title: Comparative phosphorylation site mapping from gel-derived proteins using a multidimensional ES/MS-based approach.
abstract: Understanding how phosphorylation regulates the behavior of individual proteins is critical to understanding signaling pathways. These studies usually involve knowledge of which amino acid residues are phosphorylated on a given protein and the extent of such a modification. This is often a rather difficult task in that most phosphoproteins contain multiple substoichiometric sites of phosphorylation. Here we describe the multidimensional electrospray (ES) mass spectrometry (MS)-based phosphopeptide-mapping strategy developed in our laboratory. In the first dimension of the process, phosphopeptides present in a protein digest are selectively detected and collected into fractions during on-line liquid chromatography (LC)/ES/MS, which monitors for phosphopeptide-specific marker ions. This analysis generates a phosphorylation profile that can be used to assess changes in the phosphorylation state of a protein pointing to those phosphopeptides that require further investigation. The phosphopeptide-containing fractions are then analyzed in the second dimension by nano-ES with precursor-ion scan for the marker ion m/z 79. As the final step, direct sequencing of the phosphopeptides is performed by LC/ES/MS/MS. Merits and limitations of the strategy, as well as experimental details and suggestions, are described here.
authors.affiliation: Proteomics and Biological Mass Spectrometry Laboratory, Department of Computational, Analytical and Structural Sciences, GlaxoSmithKline, King of Prussia, PA, USA.
authors.forename: Francesca
authors.initials: F
authors.lastname: Zappacosta
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Huddleston
authors.forename: Roland S
authors.initials: RS
authors.lastname: Annan
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D009862:Online Systems / Q000295:instrumentation; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction / Q000502:physiology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins