title: Electrospray ionization-induced protein unfolding.
abstract: Electrospray ionization mass spectrometry (ESI-MS) measurements were performed under a variety of solution conditions on a highly acidic sub-fragment (B3C) of the C-terminal carbohydrate-binding repeat region of Clostridium difficile toxin B, and two mutants (B4A and B4B) containing fewer acidic residues. ESI-MS measurements performed in negative ion mode on aqueous ammonium acetate solutions of B3C at low ionic strength (I < 80 mM) revealed evidence, based on the measured charge state distribution, of protein unfolding. In contrast, no evidence of unfolding was detected from ESI-MS measurements made in positive ion mode at low I or in either mode at higher I. The results of proton nuclear magnetic resonance and circular dichroism spectroscopy measurements and gel filtration chromatography performed on solutions of B3C under low and high I conditions suggest that the protein exists predominantly in a folded state in neutral aqueous solutions with I > 10 mM. The results of ESI-MS measurements performed on B3C in a series of solutions with high I at pH 5 to 9 rule out the possibility that the structural changes are related to ESI-induced changes in pH. It is proposed that unfolding of B3C, observed in negative mode for solutions with low I, occurs during the ESI process and arises due to Coulombic repulsion between the negatively charged residues and liquid/droplet surface charge. ESI-MS measurements performed in negative ion mode on B4A and B4B also reveal a shift to higher charge states at low I but the magnitude of the changes are smaller than observed for B3C.
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2.
authors.forename: Hong
authors.initials: H
authors.lastname: Lin
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Johnson
authors.forename: Luiz
authors.initials: L
authors.lastname: Eugenio
authors.forename: Kenneth K S
authors.initials: KK
authors.lastname: Ng
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D000085:Acetates / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D000085:Acetates; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D011506:Proteins; D011994:Recombinant Proteins; C031815:toxB protein, Clostridium difficile; C018824:ammonium acetate