title: Investigating Therapeutic Protein Structure with Diethylpyrocarbonate Labeling and Mass Spectrometry.
abstract: Protein therapeutics are rapidly transforming the pharmaceutical industry. Unlike for small molecule therapeutics, current technologies are challenged to provide the rapid, high-resolution analyses of protein higher order structures needed to ensure drug efficacy and safety. Consequently, significant attention has turned to developing new methods that can quickly, accurately, and reproducibly characterize the three-dimensional structure of protein therapeutics. In this work, we describe a method that uses diethylpyrocarbonate (DEPC) labeling and mass spectrometry to detect three-dimensional structural changes in therapeutic proteins that have been exposed to degrading conditions. Using β2-microglobulin, immunoglobulin G1, and human growth hormone as model systems, we demonstrate that DEPC labeling can identify both specific protein regions that mediate aggregation and those regions that undergo more subtle structural changes upon mishandling of these proteins. Importantly, DEPC labeling is able to provide information for up to 30% of the surface residues in a given protein, thereby providing excellent structural resolution. Given the simplicity of the DEPC labeling chemistry and the relatively straightforward mass spectral analysis of DEPC-labeled proteins, we expect this method should be amenable to a wide range of protein therapeutics and their different formulations.
authors.affiliation: Department of Chemistry, University of Massachusetts , Amherst, Massachusetts 01003, United States.
authors.forename: Nicholas B
authors.initials: NB
authors.lastname: Borotto
authors.affiliation: Department of Chemistry, University of Massachusetts , Amherst, Massachusetts 01003, United States.
authors.forename: Yuping
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University , Bloomington, Indiana 47405, United States.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hollingsworth
authors.affiliation: QuarryBio , Bloomington, Indiana 47404, United States.
authors.forename: John E
authors.initials: JE
authors.lastname: Hale
authors.affiliation: QuarryBio , Bloomington, Indiana 47404, United States.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Graban
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University , Bloomington, Indiana 47405, United States.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Vaughan
authors.affiliation: Department of Chemistry, University of Massachusetts , Amherst, Massachusetts 01003, United States.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Vachet
mesh_terms: D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D013006:Growth Hormone / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D001613:beta 2-Microglobulin / Q000737:chemistry*
chemical_list: D007074:Immunoglobulin G; D001613:beta 2-Microglobulin; D013006:Growth Hormone; D004047:Diethyl Pyrocarbonate