Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements

Mol Cell Proteomics. 2006 Jan;5(1):172-81. doi: 10.1074/mcp.M500135-MCP200. Epub 2005 Oct 4.


The mass spectrometric analysis of protein phosphorylation is still far from being routine, and the outcomes thereof are often unsatisfying. Apart from the inherent problem of substoichiometric phosphorylation, three arguments as to why phosphorylation analysis is so problematic are often quoted, including (a) increased hydrophilicity of the phosphopeptide with a concomitant loss during the loading onto reversed-phase columns, (b) selective suppression of the ionization of phosphopeptides in the presence of unmodified peptides, and (c) lower ionization/detection efficiencies of phosphopeptides as compared with their unmodified cognates. Here we present the results of a study investigating the validity of these three arguments when using electrospray ionization mass spectrometry. We utilized a set of synthetic peptide/phosphopeptide pairs that were quantitated by amino acid analysis. Under the applied conditions none of the experiments performed supports the notions of (a) generally increased risks of losing phosphopeptides during the loading onto the reversed-phase column because of decreased retention and (b) the selective ionization suppression of phosphopeptides. The issue of ionization/detection efficiencies of phosphopeptides versus their unphosphorylated cognates proved to be less straightforward when using electrospray ionization because no evidence for decreased ionization/detection efficiencies for phosphopeptides could be found.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid
  • DNA-Binding Proteins / analysis
  • Fluorocarbons / pharmacology
  • Peptide Mapping
  • Phosphopeptides / analysis*
  • Phosphorylation
  • Saccharomyces cerevisiae Proteins / analysis
  • Sensitivity and Specificity
  • Spectrometry, Mass, Electrospray Ionization*
  • Transcription Factors / analysis


  • DNA-Binding Proteins
  • Fluorocarbons
  • PHO4 protein, S cerevisiae
  • Phosphopeptides
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • perfluorobutyric acid