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 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.
Highlights
Complete phosphorylation mapping of protein kinases was successfully undertaken using an automated LC/ MS/MS approach
Because the workflow described here was to be applied to an on-line capillary reverse phase HPLC separation of phosphopeptides, it was logical to increase the organic content of the HPLC eluate with a postcolumn addition of organic solvent
Ser-403 (ATQAPLHpSVVQQLHGK) was significantly up-regulated after activation. This post-translational modifications (PTMs) discovery method uses the direct combination of triple quadrupole and ion trapping capabilities in a hybrid triple quadrupole linear ion trap mass spectrometer to selectively identify and sequence phosphorylated peptides at low femtomole levels
Summary
The resultant scan produces a mass spectrum that contains only the molecular ions of the phosphopeptides that are present in the sample. When ions are detected in the highly selective precursor ion scan of m/z Ϫ79, the instrument switches from negative to positive ion mode (700-ms delay), and enhanced resolution (ER) scans are performed to obtain the accurate mass of the peptides. This is followed by an MS/MS sequencing scan using the high sensitivity afforded by the linear ion trap mode. This selective phosphorylated detection was coupled with amine-reactive isobaric tagging reagents [24] (iTRAQTM, Applied Biosystems, Foster City, CA) to obtain relative phosphorylation information between the active and inactive forms of mitogen-activated protein kinase-activated protein kinase-1 (MAPKAPK-1)
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