Fluorescence-Based Detection of the Desolvation Process of Protein Ions Generated in an Aqueous Electrospray Plume

Abstract

A new experimental setup to study laser-induced fluorescence (LIF) from analytes at different locations in an electrospray plume has been developed. The high fluorescence collection efficiency (~2%) of the setup, along with a sensitive CCD detector, enabled the study of low ion concentrations (down to ~fM) in the plume. The use of small electrospray tip inner diameters (<1 μm) facilitated the fast desolvation of gaseous protein ions in an aqueous electrospray plume. Fluorescence spectra were acquired from specific locations along the plume axis in different aqueous electrospray plumes with three different analytes: a rhodamine dye and two proteins (ubiquitin and apomyoglobin) labeled with rhodamine dyes. To confirm the presence of gaseous ions, pure gas-phase fluorescence spectra were acquired in the vacuum of a modified ion trap mass spectrometer. These spectra were used to fit and confirm the presence of gaseous species in the corresponding spectra obtained from the electrospray plume. This study shows that with small inner diameter spray capillaries, gaseous protein ions generated at atmospheric pressure in an electrospray plume can be detected with fluorescence-based techniques. Fluorescence measurements can be used to study their structure in the electrospray plume as they transition from solution to gas-phase and in the early stages after desolvating from charged droplets. Other techniques can also be applied/coupled to further study gaseous biomolecular structure at ambient conditions immediately after desolvation.

Adapting a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer for Gas-Phase Fluorescence Spectroscopy Measurement of Trapped Biomolecular Ions

“…The longer wavelength emission maximum of the lower charge state of the peptide indicates a less Coulombically driven structural change in the absence of a solvent (i.e., the protein might not be totally unfolded, and its conformation remains compact), still providing some solvation for the dye label. It is likely that the solvation environments of the dyes are different in different charge states . Another explanation could be the shorter distance between the dye and the charges on the peptide backbone for the higher charge state.…”

“…It is likely that the solvation environments of the dyes are different in different charge states. 16 Another explanation could be the shorter distance between the dye and the charges on the peptide backbone for the higher charge state. The charge distribution could affect the dye's dipole moment and thus fluorescence properties.…”

“…The difficulties include low numbers of trapped ions and thus weak fluorescence, a high degree of photodissociation (PD), and the need to modify a mass spectrometer with an interface for optical excitation and detection (i.e., limited optical access) . Until now, mostly quadrupole ion traps (QITs, “Paul trap” and cylindrical) and linear ion traps (LTQs) have been modified for fluorescence experiments. ,,− Although QITs and LTQs have a higher throughput and sensitivity, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is superior in terms of m / z range, mass resolution, and extended trapping times. For example, the m / z range of a modern QIT is up to 4000 Th, while an FT-ICR MS usually offers more than 10,000 Th.…”

Cryogenic Fluorescence Spectroscopy of Ionic Fluorones in Gaseous and Condensed Phases: New Light on Their Intrinsic Photophysics

“…Furthermore, the mass selectivity allows for the possibility to probe hydrated systems (from small organic molecules to macromolecules), developing the connection between gas- and condensed-phase fluorescence properties and allowing the impact of a given solvent to be investigated in a systematic manner. , However, because solvent molecules are usually weakly bound (e.g., tens of kJ mol –1 for weakly interacting systems), cryogenic temperatures are crucial for avoiding solvent molecule evaporation. To date, we are unaware of any such gas-phase fluorescence studies, although we note efforts to study fluorescence from partially solvated ions in electrospray plumes. − …”

Atomistic Insights into the Formation of Nonspecific Protein Complexes during Electrospray Ionization

“…The release of analyte ions from ESI nanodroplets into the gas phase remains an active research area. − It is believed that in native ESI most protein ions are released via droplet evaporation to dryness, in accordance with the charged residue model (CRM). − The ion evaporation model (IEM) describes an alternative mechanism where ions are desorbed from the droplet surface. Although the IEM is invoked mostly for low molecular weight (MW) ions, ,− it can also apply to larger species such as peptides and some proteins .…”