title: Phosphospecific proteolysis for mapping sites of protein phosphorylation.
abstract: Protein phosphorylation is a dominant mechanism of information transfer in cells, and a major goal of current proteomic efforts is to generate a system-level map describing all the sites of protein phosphorylation. Recent efforts have focused on developing technologies for enriching and quantifying phosphopeptides. Identification of the sites of phosphorylation typically relies on tandem mass spectrometry to sequence individual peptides. Here we describe an approach for phosphopeptide mapping that makes it possible to interrogate a protein sequence directly with a protease that recognizes sites of phosphorylation. The key to this approach is the selective chemical transformation of phosphoserine and phosphothreonine residues into lysine analogs (aminoethylcysteine and beta-methylaminoethylcysteine, respectively). Aminoethylcysteine-modified peptides are then cleaved with a lysine-specific protease to map sites of phosphorylation. A blocking step enables single-site cleavage, and adaptation of this reaction to the solid phase facilitates phosphopeptide enrichment and modification in one step.
authors.affiliation: Program in Chemistry and Chemical Biology, University of California, San Francisco, California 94143-0446, USA.
authors.forename: Zachary A
authors.initials: ZA
authors.lastname: Knight
authors.forename: Birgit
authors.initials: B
authors.lastname: Schilling
authors.forename: Richard H
authors.initials: RH
authors.lastname: Row
authors.forename: Denise M
authors.initials: DM
authors.lastname: Kenski
authors.forename: Bradford W
authors.initials: BW
authors.lastname: Gibson
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D019606:Phosphoamino Acids / Q000737:chemistry* / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry / Q000378:metabolism; D010769:Phosphothreonine / Q000737:chemistry / Q000378:metabolism; D010770:Phosphotransferases / Q000737:chemistry* / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D014404:Tubulin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D019606:Phosphoamino Acids; D010748:Phosphopeptides; D020543:Proteome; D014404:Tubulin; D010769:Phosphothreonine; D010768:Phosphoserine; D010770:Phosphotransferases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D010450:Endopeptidases; D010447:Peptide Hydrolases