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High-Resolution HDX-MS of Cytochrome c Using Pepsin/Fungal Protease Type XIII Mixed Bed Column

Authors
  • Hamuro, Yoshitomo1, 2
  • Zhang, Terry3
  • 1 SGS Life North America, 606 Brandywine Parkway, West Chester, PA, 19380, USA , West Chester (United States)
  • 2 Janssen Pharmaceutical, 1400 McKean Road, Spring House, PA, 19477, USA , Spring House (United States)
  • 3 Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA, 95134, USA , San Jose (United States)
Type
Published Article
Journal
Journal of the American Society for Mass Spectrometry
Publisher
Springer-Verlag
Publication Date
Oct 29, 2018
Volume
30
Issue
2
Pages
227–234
Identifiers
DOI: 10.1007/s13361-018-2087-7
Source
Springer Nature
Keywords
License
Yellow

Abstract

A pepsin/FPXIII (protease from Aspergillus saitoi, type XIII) mixed bed column significantly improved the resolution of bottom-up hydrogen/deuterium exchange mass spectrometry (HDX-MS) data compared with a pepsin-only column. The HDX-MS method using the mixed bed column determined 65 amide hydrogen exchange rates out of one hundred cytochrome c backbone amide hydrogens. Different cleavage specificities of the two enzymes generated 138 unique high-quality peptic fragments, which allows fine sub-localization of deuterium. The exchange rates determined in this method are consistent within the current study as well as with the previous HDX-NMR study. High-resolution HDX-MS data can determine the exchange rate of each residue not the deuterium buildup curve of a peptic fragment. The exchange rates provide more precise and quantitative measurements of protein dynamics in a more reproducible manner. Graphical Abstractᅟ

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