Optimized NHS-amine labeling for multiplexed phosphoproteome study.

In this project, we report the detrimental effect of hydroxylamine, a quenching reagent for NHS-amine reaction, on phosphopeptides. The degree of phosphopeptide enrichment was greatly improved by a desalting method compared with a vacuum-drying method right after quenching reaction. We demonstrate that vacuum-drying in presence of hydroxylamine promotes β-elimination of phosphate groups from phosphoserine and phosphothreonine.