title: Evaluation of capillary electrophoresis-mass spectrometry for the analysis of the conformational heterogeneity of intact proteins using beta2-microglobulin as model compound.
abstract: In this work we explored the feasibility of different CE-ESI-MS set-ups for the analysis of conformational states of an intact protein. By using the same background electrolyte at quasi physiological conditions (50 mM ammonium bicarbonate, pH 7.4) a sequential optimization was carried out, initially by evaluating a sheath-liquid interface with both a single quadrupole (SQ) and a time-of-flight (TOF) mass spectrometer; then a sheathless interface coupled with high-resolution QTOF MS was considered. Beta2-microglobulin has been taken as a model, as it is an amyloidogenic protein and its conformational changes are strictly connected to the onset of a disease. The separation of two conformers at dynamic equilibrium is achieved all the way down to the MS detection. Notably, the equilibrium ratio of the protein conformers is maintained in the electrospray source after CE separation. Strengths and weaknesses of each optimized set-up are emphasized and their feasibility in unfolding studies is evaluated. In particular, ESI-TOF MS can assign protein forms that differ by 1 Da only and sheathless interfacing is best suited to preserve protein structure integrity. This demonstrates the CE-ESI-MS performance in terms of separation, detection and characterization of conformational species that co-populate a protein solution.
authors.affiliation: Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100, Pavia, Italy. Electronic address: laura.bertoletti@unipv.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Bertoletti
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU, Rue Michel Servet 1, 1211, Geneva 4, Switzerland. Electronic address: julie.schappler@unige.ch.
authors.forename: Julie
authors.initials: J
authors.lastname: Schappler
authors.affiliation: Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100, Pavia, Italy. Electronic address: raffaella.colombo@unipv.it.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Colombo
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU, Rue Michel Servet 1, 1211, Geneva 4, Switzerland. Electronic address: serge.rudaz@unige.ch.
authors.forename: Serge
authors.initials: S
authors.lastname: Rudaz
authors.affiliation: Division of BioAnalytical Chemistry, AIMMS Research Group BioMolecular Analysis, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands. Electronic address: r.haselberg@vu.nl.
authors.forename: Rob
authors.initials: R
authors.lastname: Haselberg
authors.affiliation: Division of BioAnalytical Chemistry, AIMMS Research Group BioMolecular Analysis, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands. Electronic address: e.dominguezvega@vu.nl.
authors.forename: Elena
authors.initials: E
authors.lastname: Domínguez-Vega
authors.affiliation: Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, Viale Taramelli 3b, 27100, Pavia, Italy. Electronic address: sara.raimondi@unipv.it.
authors.forename: Sara
authors.initials: S
authors.lastname: Raimondi
authors.affiliation: Division of BioAnalytical Chemistry, AIMMS Research Group BioMolecular Analysis, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands. Electronic address: g.w.somsen@vu.nl.
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
authors.affiliation: Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100, Pavia, Italy. Electronic address: ersidelo@unipv.it.
authors.forename: Ersilia
authors.initials: E
authors.lastname: De Lorenzi
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D008956:Models, Chemical; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D001613:beta 2-Microglobulin / Q000032:analysis*
chemical_list: D001613:beta 2-Microglobulin
keywords: Amyloidosis; Beta(2)-microglobulin; Capillary electrophoresis-mass spectrometry; Folding; Protein conformation