Undesirable charge-enhancement of isobaric tagged phosphopeptides leads to reduced identification efficiency

Tine E Thingholm, Giuseppe Palmisano, Frank Kjeldsen, Martin Røssel Larsen

Research output: Contribution to journalJournal articleResearchpeer-review


The study of cellular dynamics by proteomics using mass spectrometry requires a quantitation strategy that is robust, sensitive, and of sufficient resolution to deal with subtle changes in protein expression or post-translational modification. The major quantitation strategies are stable isotopic labeling of proteins and peptides for in vitro cell culture systems (stable isotope labeling using amino acids in cell culture, SILAC) or isobaric peptide labels such as isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT) for both in vitro and in vivo systems. These quantitation strategies have also been successfully applied to phosphoproteomics studies for the investigation of signal transduction pathways. Here we describe major drawbacks associated with isobaric labeling for the identification and quantitation of phosphopeptides using electrospray tandem mass spectrometry. Phosphopeptide derivatization with isobaric tags results in significantly greater charging in electrospray ionization. This reduces phosphopeptide identification efficiency with multistage activation and HCD MS/MS by more than 50% and may contribute to the discrepancy observed between identifications observed for large cell- or tissue-based data sets from labeled and nonlabeled peptide mixtures. Ammonia vapor sprayed perpendicular to the electrospray needle during ionization resulted in an overall decrease in the average charge states and a concomitant increase in phosphopeptide identifications.
Original languageEnglish
JournalJournal of Proteome Research
Issue number8
Pages (from-to)4045-52
Number of pages8
Publication statusPublished - 6. Aug 2010


  • Ammonia
  • Chromatography, Ion Exchange
  • Hela Cells
  • Humans
  • Phosphopeptides
  • Proteomics
  • Spectrometry, Mass, Electrospray Ionization
  • Staining and Labeling
  • Titanium


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