Quantifying labile protein-ligand interactions using electrospray ionization mass spectrometry.
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 (L(ref)), 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 L(ref), 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.
El-Hawiet
Amr
A
Alberta Ingenuity Centre for Carbohydrate Science, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Kitova
Elena N
EN
Liu
Lan
L
Klassen
John S
JS
D000465:Algorithms; D003208:Concanavalin A / Q000737:chemistry / Q000378:metabolism*; D005740:Gases / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D009005:Monosaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D005740:Gases; D008024:Ligands; D009005:Monosaccharides; D057127:Single-Chain Antibodies; D003208:Concanavalin A
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