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Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry

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Abstract

Reversible phosphorylation is one of the most important posttranslational modifications of cellular proteins. Mass spectrometry is a widely used technique in the characterization of phosphorylated proteins and peptides. Similar to nonmodified peptides, sequence information for phosphopeptides digested from proteins can be obtained by tandem mass analysis using either electrospray ionization or matrix assisted laser desorption/ionization (MALDI) mass spectrometry. However, the facile loss of neutral phosphoric acid (H3PO4) or HPO3 from precursor ions and fragment ions hampers the precise determination of phosphorylation site, particularly if more than one potential phosphorylation site or concensus sequence is present in a given tryptic peptide. Here, we investigated the fragmentation of phosphorylated peptides under laser-induced dissociation (LID) using a MALDI-time-of-flight mass spectrometer with a curved-field reflectron. Our data demonstrated that intact fragments bearing phosphorylated residues were produced from all tested peptides that contain at least one and up to four phosphorylation sites at serine, threonine, or tyrosine residues. In addition, the LID of phosphopeptides derivatized by N-terminal sulfonation yields simplified MS/MS spectra, suggesting the combination of these two types of spectra could provide an effective approach to the characterization of proteins modified by phosphorylation.

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Correspondence to Dongxia Wang or Robert J. Cotter.

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Wang, D., Cole, P.A. & Cotter, R.J. Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Clin Proteom 2, 133–144 (2006) doi:10.1007/BF02752496

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Key Words

  • Phosphorylation
  • MALDI
  • time-of-flight mass spectrometry
  • curved-field reflectron
  • N-terminal sulfonation
  • fragmentation