Removal of NHS-labelling by-products in proteomic samples

N-Hydroxysuccinimide (NHS) chemistry is used extensively across proteomics sample preparation. One of the most popular applications is its use in isobaric reagent based quantitation; specifically both the iTRAQ and TMT approaches. The labels are introduced onto primary Serines, tyrosines, and threonines can be labelled during TMT labelling reactions in addition to the desired amides. These peptides are typically classed as over-labelled and are disregarded for quantification. Their presence also unnecessarily increases sample complexity, which reduces identification rates. In this study, we developed a robust method to remove over-labelled peptides through systematic screening of nucleophilic aminolysis reagents and reaction conditions. The new method 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 superior identification rates and quantitation precision.