Protein kinase A phosphorylation characterized by tandem Fourier transform ion cyclotron resonance mass spectrometry

Michael J. Chalmers, Kristina Håkansson, Robert Johnson, Richard Smith, Jianwei Shen, Mark R. Emmett, Alan G. Marshall

Research output: Contribution to journalArticle

73 Scopus citations

Abstract

A microelectrospray ionization tandem Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MSn) approach for structural characterization of protein phosphorylation is described. Identification of proteolytic peptides is based solely upon mass measurement by high field (9.4 Tesla) FT-ICR MS. The location of the modification within any phosphopeptide is then established by FT-ICR MS2 and MS3 experiments. Structural information is maximized by use of electron capture dissociation (ECD) and/or infrared multiphoton dissociation (IRMPD). The analytical utility of the method is demonstrated by characterization of protein kinase A (PKA) phosphorylation. In a single FT-ICR MS experiment, 30 PKA tryptic peptides (including three phosphopeptides) were mass measured by internal calibration to within an absolute mean error of |10.7 ppm|. The location of each of the three sites of phosphorylation was then determined by MS2 and MS 3 experiments, in which ECD and IRMPD provide complementary peptide sequence information. In two out of three cases, electron irradiation of a phosphopeptide [M + nH]n+ ion produced an abundant charge-reduced [M + nH](n-1)+• ion, but few sequence-specific c and z fragment ions. Subsequent IRMPD (MS3) of the charge-reduced radical ion resulted in the detection of a large number of ECD-type ion products (c and z ions), but no b or y type ions. The utility of activated ion ECD for the characterization of tryptic phosphopeptides was then demonstrated.

Original languageEnglish (US)
Pages (from-to)970-981
Number of pages12
JournalProteomics
Volume4
Issue number4
DOIs
StatePublished - Apr 1 2004
Externally publishedYes

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Keywords

  • Electron capture dissociation
  • Electrospray ionization
  • Fourier transform ion cyclotron resonance mass spectrometry
  • Infrared multiphoton dissociation
  • Phosphorylation
  • Protein kinase A

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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