FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins

Thomas G. Watkinson, Antonio N. Calabrese, James R. Ault, Sheena E. Radford, Alison E. Ashcroft

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in solution, and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochemical oxidation of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-β-maltoside (DDM) detergent micelles. Using quantitative mass spectrometry analyses, we show that fast photochemical oxidation reveals differences in the extent of solvent accessibility of residues between the A8-35 and DDM solubilized states, providing a rationale for the increased stability of membrane proteins solubilized with amphipol compared with detergent micelles, as a result of additional intermolecular contacts. [Figure not available: see fulltext.]

Original languageEnglish
Pages (from-to)50-55
Number of pages6
JournalJournal of the American Society for Mass Spectrometry
Volume28
Issue number1
Early online date24 Jun 2016
DOIs
Publication statusPublished - 1 Jan 2017

Keywords

  • Amphipols
  • Detergents
  • Fast photochemical oxidation
  • Membrane proteins
  • Structural proteomics

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

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