Phosphatase-directed phosphorylation-site determination: A synthesis of methods for the detection and identification of phosphopeptides

Matthew P. Torres, Roopa Thapar, William F. Marzluff, Christoph H. Borchers

Research output: Contribution to journalArticle

37 Scopus citations

Abstract

Phosphopeptides can be difficult to detect and sequence by mass spectrometry (MS) due to low ionization efficiency and suppression effects in the MS mode, and insufficient fragmentation in the tandem MS (MS/MS) mode, respectively. To address this problem, we have developed a technique called Phosphatase-directed Phosphorylation-site Determination (PPD), which combines on-target phosphatase reactions, MALDI MS/MS of IMAC beads on target, and hypothesis-driven MS (HD-MS). In this method, on-target dephosphorylation experiments are conducted on IMAC-bound phosphopeptides, because dephosphorylated peptides have, in general, higher MS sensitivities than the corresponding phosphopeptides. The detected dephosphorylated peptides are sequenced by MS/MS, which identifies the potentially phosphorylated peptide and the total number of Ser, Thr, or Tyr residues that could hypothetically be phosphorylated within that peptide. On the basis of this information, a mass list containing every possible phosphorylation state of each observed peptide (where 1 HPO3 = 80 Da) is used to direct MALDI-MS/MS on the phosphorylated peptides bound to IMAC beads at each theoretical mass from the list. If the peptide is present, the resulting MS/MS spectrum reveals the exact site(s) of phosphorylation in the peptide. We have demonstrated the applicability of PPD to the detection of in vivo phosphorylation sites on the Drosophila Stem Loop Binding Protein (dSLBP), and the complementarity of this new technique to conventional MS phosphorylation site mapping methods, since the phosphorylation sites in dSLBP could not be detected by other methods.

Original languageEnglish (US)
Pages (from-to)1628-1635
Number of pages8
JournalJournal of Proteome Research
Volume4
Issue number5
DOIs
StatePublished - Sep 1 2005

Keywords

  • IMAC
  • MALDI MS/MS
  • Mass spectrometry
  • Phosphopeptides
  • Phosphoproteomics
  • SLBP
  • Stem-loop binding protein

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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