Thin-layer chromatography can resolve phosphotyrosine, phosphoserine, and phosphothreonine in a protein hydrolyzate

Journal Article · Wed Feb 15 00:00:00 EST 1989 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(89)90028-6· OSTI ID:5564607

Neufeld, E; Goren, H J; Boland, D ^[1]

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1. Univ. of Calgary, Alberta (Canada)

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. {sup 32}P-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.

• OSTI ID: 5564607
• Journal Information: Analytical Biochemistry; (USA), Vol. 177:1; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
INSULIN
RECEPTORS
PHOSPHOPROTEINS
THIN-LAYER CHROMATOGRAPHY
AUTORADIOGRAPHY
ELECTROPHORESIS
HYDROLYSIS
MAN
PHOSPHORUS 32
PHOSPHORYLATION
RATS
SERINE
SOLVENT EXTRACTION
THREONINE
TYROSINE
AMINO ACIDS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
EXTRACTION
HORMONES
HYDROXY ACIDS
ISOTOPES
LIGHT NUCLEI
LYSIS
MAMMALS
MEMBRANE PROTEINS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HORMONES
PHOSPHORUS ISOTOPES
PRIMATES
PROTEINS
RADIOISOTOPES
RODENTS
SEPARATION PROCESSES
SOLVOLYSIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques