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

Title
Neutralizing the Detrimental Effect of an N-Hydroxysuccinimide Quenching Reagent on Phosphopeptide in Quantitative Proteomics
Authors
이철주플라산트주신영권유미이진원
Issue Date
2018-03
Publisher
Analytical chemistry
Citation
VOL 90, NO 5-3023
Abstract
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 beta-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.
URI
http://pubs.kist.re.kr/handle/201004/68206
ISSN
0003-2700
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KIST Publication > Article
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