NDLI: Supercharging by m-NBA Improves ETD-Based Quantification of Hydroxyl Radical Protein Footprinting

Content Provider	Springer Nature Link
Author	Li, Xiaoyan Li, Zixuan Xie, Boer Sharp, Joshua S.
Copyright Year	2015
Abstract	Hydroxyl radical protein footprinting (HRPF) is an MS-based technique for analyzing protein structure based on measuring the oxidation of amino acid side chains by hydroxyl radicals diffusing in solution. Spatial resolution of HRPF is limited by the smallest portion of the protein for which oxidation amounts can be accurately quantitated. Previous work has shown electron transfer dissociation (ETD) to be the most reliable method for quantifying the amount of oxidation of each amino acid side chain in a mixture of peptide oxidation isomers, but efficient ETD requires high peptide charge states, which limits its applicability for HRPF. Supercharging reagents have been used to enhance peptide charge state for ETD analysis, but previous work has shown supercharging reagents to enhance charge state differently for different peptides sequences; it is currently unknown if different oxidation isomers will experience different charge enhancement effects. Here, we report the effect of m-nitrobenzyl alcohol (m-NBA) on the ETD-based quantification of peptide oxidation. The addition of m-NBA to both a defined mixture of synthetic isomeric oxidized peptides and Robo-1 protein subjected to HRPF increased the abundance of higher charge state ions, improving our ability to perform efficient ETD of the mixture. No differences in the reported quantitation by ETD were noted in the presence or absence of m-NBA, indicating that all oxidation isomers were charge-enhanced to a similar extent. These results indicate the utility of m-NBA for residue-level quantification of peptide oxidation in HRPF and other applications. Graphical Abstract ᅟ
Starting Page	1424
Ending Page	1427
Page Count	4
File Format	PDF
ISSN	10440305
Journal	Journal of The American Society for Mass Spectrometry
Volume Number	26
Issue Number	8
e-ISSN	18791123
Language	English
Publisher	Springer US
Publisher Date	2015-04-28
Publisher Institution	The American Society for Mass Spectrometry
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Hydroxyl radical protein footprinting Covalent labeling Supercharging Mass spectrometry Protein structure Protein oxidation Analytical Chemistry Biotechnology Organic Chemistry Proteomics Bioinformatics
Content Type	Text
Resource Type	Article
Subject	Spectroscopy Structural Biology

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