Events taking place during electrospray ionization (ESI) can trigger the self-assembly of various nanoclusters. These products are often dominated by magic number clusters (MNCs) that have highly symmetrical structures. The literature rationalizes the dominance of MNCs by noting their high stability. However, this argument is not necessarily adequate because thermodynamics cannot predict the outcome of kinetically controlled reactions. Thus, the mechanisms responsible for MNC dominance remain poorly understood. Molecular dynamics (MD) simulations can provide atomistic insights into self-assembly reactions, but even this approach has thus far failed to provide pertinent answers. The current work overcomes this limitation. We focused on salt clusters formed from aqueous NaCl solutions during ESI. The corresponding mass spectra are dominated by the Na 14 Cl 13 + MNC. Simulations of ESI droplets showed nonspecific association of Na + and Cl − , culminating in gaseous clusters via solvent evaporation to dryness (charged residue mechanism). These nascent clusters did not show any preference for MNCs. In mass spectrometry experiments, analyte ions undergo in-source activation prior to detection. We emulated in-source activation by heating nascent clusters in our MD runs. Heating triggered structural fluctuations and dissociation events, generating MNC-dominated product distributions. Why are MNCs preferred after in-source activation? Thermally excited clusters frequently adopt structures consisting of a preformed MNC and a stringlike protrusion that contains the surplus ions. Facile separation of these protrusions releases the MNC (Cluster hot → MNC-protrusion → MNC + protrusion). This work marks the first time that MD simulations were able to capture cluster self-assembly with subsequent "molecular pruning", generating MNC-dominated product distributions that agree with experiments.
Mass spectrometry (MS)-based techniques are widely used for probing protein structure and dynamics in solution. H/D exchange (HDX)-MS is one of the most common approaches in this context. HDX is often considered to be a "benign" labeling method, in that it does not perturb protein behavior in solution. However, several studies have reported that D 2 O pushes unfolding equilibria toward the native state. The origin, and even the existence of this protein stabilization remain controversial. Here we conducted thermal unfolding assays in solution to confirm that deuterated proteins in D 2 O are more stable, with 2−4 K higher melting temperatures than unlabeled proteins in H 2 O. Previous studies tentatively attributed this phenomenon to strengthened H-bonds after deuteration, an effect that may arise from the lower zero-point vibrational energy of the deuterated species. Specifically, it was proposed that strengthened water−water bonds (W•••W) in D 2 O lower the solubility of nonpolar side chains. The current work takes a broader view by noting that protein stability in solution also depends on water− protein (W•••P) and protein−protein (P•••P) H-bonds. To help unravel these contributions, we performed collision-induced unfolding (CIU) experiments on gaseous proteins generated by native electrospray ionization. CIU profiles of deuterated and unlabeled proteins were indistinguishable, implying that P•••P contacts are insensitive to deuteration. Thus, protein stabilization in D 2 O is attributable to solvent effects, rather than alterations of intraprotein H-bonds. Strengthening of W•••W contacts represents one possible explanation, but the stabilizing effect of D 2 O can also originate from weakened W•••P bonds. Future work will be required to elucidate which of these two scenarios is correct, or if both contribute to protein stabilization in D 2 O. In any case, the often-repeated adage that "D-bonds are more stable than H-bonds" does not apply to intramolecular contacts in native proteins.
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