Proteomic footprinting of drug-treated cancer cells as a measure of cellular vaccine efficacy for the prevention of cancer recurrence.

The comparative proteomic study of cell surfaces of native and drug-treated cancer cells was performed. To this end, cell proteomic footprinting, which reflects the mass spectrometry profiling of cell surface proteins, was applied to breast adenocarcinoma cells (MCF-7), which were untreated or treated with doxorubicin, tamoxifen, or etoposide. The footprints of drug-treated cells were compared with the footprints of untreated cells and the footprint of a randomly selected control cancer cell culture. It was found that drug-treated cells have reproducible, pronounced, and drug-specific changes in cell surface protein expression. Cytotoxicity assays, which are an in vitro model of human antitumor vaccination, revealed that the degree of these changes correlates directly with the ability of the cancer cells to escape cell death induced by a cytotoxic T-cell-mediated immune response. Moreover, cancer cells escape from the immune response was linearly approximated (R(2) equal to 0.99) with the degree by which their proteomic footprints diverged from the footprint of the targeted (native) cancer cells. From these findings, it was concluded that the design of anticancer vaccines intended to prevent cancer recurrence after primary treatment should consider the drug-specific changes in cancer cell-surface antigens. Such changes can be easily identified by cell proteomic footprinting, renewing hopes for development of efficient cellular cancer vaccines.

Cell proteomic footprint.

The authentication of mammalian cell cultures and their subpopulations is of great demand in biotechnology and cell therapy. However, current techniques are either not efficient or can be very complex and expensive. Here we report a simple and straightforward approach for authentication of biological cells and their subpopulations with high speed, high throughput, low sample cost, and high sensitivity. We discovered that cell cultures treated with protease under soft, 'non-killing' conditions release fragments of cell surface proteins, whose composition is a strong characteristic of the cells. Mass spectrometric analysis of the released fragments allows a direct comparison of the produced mass spectrum with the mass spectrum of known cells. As an example, we applied this technique to verify subpopulations of human fibroblasts with different origins and which exhibit different medical characteristics.

Effect of buffer gas on the fluorescence yield of trapped gas-phase ions.

We investigated the dependence of three different gases, helium, argon, and nitrogen, on the fluorescence signal intensity of rhodamine 6G cations in the gas phase. The method is based on laser-induced fluorescence of ions trapped in a Fourier transform ion cyclotron mass spectrometer. We found that the use of helium results in the highest fluorescence signal, while no fluorescence was detected when using argon under the same conditions.

Kinetic energy of free electrons affects MALDI positive ion yield via capture cross-section.

A method for enhancing positive analyte ion signal in MALDI is described. The idea is based on influencing the kinetic energy of free electrons emitted from the organic/metal interface. It has been recently shown that free electrons in MALDI have energies around 1 eV. This energy is close to the maximum capture cross-section of most common MALDI matrices, leading to the efficient formation of negative matrix ions. This results in the reduction of the positive analyte ion yield. The effect can be minimized by shifting the kinetic energy of the electrons away from the maximum of the matrix capture cross-section by choosing a different substrate material.

Laser-induced fluorescence of trapped gas-phase molecular ions generated by internal source matrix-assisted laser desorption/ionization in a Fourier transform ion cyclotron resonance mass spectrometer.

The combination of laser-induced fluorescence with mass spectrometry opens up new possibilities both for detection purposes and for structural studies of trapped biomolecular ions in the gas phase. However, this approach is experimentally very challenging, and only a handful of studies have been reported so far. In this contribution, a novel scheme for laser-induced fluorescence measurements of ions trapped inside a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer will be introduced. It is based on an open FT-ICR cell design, continuous wave axial excitation of the fluorescence, orthogonal photon collection by fiber optics, and single photon counting detection. Rhodamine 6G ions generated by an internal matrix-assisted laser desorption/ionization source were used to develop and test the set-up. Due to photobleaching processes, the excitation laser power and the observation time window have to be carefully optimized. An ion tomography method was used to align the excitation laser. Potential applications for studying the gas-phase structure of fluorescent biomolecular ions and for investigating fluorescence resonance energy transfer of donor-acceptor pairs will be presented.

Clear evidence of fluorescence resonance energy transfer in gas-phase ions.

Fluorescence resonance energy transfer (FRET) is a distance-sensitive method that correlates changes in fluorescence intensity with conformational changes, for example, of biomolecules in the cellular environment. Applied to the gas phase in combination with Fourier transform ion cyclotron resonance mass spectrometry, it opens up possibilities to define structural/conformational properties of molecular ions, in the absence of solvent, and without the need for purification of the sample. For successfully observing FRET in the gas phase it is important to find suitable fluorophores. In this study several fluorescent dyes were examined, and the correlation between solution-phase and gas-phase fluorescence data were studied. For the first time, FRET in the gas phase is demonstrated unambiguously.

MALDI-Fourier transform mass spectrometric and theoretical studies of donor-acceptor and donor-bridge-acceptor fullerenes.

Fourier-transform ion cyclotron resonance (FTICR) mass spectrometric studies have been performed on donor-acceptor and donor-bridge acceptor fullerene-based systems. Matrix-assisted laser desorption/ionization (MALDI) was used for ion production; both the positive and negative ion modes were utilized. In addition, collision-induced dissociation (CID) experiments were carried out to study the movement of the charge (electron or hole) upon fragmentation. The experiments are complemented by ab initio theoretical calculations yielding both molecular orbital energies and electron density distributions. It was found that the theoretical electron density map predicted the experimentally observed fragmentation correctly in every case. Both the calculations and the MS experiments may be useful in studying these and related donor-acceptor systems in view of their use for charge separation and eventually, solar energy production.

Role of electrons in laser desorption/ionization mass spectrometry.

A nonmetallic sample support for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry enhances the positive ion yield by 2 orders of magnitude and generally affects the charge balance in the desorption plume. We interpret the effects of the target material and of the sample preparation on MALDI mass spectra as a result of photoelectrons emitted upon laser irradiation of a metal target covered by a thin sample layer. These electrons are shown to play an important role in MALDI and laser desorption/ionization because they decrease the yield of positive ions, reduce ions with higher oxidation states, and affect the ion velocity distribution as well as the mass resolution. Understanding the role of these photoelectrons helps to clarify previously obscure aspects of the ion formation mechanism in MALDI.

Production and fragmentation of multiply charged ions in 'electron-free' matrix-assisted laser desorption/ionization.

An unusually large fraction of multiply charged ions is observed in 'electron-free' matrix-assisted laser desorption/ionization (MALDI). Here we investigate how the yield of multiply charged ions depends on experimental parameters in MALDI. It is found to increase if measures are taken to limit the number of electrons in the plume, for example, by using non-metallic MALDI targets or low laser pulse energies. The ionization energy of the matrix is another important parameter that affects the yield of multiply charged ions: matrices with high ionization energies lead to greater intensities of multiply charged ions. It is furthermore proposed that some of the fragment ions observed in MALDI are due to reactions of analyte with electrons in the plume. The possibility of electron capture dissociation of multiply charged ions produced by MALDI is shown.