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

Hanno Steen, Judith A. Jebanathirajah, John Rush, Nicholas Morrice, Marc W. Kirschner

    Research output: Contribution to journalArticlepeer-review

    301 Citations (Scopus)


    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.
    Original languageEnglish
    Pages (from-to)172-181
    Number of pages10
    JournalMolecular & Cellular Proteomics
    Issue number1
    Publication statusPublished - Jan 2006


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