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

Hui Min Zhang, Shaun M. McLoughlin, Stephen D. Frausto, Hengli Tang, Mark Emmett, Alan G. Marshall

Research output: Contribution to journalArticle

31 Citations (Scopus)

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 oftris(2-carboxyethyl) phosphinehydrochloride(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 solventexposed segments for mass-analyzed solution-phase H/D exchange of proteins containing disulfide bonds.

Original languageEnglish (US)
Pages (from-to)1450-1454
Number of pages5
JournalAnalytical Chemistry
Volume82
Issue number4
DOIs
StatePublished - Feb 15 2010
Externally publishedYes

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Deuterium
Disulfides
Mass spectrometry
Hydrogen
Proteolysis
Ion exchange
Proteins
Peptides
Aspergillus
Pepsin A
Urea
Peptide Hydrolases

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Simultaneous reduction and digestion of proteins with disulfide bonds for hydrogen/deuterium exchange monitored by mass spectrometry. / Zhang, Hui Min; McLoughlin, Shaun M.; Frausto, Stephen D.; Tang, Hengli; Emmett, Mark; Marshall, Alan G.

In: Analytical Chemistry, Vol. 82, No. 4, 15.02.2010, p. 1450-1454.

Research output: Contribution to journalArticle

Zhang, Hui Min ; McLoughlin, Shaun M. ; Frausto, Stephen D. ; Tang, Hengli ; Emmett, Mark ; Marshall, Alan G. / Simultaneous reduction and digestion of proteins with disulfide bonds for hydrogen/deuterium exchange monitored by mass spectrometry. In: Analytical Chemistry. 2010 ; Vol. 82, No. 4. pp. 1450-1454.
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AU - Emmett, Mark

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