Dual‐spray hydrogen/deuterium exchange (HDX) reactions: A new method of probing protein structure
Bibliographic record
Abstract
RATIONALE: Traditionally, hydrogen/deuterium exchange (HDX) reactions are done in the solution phase. This usually involves incubating the protein with a suitable deuterating agent then acidifying the solution to quench the reaction. A more efficient method may be to conduct the reaction within the ion source of a mass spectrometer and subsequently analyze the products. METHODS: Using the two electrospray emitters equipped on the Waters Synapt G1 mass spectrometer, HDX reactions were conducted within the ion source region in a controlled fashion ('dual-spray'). Peptide and protein solutions were electrosprayed through one emitter and the deuterating agent D2 O through the secondary electrospray emitter. For the relatively small peptides, Phe-Leu-Glu-Glu-Leu and oxytocin, the yield of products was calculated using deconvolution functions. Electrospray ionization (ESI) charge-state distributions and average number of deuterium exchanges were used to probe secondary and tertiary structures of ubiquitin, lysozyme, and cytochrome c in their native and unfolded states. RESULTS: Clear shifts in isotope distributions indicated HDX occurring within the ion source. By ion mobility, simultaneous deuterium exchange for two isobaric species, the oxytocin monomer and dimer, was observed. For denatured ubiquitin, the 12+ and 13+ charge states have a lower average number of exchanges relative to the lower charge states which indicates that these charge states have segments which restrict the access of D2 O. Lysozyme has a linear relationship between the charge state and the average number of exchanges, indicating that lysozyme becomes increasingly unfolded as the charge state increases. The dual-spray HDX method was paired to high-performance liquid chromatography (HPLC) to demonstrate the applicability of the technique for probing gas-phase structures in protein mixtures. CONCLUSIONS: ESI droplets formed from a secondary emitter penetrate primary ESI droplets and change the solvent composition. Dual-spray HDX is demonstrated to be a more efficient method for probing the structure of proteins than solution-phase HDX since the acid quenching step can be surpassed. Copyright © 2016 John Wiley & Sons, Ltd.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".