Method for Stabilizing Protein−Ligand Complexes in Nanoelectrospray Ionization Mass Spectrometry
Bibliographic record
Abstract
The interaction between the bovine pancrease trypsin (Tryp) and its competitive inhibitor benzamidine (1), in solution and the gas phase, is investigated using nanoflow electrospray ionization (nanoES) and Fourier transform ion cyclotron resonance mass spectrometry. In a recent study (Clark, S.M.; Konermann L. Anal. Chem. 2004, 76, 7077-7083), it was reported that the (Tryp + 1) complex could not be detected by ES-MS. Here, it is shown that, with gentle sampling conditions, it is possible to detect gaseous protonated ions of the (Tryp + 1) complex with nanoES-MS. However, the relative abundance of the detected (Tryp + 1)n+ ions is lower than expected, based on solution composition, which suggests that dissociation of (Tryp + 1)n+ ions occurs during MS sampling. The dissociation pathways and corresponding Arrhenius parameters for the protonated (Tryp + 1)n+ ions, at n = 7-9, are determined from time-resolved thermal dissociation experiments, implemented with the blackbody infrared radiative dissociation technique. The gaseous (Tryp + 1)n+ ions are found to have short lifetimes, e.g., <0.6 s, at temperatures of >100 degrees C. The use of solution additives, including polyols, carbohydrates, amino acids, and small organic molecules, to stabilize the (Tryp + 1)n+ ions during nanoES-MS analysis is investigated. Notably, the addition of imidazole to the nanoES solution is shown to preserve the (Tryp + 1)n+ ions. The Kassoc value, (1.9 +/- 0.2) x 104 M-1, determined for the (Tryp + 1) complex by the direct ES-MS method is in agreement with values determined by other analytical methods. The stabilizing effect of imidazole in nanoES-MS is further demonstrated for the interaction between carbonic anhydrase II and 5-(dimethylamino)naphthalene-1-sulfonamide. The stabilizing effect of imidazole may be due to enhanced evaporative cooling achieved by the dissociation of molecules of imidazole, bound nonspecifically, from the protein-ligand complex in the ion source.
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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) | 0.002 | 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".