Subzero Temperature Chromatography and Top-Down Mass Spectrometry for Protein Higher-Order Structure Characterization: Method Validation and Application to Therapeutic Antibodies
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- 作者：Jingxi Pan ; Suping Zhang ; Carol E. Parker ; Christoph H. Borchers
- 刊名：Journal of the American Chemical Society
- 出版年：2014
- 出版时间：September 17, 2014
- 年：2014
- 卷：136
- 期：37
- 页码：13065-13071
- 全文大小：448K
- ISSN：1520-5126
文摘
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 鈥渢op-down鈥?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 鈥渢op-down鈥?approach could not be used for studying proteins under physiological conditions鈥攖he very conditions which are often very important for preserving a protein鈥檚 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.