Liquid chromatography - Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation

Michael J. Chalmers, John P. Quinn, Greg T. Blakney, Mark Emmett, Harold Mischak, Simon J. Gaskell, Alan G. Marshall

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

A vented column, capillary liquid chromatography (LC) microelectrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR (9.4 T)) mass spectrometry (MS) approach to phosphopeptide identification is described. A dual-ESI source capable of rapid (∼200 ms) switching between two independently controlled ESI emitters was constructed. The dual-ESI source, combined with external ion accumulation in a linear octopole ion trap, allowed for internal calibration of every mass spectrum during LC. LC ESI FT-ICR positive-ion MS of protein kinase C (PKC) revealed four previously unidentified phosphorylated peptides (one within PKCα, one within PKCδ, and two within PKCζ). Internal calibration improved the mass accuracy for LC MS spectra from an absolute mean (47 peptide ions) of 11.5 ppm to 1.5 ppm. Five additional (out of eight known) activating sites of PKC phosphorylation, not detected in positive-ion experiments, were observed by subsequent negative-ion direct infusion nanoelectrospray. Extension of the method to enable infrared multiphoton dissociation of all ions in the ICR cell prior to every other mass measurement revealed the diagnostic neutral loss of H3PO4 from phosphorylated peptide ions. The combination of accurate-mass MS and MS/MS offers a powerful new tool for identifying the presence and site(s) of phosphorylation in peptides, without the need for additional wet chemical derivatization.

Original languageEnglish (US)
Pages (from-to)373-382
Number of pages10
JournalJournal of Proteome Research
Volume2
Issue number4
DOIs
StatePublished - Jul 2003
Externally publishedYes

Fingerprint

Cyclotrons
Cyclotron resonance
Phosphorylation
Liquid chromatography
Fourier Analysis
Liquid Chromatography
Protein Kinase C
Mass spectrometry
Fourier transforms
Ions
Peptides
Tandem Mass Spectrometry
Mass Spectrometry
Positive ions
Calibration
Phosphopeptides
Ionization
Negative ions
Infrared radiation

Keywords

  • Fourier transform
  • FT-ICR
  • FTMS
  • ICR
  • Infrared multiphoton dissociation
  • Ion cyclotron resonance
  • LC/MS
  • LC/MS/MS
  • MS/MS
  • Phosphorylation
  • PKC
  • Tandem mass spectrometry

ASJC Scopus subject areas

  • Genetics
  • Biotechnology
  • Biochemistry

Cite this

Liquid chromatography - Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation. / Chalmers, Michael J.; Quinn, John P.; Blakney, Greg T.; Emmett, Mark; Mischak, Harold; Gaskell, Simon J.; Marshall, Alan G.

In: Journal of Proteome Research, Vol. 2, No. 4, 07.2003, p. 373-382.

Research output: Contribution to journalArticle

Chalmers, Michael J. ; Quinn, John P. ; Blakney, Greg T. ; Emmett, Mark ; Mischak, Harold ; Gaskell, Simon J. ; Marshall, Alan G. / Liquid chromatography - Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation. In: Journal of Proteome Research. 2003 ; Vol. 2, No. 4. pp. 373-382.
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