Protein-inhibitor complexes analyzed by alkaline capillary LC-MS.
Liquid chromatography-mass spectrometry (LC-MS) has been used extensively in determination of the molecular weights of proteins, as well as covalent protein-ligand complexes. We have successfully developed LC-MS method for protein molecular weight measurement using small-bore and capillary LC-MS under acidic and basic conditions. A high pH method was critical in studying complexes that were unstable under acidic conditions. Microgram sensitivity was achieved using both methods. A protocol to study the binding mode of protein-ligand complexes under denaturing conditions was developed. These methods were applied to CP88 (a proprietary cysteine protease) inhibitors and revealed different binding modes of inhibitors to proteins that had similar non-reversible behavior in biochemical activity assays. The method also confirmed that one inhibitor studied binds to CP88 in a reversible covalent manner.
Shi
Stone D-H
SD
Pfizer Global Research & Development-La Jolla, 10770 Science Center Drive, San Diego, CA 92121, USA. stone.shi@pfizer.com
Greig
Michael J
MJ
Solowiej
James E
JE
Murray
Brion W
BW
D000085:Acetates; D000641:Ammonia; D002021:Buffers; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D003546:Cysteine Endopeptidases / Q000737:chemistry; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008970:Molecular Weight; D011485:Protein Binding; D011506:Proteins / Q000037:antagonists & inhibitors*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000085:Acetates; D002021:Buffers; D015853:Cysteine Proteinase Inhibitors; D008024:Ligands; D011506:Proteins; D000641:Ammonia; D003546:Cysteine Endopeptidases; C018824:ammonium acetate