Addressing NHS Chemistry: Efficient Quenching of Excess TMT Reagent and Reversing TMT Overlabeling in Proteomic Samples by Methylamine

N-hydroxysuccinimide (NHS) ester chemistry is used extensively across proteomics sample preparation. One of its increasingly prevalent applications is in isobaric reagent–based quantitation such as isobaric tags for relative and absolute quantitation and tandem mass tag approaches. In these methods, labeling on the primary amines of lysine residues and N termini of tryptic peptides via amide formation (N-derivatives) from corresponding NHS ester reagents is the intended reactive outcome. However, the role of NHS esters as activated carboxyls can also drive the formation of serine-, tyrosine-, and threonine-derived esters (O-derivatives). These O-derivative peptides are typically classed as overlabeled and are disregarded for quantitation, leading to loss of information and hence potential sensitivity. Their presence also unnecessarily increases sample complexity, which reduces the overall identification rates. One common approach for removing these unwanted labeling events has involved treatment with hydroxylamine. We show here that this approach is not efficient and can still leave substantial levels of unwanted overlabeled peptides. Through systematic study of nucleophilic aminolysis reagents and reaction conditions, we have now developed a robust method to efficiently remove overlabeled peptides. The new method reduces the proportion of overlabeled peptides in the sample to less than 1% without affecting the labeling rate or introducing other modifications, leading to superior identification rates and quantitation precision.