title: Fast track to a phosphoprotein sketch - MALDI-TOF characterization of TLC-based tryptic phosphopeptide maps at femtomolar detection sensitivity.
abstract: Tryptic phosphopeptide mapping by TLC on microcrystalline cellulose has been a convenient method to get a fast and highly reproducible overview of the number of phosphopeptides present in any given (32)P-labeled phosphoprotein. This method also provides an immediate presentation of the relative phosphorylation stoichiometry between individual phosphopeptides. However, so far, traditional tryptic phosphopeptide maps have not been useful for phosphoproteomics applications, as the S/N has been very poor, due to the large number of quenching substances and contaminants present on cellulose plates. In this study, we present a rapid and easy method for phosphopeptides identification from 2-D phosphopeptide maps (2-D-PPMs). We obtain improved sensitivity (femtomole levels) upon MALDI-TOF MS analysis of phosphopeptides extracted from 2-D-PPMs. Using this approach we could confidently characterize the major phosphorylation sites of in vivo and in vitro (32)P-labeled proteins.
authors.affiliation: Turku Centre for Biotechnology, University of Turku and Abo Akademi University, BioCity, Turku, Finland.
authors.forename: Vitaly
authors.initials: V
authors.lastname: Kochin
authors.forename: Susumu Y
authors.initials: SY
authors.lastname: Imanishi
authors.forename: John E
authors.initials: JE
authors.lastname: Eriksson
mesh_terms: D002855:Chromatography, Thin Layer; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes