Higher-order structural interrogation of antibodies using middle-down hydrogen/deuterium exchange mass spectrometry
Bibliographic record
Abstract
Although X-ray crystallography is the "gold standard" method for protein higher-order structure analysis, the challenges of antibody crystallization and the time-consuming data analysis involved make this technique unsuitable for routine structural studies of antibodies. In addition, crystallography cannot be used for the structural characterization of an antibody in solution, under conditions where antibody drugs are active. Intact antibodies are also too large and too complex for NMR. Top-down mass spectrometry coupled to hydrogen/deuterium exchange (HDX) is a powerful tool for high-resolution protein structural characterization, but its success declines rapidly as protein size increases. Here we report for the first time a new hybrid "middle-down" HDX approach that overcomes this limitation through enabling the nonspecific enzyme pepsin to perform specific restricted digestion at low pH prior to HPLC separation at subzero temperatures and online electron transfer dissociation (ETD). Three large specific peptic fragments (12 to 25 kDa) were observed from the heavy chain and light chain of a therapeutic antibody Herceptin, together with a few smaller fragments from the middle portion of the heavy chain. The average amino-acid resolutions obtained by ETD were around two residues, with single-residue resolution in many regions. This middle-down approach is also applicable to other antibodies. It provided HDX information on the entire light chain, and 95.3% of the heavy chain, representing 96.8% of the entire antibody (150 kDa). The structural effects of glycosylation on Herceptin were determined at close-to-single residue level by this method.
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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 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 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".