title: Optimization of the β-elimination/michael addition chemistry on reversed-phase supports for mass spectrometry analysis of O-linked protein modifications.
abstract: We previously adapted the β-elimination/Michael addition chemistry to solid-phase derivatization on reversed-phase supports, and demonstrated the utility of this reaction format to prepare phosphoseryl peptides in unfractionated protein digests for mass spectrometric identification and facile phosphorylation-site determination. Here, we have expanded the use of this technique to β-N-acetylglucosamine peptides, modified at serine/threonine, phosphothreonyl peptides, and phosphoseryl/phosphothreonyl peptides, followed in sequence by proline. The consecutive β-elimination with Michael addition was adapted to optimize the solid-phase reaction conditions for throughput and completeness of derivatization. The analyte remained intact during derivatization and was recovered efficiently from the silica-based, reversed-phase support with minimal sample loss. The general use of the solid-phase approach for enzymatic dephosphorylation was demonstrated with phosphoseryl and phosphothreonyl peptides and was used as an orthogonal method to confirm the identity of phosphopeptides in proteolytic mixtures. The solid-phase approach proved highly suitable to prepare substrates from low-level amounts of protein digests for phosphorylation-site determination by chemical-targeted proteolysis. The solid-phase protocol provides for a simple, robust, and efficient tool to prepare samples for phosphopeptide identification in MALDI mass maps of unfractionated protein digests, using standard equipment available in most biological laboratories. The use of a solid-phase analytical platform is expected to be readily expanded to prepare digest from O-glycosylated- and O-sulfonated proteins for mass spectrometry-based structural characterization.
authors.affiliation: Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Heinz
authors.initials: H
authors.lastname: Nika
authors.forename: Edward
authors.initials: E
authors.lastname: Nieves
authors.forename: David H
authors.initials: DH
authors.lastname: Hawke
authors.forename: Ruth Hogue
authors.initials: RH
authors.lastname: Angeletti
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry*; D006031:Glycosylation; D013058:Mass Spectrometry; D008970:Molecular Weight; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D010766:Phosphorylation; D059748:Proteolysis; D060327:Solid-Phase Synthesis Techniques; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010748:Phosphopeptides; D000117:Acetylglucosamine
keywords: O-GlcNAcylation; ZipTipC18 pipette tips; alkaline phosphatase; chemical-targeted proteolysis; phosphoprotein characterization; serial solid-phase derivatization