title: Influence of Sulfolane on ESI-MS Measurements of Protein-Ligand Affinities.
abstract: The results of an investigation into the influence of sulfolane, a commonly used supercharging agent, on electrospray ionization mass spectrometry (ESI-MS) measurements of protein-ligand affinities are described. Binding measurements carried out on four protein-carbohydrate complexes, lysozyme with β-D-GlcNAc-(1→4)-β-D-GlcNAc-(1→4)-β-D-GlcNAc-(1→4)-D-GlcNAc, a single chain variable fragment and α-D-Gal-(1→2)-[α-D-Abe-(1→3)]-α-D-Man-OCH3, cholera toxin B subunit homopentamer with β-D-Gal-(1→3)-β-D-GalNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal-(1→4)-β-D-Glc, and a fragment of galectin 3 and α-L-Fuc-(1→2)-β-D-Gal-(1→3)-β-D-GlcNAc-(1→3)-β-D-Gal-(1→4)-β-D-Glc, revealed that sulfolane generally reduces the apparent (as measured by ESI-MS) protein-ligand affinities. To establish the origin of this effect, a detailed study was undertaken using the lysozyme-tetrasaccharide interaction as a model system. Measurements carried out using isothermal titration calorimetry (ITC), circular dichroism, and nuclear magnetic resonance spectroscopies reveal that sulfolane reduces the binding affinity in solution but does not cause any significant change in the higher order structure of lysozyme or to the intermolecular interactions. These observations confirm that changes to the structure of lysozyme in bulk solution are not responsible for the supercharging effect induced by sulfolane. Moreover, the agreement between the ESI-MS and ITC-derived affinities indicates that there is no dissociation of the complex during ESI or in the gas phase (i.e., in-source dissociation). This finding suggests that supercharging of lysozyme by sulfolane is not related to protein unfolding during the ESI process. Binding measurements performed using liquid sample desorption ESI-MS revealed that protein supercharging with sulfolane can be achieved without a reduction in affinity.
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.
authors.forename: Yuyu
authors.initials: Y
authors.lastname: Yao
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.
authors.forename: Michele R
authors.initials: MR
authors.lastname: Richards
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada. john.klassen@ualberta.ca.
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D000818:Animals; D002645:Chickens; D002772:Cholera Toxin / Q000378:metabolism; D037502:Galectin 3 / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009113:Muramidase / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013876:Thiophenes / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D014734:Vibrio cholerae / Q000378:metabolism
chemical_list: D037502:Galectin 3; D008024:Ligands; D009844:Oligosaccharides; D011506:Proteins; D013876:Thiophenes; D025801:Ubiquitin; D002772:Cholera Toxin; D009113:Muramidase; C013693:sulfolane
keywords: ESI-MS; Liquid sample DESI-MS; Protein–ligand affinities; Supercharging