Neutralizing the Detrimental Effect of an N‑Hydroxysuccinimide Quenching Reagent on Phosphopeptide in Quantitative Proteomics

One of the most common chemistries used to label primary amines utilizes N-hydroxysuccinimide (NHS), which is also structurally incorporated in various quantitative proteomic reagents such as isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tags (TMT). In this paper we report detrimental effect of hydroxylamine, a widely used quenching reagent for excess NHS, on phosphopeptides. We found an impairment in the degree of phosphopeptide identification when hydroxylamine-quenched TMT-labeled samples were vacuum-dried and desalted compared to the nondried (just diluted) and desalted ones prior to phosphoenrichment. We have also demonstrated that vacuum-drying in the presence of hydroxylamine promotes β-elimination of phosphate groups from phosphoserine and phosphothreonine while having a minimalistic effect on phosphotyrosine. Additionally, we herein report that this negative impact of hydroxylamine could be minimized by direct desalting after appropriate dilution of quenched samples. We also found a 1.6-fold increase in the number of phosphopeptide identifications after employing our optimized method. The above method was also successfully applied to human tumor tissues to quantify over 15000 phosphopeptides from 3 mg TMT 6-plex labeled-peptides.