Thin-layer chromatography can resolve phosphotyrosine, phosphoserine, and phosphothreonine in a protein hydrolyzate.
A solution of propionic acid, 1 M ammonium hydroxide, and isopropyl alcohol (45/17.5/17.5, v/v) was the ascending solvent in the separation of phosphotyrosine, phosphothreonine, and phosphoserine by thin-layer chromatography. The immobile phase was cellulose. The relative migrations were 0.44, 0.38, and 0.2, respectively. A previously described thin-layer system consisting of isobutyric acid and 0.5 M ammonium hydroxide (50/30, v/v) gave very similar relative migrations. To determine the usefulness of thin-layer chromatography in phosphoamino acid analysis, the propionic acid/ammonium hydroxide/isopropyl alcohol solution was used to characterize phosphorylated residues in a plasma membrane protein which is a substrate for the insulin receptor kinase, in insulin receptor phosphorylated histone H2B, and in an in vivo phosphorylated 90000-Da protein from IM9 cells. 32P-labeled proteins were separated by dodecyl sulfate-gel electrophoresis, digested with trypsin, and then hydrolyzed with 6 N HCl, 2 h, 110 degrees C. Following thin-layer chromatography of the hydrolyzates and autoradiography, phosphotyrosine was detected in insulin receptor substrates, and phosphoserine and phosphothreonine were found in the in vivo-phosphorylated protein. This study supports previous reports about the practicality of thin-layer chromatography in phosphoamino acid analysis and it demonstrates that a propionic acid, ammonium hydroxide, isoprophyl alcohol solution may be a useful ascending solvent mixture for this purpose.
Neufeld
E
E
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
Goren
H J
HJ
Boland
D
D
D000818:Animals; D001345:Autoradiography; D002855:Chromatography, Thin Layer; D006801:Humans; D006868:Hydrolysis; D010750:Phosphoproteins / Q000032:analysis; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis*; D010769:Phosphothreonine / Q000032:analysis*; D019000:Phosphotyrosine; D051381:Rats; D012694:Serine / Q000031:analogs & derivatives*; D013912:Threonine / Q000031:analogs & derivatives*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D013912:Threonine; D014443:Tyrosine; D012694:Serine