Phospho-proteomics: evaluation of the use of enzymatic de-phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis

Proteomics. 2001 Feb;1(2):223-38. doi: 10.1002/1615-9861(200102)1:2<223::AID-PROT223>3.0.CO;2-B.

Abstract

Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in such proteins is a major challenge in proteomics. In the present study we evaluate the use of enzymatic de-phosphorylation in combination with differential peptide mass mapping for identification of phosphorylated peptides in peptide mixtures derived from in-gel digested phospho-proteins. Phospho-peptides could be identified provided that improved sample preparation methods prior to mass spectrometric analysis were used. An attempt to identify the proteins visualized by [32P] autoradiography in a proteomics study and their phosphorylation sites, demonstrated that protein identification was possible whereas reliable identification of the phospho-peptides requires more protein than normally available in our proteomics studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase
  • Animals
  • Binding Sites
  • Caseins / chemistry
  • Electrophoresis, Gel, Two-Dimensional
  • Humans
  • Keratins / chemistry
  • Mass Spectrometry / methods*
  • Mice
  • Ovalbumin / chemistry
  • Peptide Elongation Factor 1 / chemistry
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Peptide Mapping / methods*
  • Phosphoproteins / chemistry*
  • Phosphorylation
  • Proteome / chemistry*
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trypsin

Substances

  • Caseins
  • Peptide Elongation Factor 1
  • Peptide Fragments
  • Phosphoproteins
  • Proteome
  • Keratins
  • Ovalbumin
  • Alkaline Phosphatase
  • Trypsin