title: Phosphopeptide Enrichment by Covalent Chromatography After Solid Phase Derivatization of Protein Digests on Reversed Phase Supports.
abstract: The isolation of the phosphopeptide constituents from phosphoprotein digests is prerequisite to facilitate the mass spectrometric characterization of phosphorylation events. Here, we describe a chemical proteomics approach which combines solid phase derivatization of phosphoprotein digests with phosphopeptide enrichment by covalent chromatography. The use of the solid phase support for derivatization ensures for speed and completeness of reactions. The isolates proved highly suitable for mapping of the sites of phosphorylation by collisionally induced dissociation (CID). The method combines robustness with simplicity of operation using equipment available in biological laboratories, and may be readily extended to map the sites of O-glycosylation.
authors.affiliation: Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.|Department for Molecular and Developmental Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.
authors.forename: Heinz
authors.initials: H
authors.lastname: Nika
authors.affiliation: Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.|Department for Molecular and Developmental Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.
authors.forename: Ruth Hogue
authors.initials: RH
authors.lastname: Angeletti
authors.affiliation: Department of Systems Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. dhawke@mdanderson.org.
authors.forename: David H
authors.initials: DH
authors.lastname: Hawke
mesh_terms: D000818:Animals; D056148:Chromatography, Reverse-Phase; D006801:Humans; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D052616:Solid Phase Extraction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D057188:Workflow
chemical_list: D010748:Phosphopeptides
keywords: Covalent chromatograph y; Mass spectrometry; Multi-step solid-phase derivatization; Phosphopeptide enrich ment; Phosphorylation site determination; Reversed phase support; β-elimination /Michael addition