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

  • Yoshitomo HamuroEmail author
  • Terry Zhang
Research Article


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 Abstract


Cytochrome c Electrospray ionization Exchange rate Fungus protease XIII Hydrogen/deuterium exchange Mass spectrometry Pepsin 



Electron capture dissociation


Electron transfer dissociation


Protease from Aspergillus saitoi, type XIII


Guanidine hydrochloride


Hydrogen/deuterium exchange


Liquid chromatography


Mass spectrometry


Tandem mass spectrometry


Trifluoroacetic acid



The authors thank Stephen J. Coales for his technical support.

Supplementary material

13361_2018_2087_MOESM1_ESM.doc (342 kb)
ESM 1 (DOC 342 kb)


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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.SGS Life North AmericaWest ChesterUSA
  2. 2.Janssen PharmaceuticalSpring HouseUSA
  3. 3.Thermo Fisher ScientificSan JoseUSA

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