Surrogate H/D Detection Strategy for Protein Conformational Analysis Using MS/MS Data
Bibliographic record
Abstract
Amide hydrogen/deuterium exchange (H/DX) measurements by mass spectrometry provide a powerful tool for probing the structure and dynamics of proteins. In order to extend such measurements to complex multiprotein systems, new methods for delivering higher sensitivity and sequence coverage are required. In this study, we investigated the utility of tandem mass spectrometry (MS/MS) for providing an alternative to the conventional MS mode of operation applied to bottom-up H/DX experiments. Specifically, we aimed to determine whether differential deuteration measurements of collisionally induced dissociation (CID)-generated product ions can serve as effective surrogates for their corresponding intact peptide, thus providing an additional dimension for analysis. Replicate deuterium measurements of calmodulin (in its apo and holo forms) were obtained from peptic digests in both the MS and MS/MS domains and studied as a function of % deuteration, fragment ion selection, and contaminant level. We show that successful acquisition of MS/MS data for deuterated peptides requires controlled expansion of the isotopic envelope by limiting the range of deuterium label applied in the exchange-in reaction (< or = 50%) and that automation of ion selection via data-dependent acquisition is ultimately dependent upon peak detection algorithms. Upon full transmission of the isotopic envelope, fragment data demonstrate that all ions, with the exception of neutral loss fragment ions, return deuteration ratios reflecting the apo/holo transition that are in general agreement with values obtained from the corresponding precursor ions. The agreement is limited primarily by the ion statistics for each fragment, as the base peak in the MS/MS spectra provided the best correlation regardless of its m/z. We highlight that the freedom to select the base peak as a surrogate for the precursor ion derives from extensive H/D scrambling inducible under conventional CID conditions. When spectral interference prohibits conventional H/DX-MS measurements, we further show that the surrogate approach recovers accurate and precise per-peptide deuteration levels. Thus, a generalized strategy is presented in which CID-based automated H/DX-MS/MS acquisition can be used to extend measurements to complex protein systems, exceeding the peptide capacity of conventional H/DX-MS alone.
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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) | 0.004 | 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".