Phosphopeptide enrichment by covalent chromatography after solid phase derivatization of protein digests on reversed phase supports

Heinz Nika, Ruth Hogue Angeletti, David H. Hawke

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages31-50
Number of pages20
Volume1355
DOIs
Publication statusPublished - Jan 1 2016
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1355
ISSN (Print)10643745

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Keywords

  • Covalent chromatography
  • Mass spectrometry
  • Multi-step solid-phase derivatization
  • Phosphopeptide enrichment
  • Phosphorylation site determination
  • Reversed phase support
  • β-elimination/Michael addition

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Nika, H., Angeletti, R. H., & Hawke, D. H. (2016). Phosphopeptide enrichment by covalent chromatography after solid phase derivatization of protein digests on reversed phase supports. In Methods in Molecular Biology (Vol. 1355, pp. 31-50). (Methods in Molecular Biology; Vol. 1355). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-3049-4_3