Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry
- 353 Downloads
A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying protein—ligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (Lref), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein—ligand complex in the gas phase, is added to the solution. The fraction of P bound to Lref, which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. Affinities of two carbohydrate-binding proteins, a single chain fragment of a monoclonal antibody and the lectin concanavalin A, for monosaccharide ligands are reported and the results are shown to agree with values obtained using isothermal titration calorimetry.
- 7.Rademacher, C.; Shoemaker, G. K.; Kim, H.-S.; Zheng, R. B.; Taha, H.; Liu, C.; Nacario, R. C.; Schriemer, D. C.; Klassen, J. S.; Peters, T.; Lowary, T. L. Ligand Specificity of CS-35, a Monoclonal Antibody That Recognizes Mycobacterial Lipoarabinomannan. A Model System for Oligofuranoside-Protein Recognition. J. Am. Chem. Soc. 2007, 129, 10489–10502.CrossRefGoogle Scholar
- 8.Jorgensen, T. J. D.; Roepstorff, P.; Heck, A. J. R. Direct Determination of Solution Binding Constants for Noncovalent Complexes Between Bacterial Cell Wall Peptide Analogues and Vancomycin Group Antibiotics by Electrospray Ionization Mass Spectrometry. Anal. Chem. 1998, 70, 4427–4432.CrossRefGoogle Scholar
- 9.Jecklin, M. C.; Touboul, D.; Bovet, C.; Wortmann, A.; Zenobi, R. Which Electrospray-Based Ionization Method Best Reflects Protein-Ligand Interactions Found in Solution? A Comparison of ESI, nanoESI, and ESSI for the Determination of Dissociation Constants with Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19, 332–343.CrossRefGoogle Scholar