Ion-ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues.
Mass Spectra of charge states of folded proteins were obtained with nanospray and aqueous solution containing 20 microM the protein (ubiquitin, cytochrome c, lysozyme) and one of the NaA salts NaCl, NaI, NaAc (acetate) (1-10 mM). At very low collision activated decomposition (CAD), the mass spectra of a protein with charge z exhibited a replacement of zH+ with zNa+ and also multiple adducts of NaA. Higher CAD converts the NaA adduct peaks to Na minus H peaks. These must be due to loss of HA where the H was provided by the protein. The degree of HA loss with increasing CAD followed the order I < Cl < Ac. Significantly, the intensity of the ions with n (Na minus H) adducts showed a downward break past an n(MAX) which is equal to the number of acidic residues of the protein plus the charge of the protein. All the observations could be rationalized within the framework of the electrospray mechanism and the charge residue model, which predict that due to extensive evaporation of solvent, the solutes will reach very high concentrations in the final charged droplets. At such high concentrations, positive ions such as Na+, NH4+ form ion pairs with ionized acidic residues and the negative A- form ion pairs with ionized basic residues of the protein. Adducts of Na+, and NaA to backbone amide groups occur also. This reaction mechanism fits all the experimental observations and provides predictions that the number of acidic and basic groups at the surface of the gaseous protein that remain ionized can be controlled by the absence or presence of additives to the solution.
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Udo H
UH
Verkerk
Paul
P
Kebarle
D000085:Acetates; D019342:Acetic Acid; D024342:Amino Acids, Acidic / Q000032:analysis* / Q000737:chemistry; D024361:Amino Acids, Basic / Q000032:analysis* / Q000737:chemistry; D045304:Cytochromes c / Q000032:analysis / Q000737:chemistry; D007477:Ions; D009113:Muramidase / Q000032:analysis / Q000737:chemistry; D010316:Particle Size; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D019346:Sodium Acetate; D012965:Sodium Chloride; D012974:Sodium Iodide; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D025801:Ubiquitin / Q000032:analysis / Q000737:chemistry
D000085:Acetates; D024342:Amino Acids, Acidic; D024361:Amino Acids, Basic; D007477:Ions; D025801:Ubiquitin; D012965:Sodium Chloride; D019346:Sodium Acetate; D045304:Cytochromes c; D009113:Muramidase; D012974:Sodium Iodide; D019342:Acetic Acid; C018824:ammonium acetate