Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry.
A new method combining chemical modification and affinity purification is described for the characterization of serine and threonine phosphopeptides in proteins. The method is based on the conversion of phosphoserine and phosphothreonine residues to S-(2-mercaptoethyl)cysteinyl or beta-methyl-S-(2-mercaptoethyl)cysteinyl residues by beta-elimination/1,2-ethanedithiol addition, followed by reversible biotinylation of the modified proteins. After trypsin digestion, the biotinylated peptides were affinity-isolated and enriched, and subsequently subjected to structural characterization by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Database searching allowed for automated identification of modified residues that were originally phosphorylated. The applicability of the method is demonstrated by the identification of all known phosphorylation sites in a mixture of alpha-casein, beta-casein, and ovalbumin. The technique has potential for adaptations to proteome-wide analysis of protein phosphorylation.
Adamczyk
M
M
Department of Chemistry (9NM), Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, IL 60064-6016, USA. maceij.adamczyk@abbott.com
Gebler
J C
JC
Wu
J
J
D000595:Amino Acid Sequence; D001331:Automation; D019921:Biotinylation; D002364:Caseins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019992:Databases as Topic; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry*; D014357:Trypsin
D002364:Caseins; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010748:Phosphopeptides; D011506:Proteins; D010047:Ovalbumin; D014357:Trypsin