title: Separation and characterisation of beta2-microglobulin folding conformers by ion-exchange liquid chromatography and ion-exchange liquid chromatography-mass spectrometry.
abstract: In this work we present for the first time the use of ion-exchange liquid chromatography to separate the native form and a partially structured intermediate of the folding of the amyloidogenic protein beta2-microglobulin. Using a strong anion-exchange column that accounts for the differences in charge exposure of the two conformers, a LC-UV method is initially optimised in terms of mobile phase pH, composition and temperature. The preferred mobile phase conditions that afford useful information were found to be 35 mM ammonium formate, pH 7.4 at 25°C. The dynamic equilibrium of the two species is demonstrated upon increasing the concentration of acetonitrile in the protein sample. Then, the chromatographic method is transferred to MS detection and the respective charge state distributions of the separated conformers are identified. The LC-MS results demonstrate that one of the conformers is partially unfolded, compared with the native and more compact species. The correspondence with previous results obtained in free solution by capillary electrophoresis suggest that strong ion exchange LC-MS does not alter beta2-microglobulin conformation and maintains the dynamic equilibrium already observed between the native protein and its folding intermediate.
authors.affiliation: Department of Drug Sciences, University of Pavia, Pavia, Italy. laura.bertoletti@unipv.it
authors.forename: Laura
authors.initials: L
authors.lastname: Bertoletti
authors.forename: Luca
authors.initials: L
authors.lastname: Regazzoni
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Aldini
authors.forename: Raffaella
authors.initials: R
authors.lastname: Colombo
authors.forename: Franco
authors.initials: F
authors.lastname: Abballe
authors.forename: Gabriele
authors.initials: G
authors.lastname: Caccialanza
authors.forename: Ersilia
authors.initials: E
authors.lastname: De Lorenzi
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D011487:Protein Conformation; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D001613:beta 2-Microglobulin