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Updated: Feb 3, 2026

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity
Published on: August 4, 2009
High-Resolution HDX-MS of Cytochrome c Using Pepsin/Fungal Protease Type XIII Mixed Bed Column
Yoshitomo Hamuro1,2, Terry Zhang3
1SGS Life North America, 606 Brandywine Parkway, West Chester, PA, 19380, USA. yhamuro@its.jnj.com.
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 Abstract ᅟ.
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