title: Gas-phase intramolecular protein crosslinking via ion/ion reactions: ubiquitin and a homobifunctional sulfo-NHS ester.
abstract: Gas-phase intra-molecular crosslinking of protein ubiquitin cations has been demonstrated via ion/ion reactions with anions of a homobifunctional N-hydroxysulfosuccinimide (sulfo-NHS) ester reagent. The ion/ion reaction between multiply-protonated ubiquitin and crosslinker monoanions produces a stable, charge-reduced complex. Covalent crosslinking is indicated by the consecutive loss of 2 molecules of sulfo-NHS under ion trap collisional activation conditions. Covalent modification is verified by the presence of covalently crosslinked sequence ions produced by ion-trap collision-induced dissociation of the ion generated from the losses of sulfo-NHS. Analysis of the crosslinked sequence fragments allows for the localization of crosslinked primary amines, enabling proximity mapping of the gas-phase 3-D structures. The presence of two unprotonated reactive sites within the distance constraint of the crosslinker is required for successful crosslinking. The ability to covalently crosslink is, therefore, sensitive to protein charge state. As the charge state increases, fewer reactive sites are available and protein structure is more likely to become extended because of intramolecular electrostatic repulsion. At high charge states, the reagent shows little evidence for covalent crosslinking but does show evidence for 'electrostatic crosslinking' in that the binding of the sulfonate groups to the protein is sufficiently strong that backbone cleavages are favored over reagent detachment under ion trap collisional activation conditions.
authors: Department of Chemistry, Purdue University, West Lafayette, IN 47907-2084, USA.
authors: Ian K
authors: IK
authors: Webb
authors: Marija
authors: M
authors: Mentinova
authors: William M
authors: WM
authors: McGee
authors: Scott A
authors: SA
authors: McLuckey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004912:Erythrocytes / Q000737:chemistry; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D040901:Proteomics; D055672:Static Electricity; D013388:Succinimides / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D007477:Ions; D013388:Succinimides; D025801:Ubiquitin; C035761:N-hydroxysulfosuccinimide