title: Nonspecific protein-carbohydrate complexes produced by nanoelectrospray ionization. Factors influencing their formation and stability.
abstract: Factors influencing the formation of nonspecific protein-carbohydrate complexes during the nanoelectrospray (nanoES) process have been investigated. Protonated and deprotonated nonspecific complexes of ubiquitin (Ubq) and protonated complexes of carbonic anhydrase (CA) with carbohydrates, ranging in size from mono- to tetrasaccharide, were produced by nanoES and detected with a Fourier transform ion cyclotron resonance mass spectrometer. Both the fraction of protein engaged in nonspecific binding with the carbohydrates and the number of carbohydrates bound to the protein increase with increasing carbohydrate concentration. At a given concentration of protein and carbohydrate, nonspecific binding is favored for small (mono- and disaccharide) or hydrophilic carbohydrates over larger or more hydrophobic molecules, which tend to form gaseous monomer or cluster ions by nanoES. However, the extent of nonspecific binding is insensitive to the structure of the protein, with similar distributions of nonspecific complexes observed for both CA and Ubq. Nonspecific association is also insensitive to the charge state of the complex. A comparable degree of binding is observed for complexes in their protonated and deprotonated forms. Furthermore, the number of bound ligands can exceed significantly the charge state of the complex. Thermal dissociation experiments performed on the gaseous nonspecific complexes reveal that their kinetic stability is sensitive to both the structure of the carbohydrate (i.e., mono- < di- < tri- < tetrasaccharide) and the protein (Ubq < CA) and to the charge state, although no simple relationship between stability and charge state was identified. Taken together, the results of this study suggest that neutral protein-carbohydrate interactions (e.g., hydrogen bonds) contribute significantly and, perhaps, predominantly to the formation and stabilization of the nonspecific complexes. A strategy to minimize the formation of the nonspecific complexes, which is based on the enhancement of gaseous carbohydrate ion formation through the addition of metal salts (e.g., CaCl2) to the nanoES solution, is demonstrated.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors.forename: Weijie
authors.initials: W
authors.lastname: Wang
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D002241:Carbohydrates / Q000032:analysis / Q000737:chemistry*; D002256:Carbonic Anhydrases / Q000032:analysis / Q000737:chemistry; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D036103:Nanotechnology / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D011522:Protons; D012492:Salts / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013696:Temperature; D025801:Ubiquitin / Q000032:analysis / Q000737:chemistry
chemical_list: D002241:Carbohydrates; D011506:Proteins; D011522:Protons; D012492:Salts; D025801:Ubiquitin; D002256:Carbonic Anhydrases