Phosphoproteome analysis by in-gel isoelectric focusing and tandem mass spectrometry.

Sarka Beranova, Dominic M. Desiderio, Francesco Giorgianni

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Protein phosphorylation is central to most signaling events in eukaryotic cells. Large-scale analysis of protein phosphorylation in vivo is a highly challenging undertaking that requires powerful analytical and bioinformatics tools; numerous phosphoproteomic methodologies that use various combinations of these tools have been developed recently. This chapter describes an in-gel isoelectric focusing-liquid chromatography-tandem mass spectrometry (IEF-LC-MS/MS) analytical strategy for phosphoproteome mapping. The strategy encompasses seven steps: (1) extraction of proteins from the biological system under study (e.g., a tissue); (2) separation of the protein mixture by isoelectric focusing in an immobilized pH gradient (IPG) strip; (3) protein fixation followed by sectioning of the IPG strip; (4) digestion of the proteins in each gel section; (5) enrichment of phosphopeptides in the digests by immobilized metal ion affinity chromatography; (6) analysis of the enriched digests by LC-MS/MS; and (7) identification of the phosphopeptides/proteins through database searches, and assignment of the sites of phosphorylation in these proteins.

Original languageEnglish (US)
Pages (from-to)383-396
Number of pages14
JournalMethods in molecular biology (Clifton, N.J.)
Volume519
DOIs
StatePublished - Jan 1 2009

Fingerprint

Isoelectric Focusing
Tandem Mass Spectrometry
Gels
Phosphopeptides
Proteins
Proton-Motive Force
Phosphorylation
Protein Databases
Eukaryotic Cells
Computational Biology
Affinity Chromatography
Liquid Chromatography
Proteolysis
Metals
Ions

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

Phosphoproteome analysis by in-gel isoelectric focusing and tandem mass spectrometry. / Beranova, Sarka; Desiderio, Dominic M.; Giorgianni, Francesco.

In: Methods in molecular biology (Clifton, N.J.), Vol. 519, 01.01.2009, p. 383-396.

Research output: Contribution to journalArticle

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