effect on histidyl- and hydroxyl-containing peptides.

Here, we report a novel TMTpro labeling method that overcomes detrimental overlabeling while providing high labeling efficiency of amines. Additionally, our method is cost-effective, as it requires only half the amount of TMTpro reagents recommended by the reagent manufacturer. In a deep-scale analysis of a yeast/human two-proteome model sample, we compared our method with a typical alkaline labeling method using a reduced molar excess of TMTpro. Even at a depth of over 10,000 proteins, our method detected 23.7% more unique peptides and 8.7% more protein groups compared to the alkaline labeling method. Moreover, our method significantly improved the quantitative precision due to the reduced variability in labeling and increased protein sequence coverage. This substantially enhanced the statistical power of our method for detecting differentially abundant proteins, providing an average of 13% more yeast proteins that reached statistical significance.

We presented a novel TMTpro labeling method that overcomes the detrimental O-acylation and thus significantly improves the depth and quantitative precision for proteome analysis.