Simultaneous reduction and digestion of proteins with disulfide bonds for hydrogen/deuterium exchange monitored by mass spectrometry.
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.
Zhang
Hui-Min
HM
Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, USA.
McLoughlin
Shaun M
SM
Frausto
Stephen D
SD
Tang
Hengli
H
Emmett
Mark R
MR
Marshall
Alan G
AG
D000595:Amino Acid Sequence; D000818:Animals; D001230:Aspergillus / Q000201:enzymology; D041961:Deuterium Exchange Measurement; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D008024:Ligands; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010434:Pepsin A / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D019163:Reducing Agents / Q000494:pharmacology; D013552:Swine; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
D004220:Disulfides; D008024:Ligands; D010434:Pepsin A; D011506:Proteins; D019163:Reducing Agents; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase