From purification of large amounts of phospho-compounds (nucleotides) to enrichment of phospho-peptides using anion-exchanging resin

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Through years of practice, mass spectrometry has proven to be one of the most reliable and sensitive methods for the localization of protein phosphorylation sites. Among numerous innovative methods, affinity enrichment such as immobilized metal-ion affinity chromatography followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis appears to be the most widely chosen procedure. Here, I report a method that was originally designed for purification of large amounts of nucleotides using anion-exchanging resin but has shown the promise of enriching phosphorylated peptides. Mixtures composed of uridine monophosphate, uridine diphosphate, uridine triphosphate, and their nonphosphate compound-uridine were bottom-line separated on an anion-exchanging solid-phase extraction (SPE) column by four steps of elution with a gradient of salt concentration and pH values. The miniature form of this SPE column showed significant separation (or enrichment) of the tryptic phospho-peptides from non-phospho-peptides of the standard protein β-casein with two steps of elution (100 mM NaCl and 5% NH4OH). Furthermore, after utilization of this anion-exchanging-column enrichment followed by LC/MS/MS analysis on a quadrupole-tine of flight instrument, a new phosphorylation site (S191) in bovine chromogranin A was identified.

Original languageEnglish (US)
Pages (from-to)225-231
Number of pages7
JournalAnalytical Biochemistry
Volume357
Issue number2
DOIs
StatePublished - Oct 15 2006
Externally publishedYes

Keywords

  • Anion-exchanging chromatography
  • Enrichment
  • Mass spectrometry
  • Phosphorylation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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