Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer

Brian L. Williamson, Jason Marchese, Nicholas Morrice

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

Complete phosphorylation mapping of protein kinases was successfully undertaken using an automated LC/MS/MS approach. This method uses the direct combination of triple quadrupole and ion trapping capabilities in a hybrid triple quadrupole linear ion trap to selectively identify and sequence phosphorylated peptides. In particular, the use of a precursor ion scan of m/z -79 in negative ion mode followed by an ion trap high resolution scan (an enhanced resolution scan) and a high sensitivity MS/MS scan (enhanced product ion scan) in positive mode is a very effective method for identifying phosphorylation sites in proteins at low femtomole levels. Coupling of this methodology with a stable isotope N-terminal labeling strategy using iTRAQ™ reagents enabled phosphorylation mapping and relative protein phosphorylation levels to be determined between the active and inactive forms of the protein kinase MAPKAPK-1 in the same LC/MS run.
Original languageEnglish
Pages (from-to)337-346
Number of pages10
JournalMolecular & Cellular Proteomics
Volume5
Issue number2
DOIs
Publication statusPublished - Feb 2006

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Phosphorylation
Mass spectrometers
Ions
Proteins
Protein Kinases
Isotopes
Labeling
Negative ions
Peptides

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title = "Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer",
abstract = "Complete phosphorylation mapping of protein kinases was successfully undertaken using an automated LC/MS/MS approach. This method uses the direct combination of triple quadrupole and ion trapping capabilities in a hybrid triple quadrupole linear ion trap to selectively identify and sequence phosphorylated peptides. In particular, the use of a precursor ion scan of m/z -79 in negative ion mode followed by an ion trap high resolution scan (an enhanced resolution scan) and a high sensitivity MS/MS scan (enhanced product ion scan) in positive mode is a very effective method for identifying phosphorylation sites in proteins at low femtomole levels. Coupling of this methodology with a stable isotope N-terminal labeling strategy using iTRAQ™ reagents enabled phosphorylation mapping and relative protein phosphorylation levels to be determined between the active and inactive forms of the protein kinase MAPKAPK-1 in the same LC/MS run.",
author = "Williamson, {Brian L.} and Jason Marchese and Nicholas Morrice",
note = "dc.publisher: American Society for Biochemistry and Molecular Biology dc.description.sponsorship: We thank the production team of the Division of Signal Therapy, University of Dundee for the recombinant protein kinases. We thank the Post-Genomics and Molecular Interactions Centre facility at the University of Dundee for the Mass Spectrometry facilities and Tina Settineri and Christie Hunter for the critical reading of this manuscript. N. M. thanks Dionex (UK) and Applied Biosystems for providing the HPLC and the 4000 Q TRAP systems for this work .Funded by the Medical Research Council, UK and by Pfizer, Astra Zeneca, GlaxoSmithKline, Boehringer Ingelheim, Merck & Co., and Merck KGaA.",
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Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer. / Williamson, Brian L.; Marchese, Jason; Morrice, Nicholas.

In: Molecular & Cellular Proteomics, Vol. 5, No. 2, 02.2006, p. 337-346.

Research output: Contribution to journalArticle

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