An efficient and robust protocol for quenching TMT-labelling reactions for reversal of over-labelling at serine, threonine, and tyrosine residues

Tandem mass tags (TMT) are widely used for relative quantification of protein abundance. The labels are introduced onto primary amines of lysine residues and N-termini of tryptic peptides via amide formation upon reaction with their N-hydroxy succinimide (NHS) ester moiety. Although otherwise rare products, under the conditions required for complete labelling, seryl-, tyrosyl-, and threonyl- esters of the TMT label can form on up to 15% of peptides in addition to the expected amide derivatives. These peptides are typically classed as over-labelled and are disregarded for quantification. Their presence also unnecessarily increases sample complexity, which reduces identification rates. Hydroxylamine was shown to reverse the formation of tyrosyl esters and has since been universally used to quench TMT-labelling reactions. However, our data shows that hydroxylamine treatment has limited efficacy against seryl- or threonyl- esters. Through systematic screening of nucleophilic aminolysis reagents and reaction conditions, we developed a robust method to remove all three ester types from over-labelled peptides across a wide range of reaction scales. The new method reliably reduces the proportion of over-labelled peptides in the sample to less than 1% without affecting the labelling rate or introducing other modifications, leading to an increase in identification rates from single-shot analysis.