The determination of high-affinity protein/inhibitor binding constants by electrospray ionization hydrogen/deuterium exchange mass spectrometry.
Recently, a hydrogen/deuterium exchange method termed SUPREX (Stability of Unpurified Proteins from Rates of hydrogen/deuterium EXchange), capable of measuring protein/ligand binding constants, which utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported. Unlike more conventional approaches, SUPREX is inherently capable of measuring Kd values of tight binding ligands. Here we present a SUPREX-based method, incorporating automation and electrospray ionization (ESI)-MS, to measure Kd values for very potent inhibitors of the kinase PKCtheta. The use of ESI offers an alternative to MALDI, with the advantages of improved mass measurement precision for larger proteins, and amenability to automation. Kd values generated by this method are in good agreement with those generated by a molecular protein kinase assay.
Frego
Lee
L
Boehringer Ingelheim Pharmaceuticals, Research and Development Center, 900 Ridgebury Road, Ridgefield, CT 06877, USA.
Gautschi
Elda
E
Martin
Leslie
L
Davidson
Walter
W
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D041961:Deuterium Exchange Measurement / Q000379:methods; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D011493:Protein Kinase C