Although Electrospray ionization (ESI) is a well-established technique for producing intact gaseous ions for mass spectrometric analysis, the mechanisms driving the formation of these gaseous ions are still illusive. A better mechanistic insight will enhance our understanding of ion formation process and will help us to increase the ion yields. Specifically, in the case of biomolecules, it will give clues about the structural evolution as the analyte passes from the droplet phase to the gas phase. Such studies have attracted attention of several groups and considerable attempts have been made from both experimental and theoretical sides. It has already been shown that laser-induced fluorescence experiments in the electrospray plume at different distances give snapshots of the electrospray process from the droplet phase to the desolvated gas phase.

We have developed a setup to study laser-induced fluorescence at different distances along the electrospray axis. A unique aspect of this apparatus is also that it facilitates both wavelength and time resolved studies simultaneously from a particular spot in the plume. The light source in the experiments is a pulsed (~100 fs pulses), tunable (690-1040 nm) titanium sapphire laser, which is frequency doubled to access the UV-Vis wavelength range (345-520 nm). Tunability of laser light in this wavelength lets us probe the spectroscopic properties of biologically relevant chromophores. Additionally, the ultra-short pulses are crucial to make sense from complex fluorescence signals.

Laser-induced fluorescence of biomolecules along the electrospray axis is being studied to understand different electrospray ionization mechanisms. Proteins like apo-myoglobin are labelled with fluorescent dyes and their fluorescence is monitored as they traverse from the droplet phase to the gas phase. This gives us valuable information about structural changes in the protein and its surrounding, which in turn is a reliable indicator of the mechanism of ESI taking place. Further, studying the fluorescence along the spray axis also provides us with time scale information, which is known to be different in different mechanisms. We have already developed a setup that simultaneously allows us to record the fluorescence spectra and lifetime from the same spot in the ESI plume. The initial optical alignment and calibration have also been completed. Now the main focus is not only to study how different classes of molecules follow different ionization mechanisms, but also to probe the influence of solvent/spray conditions on the ion yield and ionization mechanism. As a next step, Fluorescence Resonance Energy Transfer (FRET) experiments are expected to give distance sensitive information that can be used to track conformational changes of electrosprayed protein along the electrospray axis.

Details of the developed setup will be presented, along with preliminary results from laser-induced fluorescence on biomolecules along the electrospray axis.

[1] Chingin, K., Frankevich, V., Balabin, Roman M., Barylyuk, K., Chen, H., Wang, R. and Zenobi, R., “Direct access to isolated biomolecules under ambient conditions.” Angew. Chem. Int. Ed., 2010, 49, 2358.