title: Subzero temperature chromatography and top-down mass spectrometry for protein higher-order structure characterization: method validation and application to therapeutic antibodies.
abstract: Characterization of the higher-order structure and structural dynamics of proteins is crucial for in-depth understanding of their functions. Amide hydrogen/deuterium exchange (HDX), monitored by mass spectrometry (MS), is now a popular technique for measuring protein higher-order structural changes. Although the proteolysis-based HDX-MS approach is most commonly used, the "top-down" approach, which fragments intact proteins directly using electron-based dissociation, is becoming an important alternative and has several advantages. However, the commonly used top-down strategies are direct-infusion based and thus can only be used with volatile buffers. This has meant that the "top-down" approach could not be used for studying proteins under physiological conditions-the very conditions which are often very important for preserving a protein's native structure and function. More complex proteins such as those with disulfide bonds present another challenge. Therefore, there is significant interest in developing novel top-down HDX methods that are applicable to all types of protein samples. In this paper, we show how top-down electron capture dissociation and subzero temperature HPLC can be combined and used for this purpose. This method keeps the back-exchange level as low as 2% and has no limitations in terms of protein type and sample solution conditions. Close to single-residue level protein structural information can be generated. The new method is validated through comparison with NMR data using calmodulin as a model protein. Its capability of determining structural changes in therapeutic antibodies (Herceptin) is also demonstrated.
authors.affiliation: University of Victoria-Genome British Columbia Proteomics Centre , Vancouver Island Technology Park, 3101-4464 Markham St., Victoria, British Columbia V8Z 7X8, Canada.
authors.forename: Jingxi
authors.initials: J
authors.lastname: Pan
authors.forename: Suping
authors.initials: S
authors.lastname: Zhang
authors.forename: Carol E
authors.initials: CE
authors.lastname: Parker
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D000577:Amides / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003080:Cold Temperature; D041961:Deuterium Exchange Measurement / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D000068878:Trastuzumab
chemical_list: D000577:Amides; D061067:Antibodies, Monoclonal, Humanized; D002147:Calmodulin; D011994:Recombinant Proteins; D000068878:Trastuzumab