title: Affinity capture of biotinylated proteins at acidic conditions to facilitate hydrogen/deuterium exchange mass spectrometry analysis of multimeric protein complexes.
abstract: Characterization of conformational and dynamic changes associated with protein interactions can be done by hydrogen/deuterium exchange mass spectrometry (HDX-MS) by comparing the deuterium uptake in the bound and unbound state of the proteins. Investigation of local hydrogen/deuterium exchange in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads prior to the HDX-MS experiment. However, when studying protein complexes of more than two proteins, immobilization can possibly introduce steric limitations to the interactions. Here, we present a method based on the high affinity biotin-streptavidin interaction that allows selective capture of biotinylated proteins even under the extreme conditions for hydrogen/deuterium exchange quenching i.e. pH 2.5 and 0 °C. This biotin-streptavidin capture strategy allows hydrogen/deuterium exchange to occur in proteins in solution and enables characterization of specific proteins in heteromultimeric protein complexes without interference of peptides originating from other interaction partners in the complex. The biotin-streptavidin strategy has been successfully implemented in a model system with two recombinant monoclonal antibodies that target nonoverlapping epitopes on the human epidermal growth factor receptor (EGFR). We present a workflow for biotinylation and characterization of recombinant antibodies and demonstrate affinity capture of biotinylated antibodies under hydrogen/deuterium exchange quench conditions by the biotin-streptavidin strategy.
authors.affiliation: Symphogen A/S, Elektrovej Building 375, DK-2800 Lyngby, Denmark.
authors.forename: Pernille Foged
authors.initials: PF
authors.lastname: Jensen
authors.forename: Thomas J D
authors.initials: TJ
authors.lastname: Jørgensen
authors.forename: Klaus
authors.initials: K
authors.lastname: Koefoed
authors.forename: Frank
authors.initials: F
authors.lastname: Nygaard
authors.forename: Jette Wagtberg
authors.initials: JW
authors.lastname: Sen
mesh_terms: D000818:Animals; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D041961:Deuterium Exchange Measurement / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism; D013696:Temperature
chemical_list: D011506:Proteins; D001710:Biotin; D019809:Streptavidin