Rapid separation of phosphoamino acids including the phosphohistidines by isocratic high-performance liquid chromatography of the orthophthalaldehyde derivatives.
Amino acids were derivatized with orthophthalaldehyde and separated by high-performance liquid chromatography on a polymer-based reverse-phase column (Hamilton PRP-1) at pH 7.2 using isocratic elution with 14.3 mM sodium phosphate, 1.1% tetrahydrofuran, 6.6% acetonitrile. Phosphorylated amino acids were eluted with baseline resolution in the following order: 1-phosphohistidine, phosphoserine, 3-phosphohistidine, phosphotyrosine, phosphothreonine, and phosphoarginine. Each of the phosphoamino acids was separated from its parent amino acid but aspartate and glutamate eluted in the same region as the phosphoamino acids. The sensitivity is in the picomole range and the separation time, injection to injection, is 15 min. The linearity for phosphothreonine extends at least from 30 pmol to 30 nmol. Quantitation by radioactivity is good for each of the phosphoamino acids except in the case of [1-32P]phosphohistidine, which coelutes with inorganic phosphate.
Carlomagno
L
L
Huebner
V D
VD
Matthews
H R
HR
D000447:Aldehydes; D000596:Amino Acids / Q000302:isolation & purification*; D001120:Arginine / Q000031:analogs & derivatives / Q000302:isolation & purification; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006639:Histidine / Q000031:analogs & derivatives* / Q000302:isolation & purification; D009943:Organophosphorus Compounds / Q000302:isolation & purification; D010768:Phosphoserine / Q000302:isolation & purification; D010769:Phosphothreonine / Q000302:isolation & purification; D019000:Phosphotyrosine; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives / Q000302:isolation & purification; D009764:o-Phthalaldehyde
D000447:Aldehydes; D000596:Amino Acids; D002021:Buffers; D009943:Organophosphorus Compounds; D010769:Phosphothreonine; C015441:phospho-L-arginine; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine; D006639:Histidine; D009764:o-Phthalaldehyde; D001120:Arginine; C005436:phosphohistidine