Use of hydrophilic interaction chromatography for the study of tyrosine protein kinase specificity.
A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [32P]phosphorylated peptide, unreacted [gamma-32P]ATP, and 32P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase.
Boutin
J A
JA
Département de Cancérologie Expérimentale, Institut de Recherches Servier, Suresnes, France.
Ernould
A P
AP
Ferry
G
G
Genton
A
A
Alpert
A J
AJ
D000595:Amino Acid Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005755:Gastrins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D005755:Gastrins; D009363:Neoplasm Proteins; C008530:minigastrin; D011505:Protein-Tyrosine Kinases