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 Emmett, Alan G. Marshall

Research output: Contribution to journalArticle

70 Citations (Scopus)

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 2004
Externally publishedYes

Fingerprint

Cyclotrons
Cyclotron resonance
Phosphorylation
Fourier Analysis
Cyclic AMP-Dependent Protein Kinases
Mass spectrometry
Phosphopeptides
Mass Spectrometry
Fourier transforms
Ions
Electrons
Infrared radiation
Electron irradiation
Electrospray Ionization Mass Spectrometry
Experiments
Calibration
Ionization

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Protein kinase A phosphorylation characterized by tandem Fourier transform ion cyclotron resonance mass spectrometry. / Chalmers, Michael J.; Håkansson, Kristina; Johnson, Robert; Smith, Richard; Shen, Jianwei; Emmett, Mark; Marshall, Alan G.

In: Proteomics, Vol. 4, No. 4, 04.2004, p. 970-981.

Research output: Contribution to journalArticle

Chalmers, Michael J. ; Håkansson, Kristina ; Johnson, Robert ; Smith, Richard ; Shen, Jianwei ; Emmett, Mark ; Marshall, Alan G. / Protein kinase A phosphorylation characterized by tandem Fourier transform ion cyclotron resonance mass spectrometry. In: Proteomics. 2004 ; Vol. 4, No. 4. pp. 970-981.
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N2 - 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.

AB - 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.

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