Irreversible thermal denaturation of cytochrome <i>c</i> studied by electrospray mass spectrometry
Bibliographic record
Abstract
This work uses electrospray ionization mass spectrometry (ESI-MS) in conjunction with hydrogen/deuterium exchange (HDX) and optical spectroscopy for characterizing the solution-phase properties of cytochrome c (cyt c) after heat exposure. Previous work demonstrated that heating results in irreversible denaturation for a subpopulation of proteins in the sample. However, that study did not investigate the physical reasons underlying this interesting effect. Here we report that the formation of oxidative modifications at elevated temperature plays a key role for the observed behavior. Tryptic digestion followed by tandem mass spectrometry is used to identify individual oxidation sites. Trp59 and Met80 are among the modified amino acids. In native cyt c both of these residues are buried deep within the protein structure, such that covalent modifications would be expected to be particularly disruptive. ESI-MS analysis after heat exposure results in a bimodal charge-state distribution. Oxidized protein appears predominantly in charge states around 11+, whereas a considerably lower degree of oxidation is observed for the 7+ and 8+ peaks. This finding confirms that different oxidation levels are associated with different solution-phase conformations. HDX measurements for different charge states are complicated by peak distortions arising from oxygen adduction. Nonetheless, comparison with simulated peak shapes clearly shows that the HDX properties are different for high- and low-charge states, confirming that interconversion between unfolded and folded conformers is blocked in solution. In addition to oxidation, partial aggregation upon heat exposure likely contributes to the formation of irreversibly denatured protein.
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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 | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.002 |
| Bibliometrics | 0.000 | 0.002 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| 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".