title: Phospho-proteomics: evaluation of the use of enzymatic de-phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis.
abstract: Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in such proteins is a major challenge in proteomics. In the present study we evaluate the use of enzymatic de-phosphorylation in combination with differential peptide mass mapping for identification of phosphorylated peptides in peptide mixtures derived from in-gel digested phospho-proteins. Phospho-peptides could be identified provided that improved sample preparation methods prior to mass spectrometric analysis were used. An attempt to identify the proteins visualized by [32P] autoradiography in a proteomics study and their phosphorylation sites, demonstrated that protein identification was possible whereas reliable identification of the phospho-peptides requires more protein than normally available in our proteomics studies.
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Proteome Analysis, University of Southern Denmark, Denmark.
authors.forename: M R
authors.initials: MR
authors.lastname: Larsen
authors.forename: G L
authors.initials: GL
authors.lastname: Sørensen
authors.forename: S J
authors.initials: SJ
authors.lastname: Fey
authors.forename: P M
authors.initials: PM
authors.lastname: Larsen
authors.forename: P
authors.initials: P
authors.lastname: Roepstorff
mesh_terms: D000469:Alkaline Phosphatase; D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007633:Keratins / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D010047:Ovalbumin / Q000737:chemistry; D020648:Peptide Elongation Factor 1 / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods*; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D020543:Proteome / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
chemical_list: D002364:Caseins; D020648:Peptide Elongation Factor 1; D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; D007633:Keratins; D010047:Ovalbumin; D000469:Alkaline Phosphatase; D014357:Trypsin