title: Characterizing protein structure in amorphous solids using hydrogen/deuterium exchange with mass spectrometry.
abstract: Elucidating protein structure in amorphous solids is central to the rational design of stable lyophilized protein drugs. Hydrogen/deuterium (H/D) exchange with electrospray ionization mass spectrometry was applied to lyophilized powders containing calmodulin (17 kDa) and exposed to D(2)O vapor at controlled relative humidity (RH) and temperature. H/D exchange was influenced by RH and by the inclusion of calcium chloride and/or trehalose in the solid. The effects were not exhibited uniformly along the protein backbone but occurred in a site-specific manner, with calcium primarily influencing the calcium-binding loops and trehalose primarily influencing the alpha-helices. The results demonstrate that the method can provide quantitative and site-specific structural information on proteins in amorphous solids and on changes in structure induced by protein cofactors and formulation excipients. Such information is not readily available with other techniques used to characterize proteins in the solid state, such as Fourier transform infrared, Raman, and near-infrared spectroscopy.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA.
authors.forename: Yunsong
authors.initials: Y
authors.lastname: Li
authors.forename: Todd D
authors.initials: TD
authors.lastname: Williams
authors.forename: Richard L
authors.initials: RL
authors.lastname: Schowen
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Topp
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium / Q000737:chemistry; D005612:Freeze Drying; D006859:Hydrogen / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D033362:Powder Diffraction; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D002147:Calmodulin; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D006859:Hydrogen; D003903:Deuterium