title: Identification and relative quantification of phosphopeptides by a combination of multi-protease digestion and isobaric labeling.
abstract: BACKGROUND
  The identification and the determination of the extent of protein phosphorylation are major prerequisites for the comparative analysis of this important posttranslational modification of proteins in different biological situations. High sequence coverages and the availability of straightforward quantification methods are necessary to achieve these goals.
  
  
  METHODS
  Phosphoproteins and non-phosphorylated analogues separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were digested using four different proteases (trypsin, chymotrypsin, elastase and GluC) and the digests were isobarically labeled using eight-plex iTRAQ. The combined labeled digests were subsequently enriched using titanium dioxide and both the phosphorylated and non-phosphorylated fractions were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The influence of different data analysis algorithms (Percolator or False Discovery Rate) on the outcome of analysis was investigated.
  
  
  RESULTS
  Almost complete sequence coverage could be achieved upon application of a multi-protease approach. The formation of peptides of different lengths and physicochemical properties allowed the identification and the mapping of all phosphorylation sites in the investigated model proteins. The introduction of isobaric labels allowed quantification of different peptides of the same phosphorylation site with more than one peptide, leading to significantly improved statistical confidence.
  
  
  CONCLUSIONS
  A workflow for the straightforward comparative analysis of protein phosphorylation in samples of low complexity, e.g. isolated proteins, was developed. The workflow is transferable to other posttranslational modifications.
authors.affiliation: Institut für Experimentelle Medizin - AG Systematische Proteomforschung und Bioanalytik, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Linke
authors.affiliation: Institut für Experimentelle Medizin - AG Systematische Proteomforschung und Bioanalytik, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany.
authors.forename: Tomas
authors.initials: T
authors.lastname: Koudelka
authors.affiliation: Institut für Experimentelle Medizin - AG Systematische Proteomforschung und Bioanalytik, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Becker
authors.affiliation: Institut für Experimentelle Medizin - AG Systematische Proteomforschung und Bioanalytik, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D002364:Caseins; D010748:Phosphopeptides; D010447:Peptide Hydrolases