Quantification of protein-ligand interactions by mass spectrometry, titration, and H/D exchange: PLIMSTEX.
Protein-ligand binding and the concomitant conformational change in the protein are of crucial importance in biophysics and drug design. We report a novel method to quantify protein-ligand interactions in solution by mass spectrometry, titration, and H/D exchange (PLIMSTEX). The approach can determine the conformational change, binding stoichiometry, and affinity in protein-ligand interactions including those that involve small molecules, metal ions, and peptides. Binding constants obtained by PLIMSTEX for four model protein-ligand systems agree with K values measured by conventional methods. At higher protein concentration, the method can be used to determine quickly the binding stoichiometry and possibly the purity of proteins. Taking advantage of concentrating the protein on-column and desalting, we are able to use different concentrations of proteins, buffer systems, salts, and pH in the exchange protocol. High picomole quantities of proteins are sufficient, offering significantly better sensitivity than that of NMR and X-ray crystallography. Automation could make PLIMSTEX a high throughput method for library screening, drug discovery, and proteomics.
Zhu
Mei M
MM
Resource for Mass Spectrometry, Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
Rempel
Don L
DL
Du
Zhaohui
Z
Gross
Michael L
ML
D000818:Animals; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D003903:Deuterium; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008555:Melitten / Q000737:chemistry / Q000378:metabolism; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019301:Oleic Acid / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013552:Swine; D017186:Titrimetry; D025521:Tumor Suppressor Proteins
D002147:Calmodulin; D002352:Carrier Proteins; C107608:FABP7 protein, human; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D008024:Ligands; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D025521:Tumor Suppressor Proteins; D006153:Guanosine Diphosphate; D008555:Melitten; D019301:Oleic Acid; D003903:Deuterium; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)