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

Abstract

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. he study of biomolecular conformation in the gas phase has attracted great attention because it opens possibilities to compare gas-phase and solution-phase structures and to understand the effect of the solvent on a molecule. Because matrix-assisted laser desorption/ionization (MALDI) [1] and electrospray ionization (ESI) [2] typically generate unsolvated ions without any adducts, their structure also provides an ideal model system for theoretical calculations of conformations. A major question today is whether singly or multiply charged biomolecular ions produced by soft ionization methods retain their native (or at least a "native-like"), active conformation in the gas phase. This is often assumed but needs to be proven rigorously.Mass spectrometry has many attractive features for studying biomolecules in the gas phase, including the capability of the isolation of ions and elimination of unwanted species before detection and a positive identification of the molecule from the exact mass or from tandem mass spectrometry (MS/MS) data. There are mass spectrometric methods available for obtaining conformational information of molecules in the gas phase: blackbody infrared radiative dissociation where ions undergo unimolecular dissociation by exchanging their energy with the surroundings by absorption and emission of infrared photons [3], collision-induced dissociation where ions are dissociated as a result of interaction with a target neutral species [4], hydrogendeuterium exchange where hydrogen atoms of the protein are exchanged with the deuterium atoms from a solution [5], covalent or noncovalent tagging of biomolecules based on the surface accessibility of specific moieties [6,7], and ion mobility spectrometry where the measured cross section of the molecule is compared with a theoretically calculated cross section [8]. However, generally speaking, these methods yield only indirect information about the gas-phase conformation, which is often derived from either the overall cross section of the molecule, from a fragmentation pattern, or from a dissociation rate.Recently, the efficient trapping capabilities of mass spectrometry have been coupled with the high sensitivity of fluorescence detection to provide structural and spectroscopic information of molecules in the gas phase. The general idea...

Electron photodetachment dissociation of DNA anions with covalently or noncovalently bound chromophores

“…As FRET efficiency depends on the donor-acceptor distance (in 1/r 6 , FRET is commonly used to probe the conformation of biomolecules in solution, including nucleic acids [53]. Several groups developed specialized instrumentation to allow fluorescence measurements in trap mass spectrometers [54 -56], and probing gasphase ion conformation either by measuring changes in the donor fluorescence [45,57], or by measuring the acceptor fluorescence [58]. Here we propose using a fluorophore as the donor and a quencher as the acceptor, in a configuration known as a "molecular beacon" [59,60].…”

Fluorescence and photodissociation of rhodamine 575 cations in a quadrupole ion trap

“…In gas-phase work, Parks and coworkers [7][8][9][10] and Zenobi and coworkers [11,12] investigated the fluorescence of trapped molecular ions commonly used as fluorescent molecular probes of biomolecules. Both groups attached fluorescent probes to peptide cations for the purposes of investigating gas-phase FRET [8,11].Chromophore and fluorophore labeling experiments both rely on energy transfer from the absorbing moiety to another part of the biomolecule, either via intramolecular vibrational energy transfer or via radiative or electronic interactions. To understand the initial absorption process and the efficiency of photoactivation, it is of interest to investigate the photophysics and chemical dynamics of the absorption, fluorescence, and dissociation processes in the fluorescent dye molecules themselves in the gas phase.…”

“…The resulting decays obtained by both methods are compared as a function of pressure, laser fluence, and irradiation time. The mechanism of rhodamine cation photodissociation at 488 nm is discussed.Several other groups have directly measured fluorescence from ions in various traps [7,11,[13][14][15][16]. Zenobi et al.…”

“…Fluorophore labeling is also used in fluorescence resonant energy transfer (FRET) experiments, in which the acceptor/donor interactions of two attached fluorophores depend on the distances between them and therefore can provide conformational information [3][4][5][6]. In gas-phase work, Parks and coworkers [7][8][9][10] and Zenobi and coworkers [11,12] investigated the fluorescence of trapped molecular ions commonly used as fluorescent molecular probes of biomolecules. Both groups attached fluorescent probes to peptide cations for the purposes of investigating gas-phase FRET [8,11].…”

“…In gas-phase work, Parks and coworkers [7][8][9][10] and Zenobi and coworkers [11,12] investigated the fluorescence of trapped molecular ions commonly used as fluorescent molecular probes of biomolecules. Both groups attached fluorescent probes to peptide cations for the purposes of investigating gas-phase FRET [8,11].…”