Can we correlate ion mobility mass spectrometry data with native solution structures? A crosslinking approach

Introduction:
The structural characterization of biomolecules is of prime importance in the understanding of biological processes at the molecular level. In this context, the development of soft ionization techniques, namely electrospray ionization, and the advent of ion mobility (IM) separation tools open new perspectives for the analysis of large intact molecular ions by mass spectrometry (MS). Previous (IM-)MS experiments emphasized that the solution phase structures may be (partially) preserved in the gas phase under soft ionization conditions. However, several factors (e.g. coulombic interactions, accelerating voltages) have an influence on the conformation of biomolecular ions in the gas phase and should be carefully considered. Consequently, some fundamental questions remain elusive: “Can we correlate ion mobility mass spectrometry data to native solution structures?”
Methods:
To bring innovative answers to these longtime debated questions, we here propose an early stage study targeting constrained “native-like” conformations in the gas phase. To this end, we capitalize on a crosslinking approach to establish chemical links between lysine residues located in close spatial proximity within the protein. The so-created covalent network is here used as a scaffold to prevent the collapsing and unfolding of biomolecular ions once desolvated in the gas phase. These “frozen” native solution conformations are then interrogated using ion mobility as implemented on the commercial SYNAPT G2 HDMS spectrometer.
Preliminary data:
Practically, we used cytochrome c as a model system that we crosslinked with BS3 linkers. Using ion mobility, we monitored the evolution of the collision cross section (CCS) quantity, a rotationally averaged 2D projection of the ion conformation, for each charge state as function of the number of covalent intramolecular linkers. These data were readily compared with those obtained in similar conditions for the non-crosslinked cytochrome c as well as with a benchmark corresponding to the native solution conformations as resolved by NMR spectroscopy. Our results highlight that the crosslinked cytochrome c adopts more compact conformations in the gas phase, closer to values monitored for the native solution conformation, compared to its non-crosslinked homologue. In addition, we found that a critical number of intramolecular linkers were required to prevent structural unfolding from Coulomb repulsions.
Novel aspect:
Bring innovative answers to the longtime debated native MS questions based on an alternative application of intramolecular covalent linkers.