title: Decharging of globular proteins and protein complexes in electrospray.
abstract: Electrospray ionization mass spectrometry (ESI-MS) is a valuable tool in structural biology for investigating globular proteins and their biomolecular interactions. During the electrospray ionization process, proteins become desolvated and multiply charged, which may influence their structure. Reducing the net charge obtained during the electrospray process may be relevant for studying globular proteins. In this report we demonstrate the effect of a series of inorganic and organic gas-phase bases on the number of charges that proteins and protein complexes attain. Solution additives with very strong gas-phase basicities (GB) were identified among the so-called "proton sponges". The gas-phase proton affinities (PA) of the compounds that were added to the aqueous protein solutions ranged from 700 to 1050 kJ mol(-1). Circular dichroism studies showed that in these solutions the proteins retain their globular structures. The size of the proteins investigated ranged from the 14.3 kDa lysozyme up to the 800 kDa tetradecameric chaperone complex GroEL. Decharging of the proteins in the electrospray process by up to 60 % could be achieved by adding the most basic compounds rather than the more commonly used ammonium acetate additive. This decharging process probably results from proton competition events between the multiply protonated protein ions and the basic additives just prior to the final desolvation. We hypothesize that such globular protein species, which attain relatively few charges during the ionization event, obtain a gas-phase structure that more closely resembles their solution-phase structure. Thus, these basic additives can be useful in the study of the biologically relevant properties of globular proteins by using mass spectrometry.
authors.affiliation: Department of Biomolecular Mass Spectrometry, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
authors.forename: M Isabel
authors.initials: MI
authors.lastname: Catalina
authors.forename: Robert H H
authors.initials: RH
authors.lastname: van den Heuvel
authors.forename: Esther
authors.initials: E
authors.lastname: van Duijn
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry; D018834:Chaperonin 60 / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013329:Structure-Activity Relationship
chemical_list: D018834:Chaperonin 60; D011506:Proteins; D012996:Solutions; D000594:Amino Acid Oxidoreductases; C042233:glutamate synthase (ferredoxin); D009113:Muramidase