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

ABSTRACT: The advent of TMTpro reagents expands the sample multiplexing capacity and enables quantification of up to 18 samples in a multiplexed manner. Similar to classic TMT reagents, TMTpro reagents contain a tertiary amine group in their mole-cules, which markedly enhances their reactivity toward hydroxyl groups and thus results in overlabeling of serine, threo-nine and tyrosine residues. Overlabeling significantly compromises the proteome analysis in terms of sensitivity and precision. Here, we report a novel TMTpro labeling method that overcomes the detrimental overlabeling while delivering efficient labeling for amines. Additionally, our method is cost-effective as it requires only half the amount of TMTpro reagents recommended by the reagent manufacturer. We compared our method with the standard method (provided by the TMTpro manufacturer) in a deep-scale analysis of a yeast/human two-proteome model sample. Even at the depth of over 10000 proteins, our method detected 36.2% more unique peptides and 11.5% more protein groups than the standard method. Moreover, our method considerably improves the quantitative precision due to the reduced variability in label-ing and increased protein sequence coverage. This substantially enhanced the statistical power of our method for detect-ing differentially abundant proteins, providing an average of 16% more yeast proteins reaching statistical significance.

ORGANISM(S): Homo Sapiens Saccharomyces Cerevisiae

SUBMITTER: Rijing Liao  

PROVIDER: PXD043778 | iProX | Tue Dec 19 00:00:00 GMT 2023

REPOSITORIES: iProX