Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry

Rapid Commun Mass Spectrom. 2000;14(17):1600-6. doi: 10.1002/1097-0231(20000915)14:17<1600::AID-RCM68>3.0.CO;2-V.

Abstract

On-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization-mass spectrometry (IMAC/CE/ESI-MS) offers selective preconcentration of phosphorylated peptides with identification of the phosphorylated amino acid(s). The preconcentration provides low concentration limits of detection and capillary electrophoresis separates the peptides. Recently, we reported a fast, simple, and sensitive on-line IMAC/CE/ESI-MS/MS method for the determination of phosphopeptides at low-pmole levels. That work is expanded here by use of multiple stage tandem mass spectrometry (MS(n), n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI-MS(n) is demonstrated by the analysis of tryptic digests of alpha- and beta-casein and in-gel tryptic digests of beta-casein.

MeSH terms

  • Amino Acid Sequence
  • Caseins / chemistry
  • Chromatography, Affinity
  • Electrophoresis, Capillary
  • Electrophoresis, Polyacrylamide Gel
  • Hydrolysis
  • Mass Spectrometry
  • Molecular Sequence Data
  • Online Systems
  • Peptide Mapping
  • Phosphorylation
  • Proteins / chemistry*
  • Trypsin

Substances

  • Caseins
  • Proteins
  • Trypsin