Uncovering the Pathway of Serine Octamer Magic Number Cluster Formation during Electrospray Ionization: Experiments and Simulations

Electrospray ionization (ESI) of serine (Ser) solution generates Ser₈H⁺ as an abundant magic number cluster. ESI clustering of most other solutes yields nonspecific stoichiometries. It is unclear why Ser₈H⁺ dominates in the case of Ser, and how Ser₈H⁺ forms during ESI. Even the location of Ser₈H⁺ formation is contentious (in solution, in ESI droplets, or elsewhere). Here we unravel key aspects of the l-Ser₈H⁺ formation pathway. Harsh ion sampling conditions promote the collision-induced dissociation (CID) of regular ESI analytes. Unexpectedly, Ser₈H⁺ was seemingly resistant against CID during ion sampling, despite its extremely low tandem mass spectrometry (MS/MS) stability. This unusual behavior reveals that Ser₈H⁺ forms during ion sampling. We propose the following pathway: (1) Nonspecific Ser clusters are released when ESI droplets evaporate to dryness. These initial clusters cover a wide size range, from a few Ser to hundreds or thousands of monomers. (2) The clusters undergo dissociation during ion sampling, mostly via successive loss of neutral monomers. For any source activation voltage, there is a subpopulation of clusters for which this CID cascade tends to terminate at the octamer level, culminating in Ser₈H⁺-dominated product distributions. Mobile proton molecular dynamics simulations were used to model the entire pathway. Ser₈H⁺ structures formed in these simulations were consistent with ion mobility experiments. The most compact structures resembled the model of [Scutelnic, V. <i>J. Am. Chem. Soc.</i> 2018, 140, 7554-7560], with numerous intermolecular salt bridges and H-bonds. Our findings illustrate how the interplay of association and dissociation reactions across phase boundaries can culminate in magic number clusters.