Separation and quantitative analysis of O-linked phosphoamino acids by isocratic high-performance liquid chromatography of the 9-fluorenylmethyl chloroformate derivatives.
Phosphoamino acids derivatized with 9-fluorenylmethyl chloroformate were separated on an anion-exchange column (Partisil 10 SAX) at pH 3.90 using an isocratic elution with 10.0 mM potassium phosphate, 1.0% tetrahydrofuran, and 55% methanol. Phosphoamino acids were eluted with baseline resolution in the following order: phosphotyrosine, phosphothreonine, and phosphoserine. Each phosphoamino acid was separated from its parent amino acid, dicarboxylic amino acids, sugaramine phosphates, as well as the other common amino acids. The turn-around time from injection to injection was 35 min. The linearity for all three O-linked phosphoamino acids extended from 0.5-1000 pmol and has been shown to be directly applicable to the analysis of isolated phosphoproteins.
D000596:Amino Acids / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005449:Fluorenes; D007475:Ion Exchange Resins; D009943:Organophosphorus Compounds / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000302:isolation & purification; D010768:Phosphoserine / Q000302:isolation & purification; D010769:Phosphothreonine / Q000302:isolation & purification; D019000:Phosphotyrosine; D014443:Tyrosine / Q000031:analogs & derivatives / Q000302:isolation & purification
D000596:Amino Acids; D005449:Fluorenes; D007475:Ion Exchange Resins; D009943:Organophosphorus Compounds; D010750:Phosphoproteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine