Exploring Fluorescence and Fragmentation of Ions Produced by Electrospray Ionization in Ultrahigh Vacuum
Supplementary material
Konstantin Chingin1, Huanwen Chen2, Gerardo Gamez1 and Renato Zenobi1*
1Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland
E-mail:
2Applied Chemistry Department, East China Institute of Technology, Fuzhou, China
Prepared for Journal of the American Society for Mass Spectrometry
Keywords: Fluorescence, Mass Spectrometry, Photofragmentation, ultrahigh vacuum, rhodamine 6G, FTICR
* Corresponding author:
Prof. Dr. Renato Zenobi
Chemistry Department and Applied Biosciences
ETH Zürich, CH-8093 Zürich, Switzerland
Fax: (+) 41-44-632-1292
E-mail:
Figure S1. Sequence of events in a typical experiment.
Design features
The very first version of the ESI-compatible LIF collection setup engineered in our laboratory[1] was quite different from the one described in the experimental section. It is worthy to highlight some general issues one has to pay attention to when designing a LIF detection experiment in vacuum.
In practice, it is extremely difficult to dispose of the whole laser beam after it has passed through the ion cloud in the ICR cell. The amount of laser light absorbed is limited by the capacity of the beam dump and by the beam divergence. As a result, a part of the laser beam is scattered inside the cell. Therefore, it is first of all important that no fluorescing materials are used in the cell assembly. Spurious signals originating from such materials can spectrally overlap with the ion fluorescence and thus cannot be easily filtered out later. An example of a fluorescing material is PEEK plastic, which is often used as an isolator in ICR cells. One feature of our setup which yielded an appreciable improvement was minimizing the PEEK content in the cell assembly.
Generally speaking, the earlier the scattered light is suppressed the less spurious fluorescence it can produce. In our setup it was particularly important that the major part of the scattered laser light was blocked before it reached the collection fiber because the fiber itself was found to produce non-negligible fluorescence, which substantially compromised the detection of the ion fluorescence. For this reason, an optical filter stage was introduced in the vacuum chamber. Also, when blocking the laser scattered light, interference filters were found to be better compared to absorbing colored glasses as the latter typically produce spurious fluorescence as well.
The distance between the collimating and the focusing lens was 10 cm to make sure that only the well collimated part of light passed both lenses and the filter and finally reached the fiber. Of course, a shorter distance between the lenses would allow for a bit more efficient fluorescence collection. In this case, however, the light passing through the lenses would not be so well collimated, and the edge filter performance would be much worse because it has a strong angle dependence.
Figure S2 shows how the background spectrum (no ions in the cell) changed after all the issues described above were taken into consideration. The peak at 488 nm corresponds to the remaining laser line. The broad bands to the right of the laser line in the range of ca. 500-700 nm on the first spectrum correspond to various spurious fluorescence sources that were discussed above. Clearly, the ion fluorescence signal-to-background ratio would be greatly affected by this spectrally overlapping background signal. The second background spectrum corresponds to our latest setup. Virtually no spurious fluorescence can be observed. The laser light however is not blocked completely, thus, the second filter is used between the fiber and the PMT in the photon counting mode (Figure 1 in the main text).
As discussed in the Experimental Section, the laser beam path was aligned when the cell was outside of the vacuum chamber. Fine adjustment was performed after the cell had been installed to maximize the fluorescence signal.
In the very first version of our setup a fiber was used to deliver the laser light directly into the ICR cell in order to avoid the troublesome alignment procedure[1]. However, the fiber was found to produce very strong fluorescence signal induced by the high laser power coupled. Thus, a laser-line filter(LL01-488-12.5, MaxLine®, Semrock, USA) was placed right after the fiber to get rid of this spurious fluorescence signal but it resulted in perturbation of the beam shape leading to stronger laser light scattering inside the cell. Thus, this approach was abandoned in favor of steering the laser beam into the cell through a window followed by a series of baffles.
Figure S2. Evolution of the background frequency spectrum (from the left to the right spectrum) as a result of modifying the experimental setup.
Figure S3. Dependence of the fluorescence signal on the intensity of the molecular peak for Rhodamine 6G ions in the mass spectrum. A second-order polynomial (black curve) provides a good fit of the experimental data (y = 12,199x – 0,0187x2 – 8,302; R2 = 0,9954). Deviation from the linear dependence (red line) can only be seen for high ion densities inside the ICR cell.
Figure S4. Dependence of the attainable fluorescence signal on the pressure of helium gas inside the ICR cell (adopted from ref. [7]).
Outlook
Here we briefly discuss some of the issues that will be addressed in future studies using our unique platform.
Fluorescence in ICR
Recent evidence based on molecular dynamics (MD)[2, 3] simulations indicates that the structure of a protein in solution remains essentially intact until desolvation is nearly complete[4]. It is therefore of great interest to experimentally explore how conformation changes as the last water molecules are evaporated in order to bridge solution and gas phase data. Recently, partially hydrated peptides and proteins have been observed in Fourier Transform ion cyclotron resonance (FTICR) experiments with ESI ionization, the extent of hydration being strongly dependent on experimental parameters, such as capillary temperature, auxiliary gas flow, solvent used[5, 6]. FTICR is celebrated for its ultra high mass resolution, selectivity and capability of long ion storage times, which makes it a powerful tool for studies of properties and structures of ionic clusters in the gas phase. Owing to the very low operating pressures in ICR experiments (10-8 – 10-10 mbar), energy exchange of ions with buffer gas is minimal. This allows one to either ‘freeze’ the extent of protein ion hydration or to promote further dehydration in a controllable way, e.g. by blackbody infrared radiation[5].
Also, it is known that properties of fluorescent molecules, such as emission / absorption spectra, quantum yield, differ significantly in solution and gas phase[7-9]. It is therefore of great interest to be able to follow how fluorescence properties of gas phase ions are changing upon gradual solvation inside an ICR cell.
Photodissociation in ICR
As discussed before, fast photodissociation is a significant factor limiting fluorescence detection efficiency in high vacuum. In the absence of vibrational cooling by collisions with buffer gas molecules, ions rapidly gain internal energy in successive fluorescence photon absorption / emission cycles or through internal conversion from the excited electronic state. This energy is then released through different fragmentation channels. If a chromophore is used to tag a large protein molecule, then this vibrational energy can be delocalized over all the degrees of freedom of the entire protein. If photofragmentation occurs before complete vibrational energy redistribution (IVR) takes place, then it should be characteristic of the chromophore’s local microenvironment, which can be used for probing the gas-phase ion structure. For instance, Bossio et al. showed decarboxylation of the polypeptide C terminus in an ICR spectrometer when a UV chromophore at the N terminus was in close proximity due to the flexibility of the peptide chain[10]. Gabelica et al. studied electron photodetachment dissociation of gas-phase DNA anions activated at 260 nm (DNA excitation)[11] and DNA anions labeled with different chromophores activated at >300 nm (chromophore excitation) in a quadrupole ion trap[12]. Radicals produced were shown to keep no memory of the activation method by comparing their MS3 fragmentation, suggesting that complete IVR took place[12]. Therefore, in order to perform localized dissociation controlled by the position of a chromophore, it is important that the rate of de-excitaition by IVR and by inelastic collisions with a bath gas of a biomolecule is slow compared to vibrational energy accumulation by the chromophore due to internal conversion of its electronic energy. As discussed earlier, the lower the background pressure the faster the ion vibrational energy is accumulated as it is not dissipated due to collisions with neutrals. Thus, it is believed that conformation-specific fragmentation could be easier to achieve in an ICR spectrometer compared to ion traps. Also, this process could explain why complete IVR was always found in studies on the electron photodetachment dissociation in a quadrupole ion trap[12].
References
1. L. Scharfenberg, Fluorescence Detection and Resonance Energy Transfer of Trapped Molecular Ions, in Department of Chemistry and Applied Biosciences. 2005, ETH: Zurich. p. 93.
2. J.A. Mccammon, B.R. Gelin, and M. Karplus, Dynamics of Folded Proteins, Nature, 5612 (1977) 585-590.
3. M. Karplus and J.A. McCammon, Molecular dynamics simulations of biomolecules, Nature Structural Biology, 9 (2002) 646-652.
4. M.Z. Steinberg, K. Breuker, R. Elber, and R.B. Gerber, The dynamics of water evaporation from partially solvated cytochrome c in the gas phase, Phys. Chem. Chem. Phys., 33 (2007) 4690-4697.
5. S.W. Lee, P. Freivogel, T. Schindler, and J.L. Beauchamp, Freeze-dried biomolecules: FT-ICR studies of the specific solvation of functional groups and clathrate formation observed by the slow evaporation of water from hydrated peptides and model compounds in the gas phase, J. Am. Chem. Soc., 45 (1998) 11758-11765.
6. S.E. Rodriguez-Cruz, J.S. Klassen, and E.R. Williams, Hydration of gas-phase ions formed by electrospray ionization, J. Am. Soc. Mass Spectr., 10 (1999) 958-968.
7. M. Dashtiev, V. Azov, V. Frankevich, L. Scharfenberg, and R. Zenobi, Clear evidence of fluorescence resonance energy transfer in gas-phase ions, J. Am. Soc. Mass Spectr., 9 (2005) 1481-1487.
8. K.C. Wright and M.W. Blades. Fluorescence Emission Spectroscopy of Trapped Molecular Ions. in 51st ASMS Conference on Mass Spectrometry and Allied Topics. 2003. Montreal, Canada.
9. N.A. Sassin, S.C. Everhart, B.B. Dangi, K.M. Ervin, and J.I. Cline, Fluorescence and Photodissociation of Rhodamine 575 Cations in a Quadrupole Ion Trap, J. Am. Soc. Mass Spectr., 1 (2009) 96-104.
10. R.E. Bossio, R.R. Hudgins, and A.G. Marshall, Gas phase photochemistry can distinguish different conformations of unhydrated photoaffinity-labeled peptide ions, J. Phys. Chem. B, 14 (2003) 3284-3289.
11. V. Gabelica, T. Tabarin, R. Antoine, F. Rosu, I. Compagnon, M. Broyer, E. De Pauw, and P. Dugourd, Electron photodetachment dissociation of DNA polyanions in a quadrupole ion trap mass spectrometer, Anal. Chem., 18 (2006) 6564-6572.
12. V. Gabelica, F. Rosu, E. De Pauw, R. Antoine, T. Tabarin, M. Broyer, and P. Dugourd, Electron photodetachment dissociation of DNA anions with covalently or noncovalently bound chromophores, J. Am. Soc. Mass Spectr., 11 (2007) 1990-2000.
1