Accessing the higher order structures of biomolecules in solution from the gas phase ? The coupling of capillary electrophoresis and ion mobility mass spectrometry to the rescue

The fine characterization of samples requires identification and quantification of the content, the determination of the stoichiometry and connectivities (primary structures) and interaction domains (higher order structures) of (supra)molecular assembly. The elucidation of structure-activity relationships holds paramount significance in evaluating and comprehending the underlying processes involved in various applications such as therapeutic oligonucleotides (ONs), aptamers, functional foods, and bio-active compounds, including pre and probiotics. Mass spectrometry plays a pivotal role in the comprehensive analysis of organic samples, even at minute trace levels. The exploration of higher order structures relies on numerous analytical methods tailored for relatively pure and concentrated samples, typically utilizing spectroscopy methods in solution. However, the utilization of mass spectrometry (MS) and ion mobility mass spectrometry (IM-MS), both of which are vacuum-based methods, is increasingly important, primarily due to its fast screening capabilities for trace amounts in non-pure samples. It is highly tempting to extrapolate data obtained from mass spectrometry (MS) and ion mobility mass spectrometry (IM-MS) to deduce structural information in solution based on the assumptions of native mass spectrometry. However, it is imperative to validate these assumptions for each investigated model to ensure their applicability and accuracy. Nonetheless, there is still a way to circumvent these problems and create a shortcut. Here we propose the concomitant use of the different modes of Capillary Electrophoresis (CE) on-line coupled with IM-MS. CE is a separation method in solution presenting different operation modes which separates analytes according to the shapes, averaged charge states, and hydrodynamic radii (capillary zone electrophoresis, CZE), the relative electrophoretic mobilities (µe) in solution (transient isotachophoresis, t-ITP), or even on the affinity and binding constant of host-guest systems (kinetic capillary electrophoresis, KCE). Different models, e.g. peptides, naturally occurring and chemically modified oligonucleotides, and G-quadruplexes were investigated by CZE, t-ITP, and KCE coupled with ion mobility mass spectrometry, energy-resolved collision induced dissociation (breakdown curve and V50), and collision induced unfolding (CIU). Advantages and drawbacks of CE-IM-MS coupling will be addressed from the analytical point of view. The adequacy of parameters extracted from CE experiments and the ion mobility constant (K) obtained from IM-MS will be also addressed from the physical chemistry point of view.
Additionally, we also propose the used of matrix assisted laser desorption ionization and in-source decay (MALDI-ISD) to solve issues concerning the sequencing of (chemically modified) therapeutics ONs frequently observed using the classical collision induced dissociation (CID) fragmentation method. The use of thin layer chromatography (TLC) for the separation of these ONs in mixtures coupled to the use of MALDI-ISD and mass spectrometry imaging detection of these TLC plates is also proposed as innovative impurities profiling methods.