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Anal Chem. 2018 Mar 6;90(5):3079-3082. doi: 10.1021/acs.analchem.7b03655. Epub 2018 Feb 1.

Top-Down Hydrogen-Deuterium Exchange Analysis of Protein Structures Using Ultraviolet Photodissociation.

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University of Victoria -Genome British Columbia Proteomics Centre , No. 3101-4464 Markham Street, Vancouver Island Technology Park , Victoria , British Columbia V8Z 7X8 , Canada.
Thermo Fisher Scientific , 355 River Oaks Parkway , San Jose , California 95134 , United States.
Department of Biochemistry and Microbiology , University of Victoria , Petch Building, Room 270d, 3800 Finnerty Road , Victoria , British Columbia V8P 5C2 , Canada.
Gerald Bronfman Department of Oncology, Jewish General Hospital , McGill University , 3755 Côte Ste-Catherine Road , Montreal , Quebec H3T 1E2 , Canada.
Proteomics Centre, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital , McGill University , 3755 Côte Ste-Catherine Road , Montreal , Quebec H3T 1E2 , Canada.


Top-down hydrogen-deuterium exchange (HDX) analysis using electron capture or transfer dissociation Fourier transform mass spectrometry (FTMS) is a powerful method for the analysis of secondary structure of proteins in solution. The resolution of the method is a function of the degree of fragmentation of backbone bonds in the proteins. While fragmentation is usually extensive near the N- and C-termini, electron capture (ECD) or electron transfer dissociation (ETD) fragmentation methods sometimes lack good coverage of certain regions of the protein, most often in the middle of the sequence. Ultraviolet photodissociation (UVPD) is a recently developed fast-fragmentation technique, which provides extensive backbone fragmentation that can be complementary in sequence coverage to the aforementioned electron-based fragmentation techniques. Here, we explore the application of electrospray ionization (ESI)-UVPD FTMS on an Orbitrap Fusion Lumos Tribrid mass spectrometer to top-down HDX analysis of proteins. We have incorporated UVPD-specific fragment-ion types and fragment-ion mixtures into our isotopic envelope fitting software (HDX Match) for the top-down HDX analysis. We have shown that UVPD data is complementary to ETD, thus improving the overall resolution when used as a combined approach.

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