Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry
- 360 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.
KeywordsLigand Complex Electrospray Ionization Mass Spectrometry Isothermal Titration Calorimetry Reference Ligand Single Chain Fragment
- 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