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Molecular & Cellular Proteomics 5:172-181, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Phosphorylation Analysis by Mass Spectrometry

Myths, Facts, and the Consequences for Qualitative and Quantitative Measurements^*

Hanno Steen^,,¶, Judith A. Jebanathirajah^,||,**, John Rush, Nicolas Morrice and Marc W. Kirschner

From the Department of Pathology, Harvard Medical School and Children’s Hospital, Boston, Massachusetts 02115, Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, ^|| Neurobiology Program, Children’s Hospital, Boston, Massachusetts 02115, ^** MDS Sciex, Concord, Ontario L4K 4V8, Canada, Cell Signaling Technology, Beverly, Massachusetts 01915, and MRC Protein Phosphorylation Unit, University of Dundee, Dundee, United Kingdom DD1 5EH

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.

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