title: Characterization of protein phosphorylation by mass spectrometry using immobilized metal ion affinity chromatography with on-resin beta-elimination and Michael addition.
abstract: A protocol combining immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition has been developed for enhanced analysis of protein phosphorylation. Immobilized metal ion affinity chromatography was initially used to enrich for phosphorylated peptides. Beta-elimination, with or without concurrent Michael addition, was then subsequently used to simultaneously elute and derivatize phosphopeptides bound to the chromatography resin. Derivatization of the phosphate facilitated the precise determination of phosphorylation sites by MALDI-PSD/LIFT tandem mass spectrometry, avoiding complications due to ion suppression and phosphate lability in mass spectrometric analysis of phosphopeptides. Complementary use of immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition in this manner circumvented several inherent disadvantages of the individual methods. In particular, (i) the protocol discriminated O-linked glycosylated peptides from phosphopeptides prior to beta-elimination/Michael addition and (ii) the elution of peptides from the chromatography resin as derivatized phosphopeptides distinguished them from unphosphorylated species that were also retained. The chemical derivatization of phosphopeptides greatly increased the information obtained during peptide sequencing by mass spectrometry. The combined protocol enabled the detection and sequencing of phosphopeptides from protein digests at low femtomole concentrations of initial sample and was employed to identify novel phosphorylation sites on the cell adhesion protein p120 catenin and the glycoprotein fetuin.
authors.affiliation: The Ludwig Institute for Cancer Research, Cruciform Building, Gower Street, London WC1E 6BT, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thompson
authors.forename: Sarah R
authors.initials: SR
authors.lastname: Hart
authors.forename: Clemens
authors.initials: C
authors.lastname: Franz
authors.forename: Karin
authors.initials: K
authors.lastname: Barnouin
authors.forename: Anne
authors.initials: A
authors.lastname: Ridley
authors.forename: Rainer
authors.initials: R
authors.lastname: Cramer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D051177:Catenins; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D005290:Ferric Compounds / Q000737:chemistry*; D051379:Mice; D009532:Nickel / Q000737:chemistry*; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D000509:alpha-Fetoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000095002:Delta Catenin
chemical_list: D051177:Catenins; D015815:Cell Adhesion Molecules; D005290:Ferric Compounds; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000509:alpha-Fetoproteins; D009532:Nickel; D000095002:Delta Catenin