Subzero Temperature Chromatography and Top-Down Mass Spectrometry for Protein Higher-Order Structure Characterization: Method Validation and Application to Therapeutic Antibodies
Bibliographic record
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.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".