Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry.
Consecutive enzymatic reactions of analytes which are affinity bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, beta-casein and alpha-casein, as well as one phosphopeptide from a kinase reaction mixture. Agarose loaded with either Fe3+ or Ga3+ was used to isolate phosphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+-loaded agarose was used to isolate phosphopeptides. The selectivity for monophosphorylated peptides, however, was better with Fe3+-loaded agarose. This technique is easy to use and has the ability to analyze extremely complicated phosphopeptide mixtures. Moreover, it eliminates the need for prior high-performance liquid chromatography separation or radiolabeling, thus greatly simplifying the sample preparation.
Zhou
W
W
Laboratory of Structural Biology, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Merrick
B A
BA
Khaledi
M G
MG
Tomer
K B
KB
D000595:Amino Acid Sequence; D002268:Carboxypeptidases; D002364:Caseins / Q000737:chemistry; D043402:Cathepsin A; D002846:Chromatography, Affinity; D005708:Gallium; D006868:Hydrolysis; D007202:Indicators and Reagents; D007501:Iron; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D002364:Caseins; D007202:Indicators and Reagents; D008670:Metals; D010748:Phosphopeptides; D005708:Gallium; D007501:Iron; D002268:Carboxypeptidases; D043402:Cathepsin A; D014357:Trypsin