Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer.
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 iTRAQtrade mark 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.
Williamson
Brian L
BL
Discovery Proteomics Small Molecule Research Center, Applied Biosystems, Framingham, Massachusetts 01701, USA.
Marchese
Jason
J
Morrice
Nicholas A
NA
D000595:Amino Acid Sequence; D001331:Automation; D002853:Chromatography, Liquid; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism
D010748:Phosphopeptides; D011506:Proteins; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D038362:Glycogen Synthase Kinase 3