Liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation.
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 (approximately 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 PKC(alpha), one within PKC(delta), and two within PKC(zeta)). 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.
Chalmers
Michael J
MJ
National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-3706, USA.
Quinn
John P
JP
Blakney
Greg T
GT
Emmett
Mark R
MR
Mischak
Harold
H
Gaskell
Simon J
SJ
Marshall
Alan G
AG
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid; D017357:Cyclotrons; D030562:Databases, Protein; D001330:Electronic Data Processing; D005583:Fourier Analysis; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D018411:Spodoptera; D000099221:Protein Kinase C zeta
D010446:Peptide Fragments; D010748:Phosphopeptides; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D000099221:Protein Kinase C zeta; C497259:Prkcd protein, mouse; C496630:Prkca protein, mouse