Simultaneous reduction and digestion of proteins with disulfide bonds for hydrogen/deuterium exchange monitored by mass spectrometry.

Analytical Chemistry
Hui-Min ZhangAlan G Marshall

Abstract

Proteolyzed peptides provide the basis for mass-analyzed hydrogen/deuterium exchange (HDX) for mapping solvent access to various segments of solution-phase proteins. Aspergillus saitoi protease type XIII and porcine pepsin can generate peptides of overlapping sequences and high sequence coverage. However, if disulfide bonds are present, proteolysis can be severely limited, particularly in the vicinity of the disulfide linkage(s). Disulfide bonds cannot be reduced before or during the H/D exchange reaction without affecting the protein higher-order structure. Here, we demonstrate simultaneous quench/digestion/reduction following H/D exchange, for subsequent mass analysis. Proteolysis is conducted in the presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) and urea, and all other steps of the H/D exchange and analysis are maintained. This method yields dramatically increased sequence coverage and localization of solvent-exposed segments for mass-analyzed solution-phase H/D exchange of proteins containing disulfide bonds.

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Citations

Apr 6, 2012·Journal of the American Society for Mass Spectrometry·Roxana E Iacob, John R Engen
Feb 23, 2012·Nature Communications·Xiaoling XuXiang-Lei Yang
May 4, 2011·Proceedings of the National Academy of Sciences of the United States of America·Pengfei FangMin Guo
May 3, 2012·The Journal of Biological Chemistry·Peter S LeePaul Schimmel
Nov 12, 2013·Nature Chemical Biology·Dae Gyu KimSunghoon Kim
Apr 15, 2011·Journal of Pharmaceutical Sciences·Damian HoudeJohn R Engen
Oct 16, 2012·Biochimica Et Biophysica Acta·Joomi AhnJohn R Engen
Jun 7, 2015·Annual Review of Analytical Chemistry·John R Engen, Thomas E Wales
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Sep 27, 2018·The Journal of Biological Chemistry·Yiming XiaoDing Xu
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Dec 22, 2010·Chemical Society Reviews·Lars KonermannYu-Hong Liu
Jul 28, 2020·Biomedicines·Dominic NarangDerek J Wilson
Mar 22, 2019·Scientific Reports·Cristina PuchadesAdrian Apetri
Nov 26, 2013·Biochemistry·David BalchinHeini W Dirr
Dec 7, 2013·Analytical Chemistry·Lars KonermannModupeola A Sowole

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