Nonspecific Protein-Carbohydrate Complexes Produced by Nanoelectrospray Ionization. Factors Influencing Their Formation and Stability

摘要

Factors influencing the formation of nonspecific proteincarbohydrate 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 carbohydfates, ranging in size from mono- to tetrssaccharide, 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, nonspecilic binding is favored for small (mono- and disaccharide) or hydrophilic carbohydrates over larger or more hydrophobic molecules, which t6nd 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 idenuned. Taken together, the results of this study subest that neutral protein-carbohydrate nit6rachons (e.g., hydrogen bonds) contribute significantly and, perhaps, predominantly to the formation and stabilization of the nonspecific complexes. A strstegy 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., CaCl_2) to the nanoES solution, is demonstrsted.