Characterization of green fluorescent proteins by 193 nm ultraviolet photodissociation mass spectrometry

Joe R. Cannon, Christien Kluwe, Andrew Ellington, Jennifer S. Brodbelt

Research output: Contribution to journalArticlepeer-review

Abstract

We investigate the utility of 193 nm ultraviolet photodissociation (UVPD) in comparison to CID, higher energy CID (HCD), and electron transfer dissociation (ETD) for top down fragmentation of highly homologous green fluorescent proteins (GFP) in the gas phase. Several GFP variants were constructed via mutation of surface residues to charged moieties, demonstrating different pIs and presenting a challenge for identification by mass spectrometry. Presented is a comparison of fragmentation techniques utilized for top down characterization of four variants with varying levels of surface charge. UVPD consistently resulted in identification of more fragment ions relative to other MS/MS methods, allowing higher confidence identification. In addition to the high number of fragment ions, the sites of fragmentation were more evenly spread throughout the protein backbone, which proved key for localizing the point mutations.

Original languageEnglish (US)
Pages (from-to)1165-1173
Number of pages9
JournalProteomics
Volume14
Issue number10
DOIs
StatePublished - May 2014
Externally publishedYes

Keywords

  • Green fluorescent protein
  • Orbitrap
  • Technology
  • Top-down
  • Ultraviolet photodissociation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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