Subzero temperature chromatography and top-down mass spectrometry for protein higher-order structure characterization: method validation and application to therapeutic antibodies

Jingxi Pan, Suping Zhang, Carol E. Parker, Christoph H. Borchers

Research output: Contribution to journalArticlepeer-review

33 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)13065-13071
Number of pages7
JournalJournal of the American Chemical Society
Volume136
Issue number37
DOIs
Publication statusPublished - 17 Sep 2014
Externally publishedYes

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