title: Quantifying ligand binding to large protein complexes using electrospray ionization mass spectrometry.
abstract: An electrospray ionization mass spectrometry (ESI-MS) method for quantifying protein-ligand complexes that cannot be directly detected by ESI-MS is described. The proxy protein ESI-MS method combines direct ESI-MS binding measurements with competitive protein-ligand binding. To implement the method, a proxy protein (P(proxy)), which interacts specifically with the ligand of interest with known affinity and can be detected directly by ESI-MS, is used to quantitatively monitor the extent of ligand binding to the protein of interest. A mathematical framework for establishing the association constant (K(a)) for protein-ligand binding by the proxy protein ESI-MS method, implemented with a P(proxy) containing a single ligand binding site, is given. A modified form of the proxy protein ESI-MS method, which accounts for real-time changes in ligand concentration, is also described. The reliability of these methods is demonstrated for the interactions between the 180 kDa wildtype homotrimeric tailspike protein of the bacteriophage P22 and its endorhamnosidase point mutant (D392N) with its ligands comprising two and three O-antigen repeats from Salmonella enterica serovar Typhimurium: octasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](2)) and dodecasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](3)). A 27 kDa single chain antibody, which binds to both ligands, served as P(proxy). The results of binding measurements performed at 10 and 25 °C are in excellent agreement with K(a) values measured previously using a fluorescence quenching assay.
authors.forename: Amr
authors.initials: A
authors.lastname: El-Hawiet
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.forename: Denis
authors.initials: D
authors.lastname: Arutyunov
authors.forename: David J
authors.initials: DJ
authors.lastname: Simpson
authors.forename: Christine M
authors.initials: CM
authors.lastname: Szymanski
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D017100:Bacteriophage P22 / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019081:O Antigens / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D019779:Salmonella enterica / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D019081:O Antigens; D014764:Viral Proteins; D006026:Glycoside Hydrolases; C041312:endorhamnosidase