Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses.
A method has been developed that utilizes phosphoprotein isotope-coded affinity tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins. The PhIAT labeling approach involves hydroxide ion-mediated beta-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D0) or four alkyl deuteriums (EDT-D4) followed by biotinylation of the EDT-D0/D4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, was synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process. The PhIAT labeling approach was initially demonstrated using the model phosphoprotein beta-casein. After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using capillary reversed-phase liquid chromatography-mass spectrometry. PhIAT-labeled beta-casein peptides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified. The PhIAT labeling method was also applied to a yeast protein extract. The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins.
Goshe
M B
MB
Environmental and Molecular Sciences Laboratory, Richland, Washington 99352, USA.
Conrads
T P
TP
Panisko
E A
EA
Angell
N H
NH
Veenstra
T D
TD
Smith
R D
RD
D000345:Affinity Labels / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D007202:Indicators and Reagents; D007554:Isotopes; D010748:Phosphopeptides / Q000032:analysis / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000737:chemistry*; D020543:Proteome; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D000345:Affinity Labels; D007202:Indicators and Reagents; D007554:Isotopes; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome