Protomers of the green and cyan fluorescent protein chromophores investigated using action spectroscopy.

The photophysics of biochromophore ions often depends on the isomeric or protomeric distribution, yet this distribution, and the individual isomer contributions to an action spectrum, can be difficult to quantify. Here, we use two separate photodissociation action spectroscopy instruments to record electronic spectra for protonated forms of the green (pHBDI+) and cyan (Cyan+) fluorescent protein chromophores. One instrument allows for cryogenic (T = 40 ± 10 K) cooling of the ions, while the other offers the ability to perform protomer-selective photodissociation spectroscopy. We show that both chromophores are generated as two protomers when using electrospray ionisation, and that the protomers have partially overlapping absorption profiles associated with the S1 ← S0 transition. The action spectra for both species span the 340-460 nm range, although the spectral onset for the pHBDI+ protomer with the proton residing on the carbonyl oxygen is red-shifted by ≈40 nm relative to the lower-energy imine protomer. Similarly, the imine and carbonyl protomers are the lowest energy forms of Cyan+, with the main band for the carbonyl protomer red-shifted by ≈60 nm relative to the lower-energy imine protomer. The present strategy for investigating protomers can be applied to a wide range of other biochromophore ions.

First-Principles Modeling of Preferential Solvation in Mixed-Modifier Differential Mobility Spectrometry.

Differential mobility spectrometry (DMS) separates ions based on mobility differences between high and low electric field conditions. To enhance resolution, solvents such as water and acetonitrile are often used to modify the collision environment and take advantage of differing dynamic clustering behavior between analytes that coelute in hard-sphere environments (e.g., N2). When binary solvent mixtures are used to modify the DMS environment, one solvent can have a dominant influence over the other with respect to ion trajectories. For example, for quinoline derivatives, a 9:1 water:acetonitrile solvent mixture exhibited identical behavior to an environment containing only acetonitrile as a modifier. It was hypothesized that this effect arises due to the significantly different binding strengths of the two solvents. Here, we utilize a first-principles model of DMS to study analytes in single and binary solvent mixtures and explore the effects governing the dominance of one solvent over the other. Computed DMS dispersion curves of quinoline derivatives are in excellent agreement with those measured experimentally. For mixed-modifier environments, the predicted cluster populations show a clear preferential solvation of ions with the stronger binding solvent. The influence of ion-solvent binding energies, solvent concentration, and solvent molecule size is discussed in the context of the observed DMS behavior. This work can guide the usage of binary solvent mixtures for improving ion separations in cases where compounds coelute in pure N2 and in single-solvent modifier environments. Moreover, our results indicate that binary solvent mixtures can be used to create a relative scale for solvent binding energies.