From purification of large amounts of phospho-compounds (nucleotides) to enrichment of phospho-peptides using anion-exchanging resin.
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 beta-casein with two steps of elution (100mM NaCl and 5% NH(4)OH). 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.
Zhang
Kangling
K
Mass Spectrometry Facility, Department of Chemistry, University of California, Riverside, CA 92521, USA.
D000818:Animals; D000837:Anion Exchange Resins / Q000737:chemistry*; D000838:Anions / Q000737:chemistry; D002247:Carbon Isotopes; D002417:Cattle; D002846:Chromatography, Affinity; D009711:Nucleotides / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000837:Anion Exchange Resins; D000838:Anions; D002247:Carbon Isotopes; D009711:Nucleotides; D010750:Phosphoproteins