Development of an electrochemical oxidation method for probing higher order protein structure with mass spectrometry

Carlee McClintock, Vilmos Kertesz, Robert L. Hettich

Research output: Contribution to journalArticlepeer-review

51 Scopus citations

Abstract

We report here the novel use of electrochemistry to generate covalent oxidative labels on intact proteins in both non-native and physiologically relevant solutions as a surface mapping probe of higher order protein structure. Two different working electrode types were tested across a range of experimental parameters including voltage, flow rate, and solution electrolyte composition to affect the extent of oxidation on intact proteins, as measured both on-line and off-line with mass spectrometry. Oxidized proteins were collected off-line for proteolytic digestion followed by LC-MS/MS analysis. Peptide MS/MS data were searched with the InsPecT scoring algorithm for 46 oxidative mass shifts previously reported in the literature. Preliminary data showed reasonable agreement between amino acid solvent accessibility and the resulting oxidation status of these residues in aqueous buffer, while more buried residues were found to be oxidized in non-native solution. Our results indicate that electrochemical oxidation using a boron-doped diamond electrode has the potential to become a useful and easily accessible tool for conducting oxidative surface mapping experiments.

Original languageEnglish
Pages (from-to)3304-3317
Number of pages14
JournalAnalytical Chemistry
Volume80
Issue number9
DOIs
StatePublished - May 1 2008

Funding

FundersFunder number
National Institute of General Medical SciencesR01GM070754

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