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

Tine E. Thingholm, Giuseppe Palmisano, Frank Kjeldsen, Martin R. Larsen

Research output: Contribution to journalArticle

82 Citations (Scopus)

Abstract

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
Pages (from-to)4045-4052
Number of pages8
JournalJournal of Proteome Research
Volume9
Issue number8
DOIs
Publication statusPublished - Aug 6 2010

Fingerprint

Phosphopeptides
Labeling
Cell culture
Peptides
Mass spectrometry
Cell Culture Techniques
Isotope Labeling
Electrospray ionization
Signal transduction
Post Translational Protein Processing
Tandem Mass Spectrometry
Ammonia
Isotopes
Needles
Proteomics
Ionization
Labels
Signal Transduction
Mass Spectrometry
Proteins

Keywords

  • Isobaric tags
  • iTRAQ
  • Mass spectrometry
  • Phosphorylation
  • Quantitation
  • TMT

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

Undesirable charge-enhancement of isobaric tagged phosphopeptides leads to reduced identification efficiency. / Thingholm, Tine E.; Palmisano, Giuseppe; Kjeldsen, Frank; Larsen, Martin R.

In: Journal of Proteome Research, Vol. 9, No. 8, 06.08.2010, p. 4045-4052.

Research output: Contribution to journalArticle

Thingholm, Tine E. ; Palmisano, Giuseppe ; Kjeldsen, Frank ; Larsen, Martin R. / Undesirable charge-enhancement of isobaric tagged phosphopeptides leads to reduced identification efficiency. In: Journal of Proteome Research. 2010 ; Vol. 9, No. 8. pp. 4045-4052.
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