Gas-phase Structures of Biomolecular Ions

The way in which an analyte is ionized by ESI can depend on the distribution of ionizable groups within the analyte, its size and shape, the composition of the solvent used for ESI, and on whether other additives (e.g., so-called supercharging agents) are present. We have developed fluorescence-based methods to study the mechanisms of the ESI process, using solvatochromic fluorescent probes. Dispersed and time-resolved fluorescence measurements of the fluorescing species present inside the electrospray plume can provide snapshots of their chemical environments, including whether they are solvated or desolvated, their ionization state, and whether they have retained their solution-phase structures. These experiments promise to give not only fundamental insights into the ESI process but will render the ESI method a more versatile tool in analysis.

An important question in the biological mass spectrometry community is whether, and under what conditions, the ionized biological molecules inside the mass spectrometer resemble their states present in solution. While in the condensed phase one can obtain detailed NMR and X-ray crystallographic structural data, not a single technique exists that yields such high-resolution structural information in the gas phase. Our group is developing fluorescence-based structural probes to examine the structures of trapped gaseous biomolecular ions and their noncovalent complexes. Among the techniques available, FRET is the most intriguing, as it provides a way to extract nanometer-scale distance information in gaseous biomolecules. For these experiments, we use a quadrupole ion trap and a FT-ICR mass spectrometer, which have been modified in-house for fluorescence measurements. Ion mobility is also employed for conformational selection of analytes.

We recently reported the first evidence of transition metal ion FRET (tmFRET) in the gas phase and its application to measure short (10–40 Å) biomolecular backbone distances. The measured FRET efficiencies in rhodamine dye (donor) labeled helical peptides complexed with Cu²⁺ ions (acceptor) decreased with increasing donor - acceptor distances, confirming the occurrence of tmFRET. The distances estimated for similar peptide sequences from the FRET efficiencies were consistently longer in the gas phase compared to those reported in solution, indicating an expanded structure and a possible loss of helicity. Additionally, experiments are performed inside an ESI plume to probe the transition from solution phase to gas phase and its effects on the biomolecular structure.