Fluorescence Resonance Energy Transfer in Gaseous, Mass-Selected Polyproline Peptides

Abstract

Despite the many successes of mass spectrometry in the analysis of biological samples, the need to better understand the correlation between condensed-phase properties and those of electrospray species remains. In particular, the link between structures in the condensed phase and in the gaseous environment of the mass spectrometer is still elusive. Here, we show that fluorescence resonance energy transfer (FRET) can be used to probe the conformations of gaseous biopolymers which are formed by electrospray ionization (ESI) and manipulated in a quadrupole ion trap mass spectrometer. A rhodamine dye pair suitable for gas-phase FRET is characterized. Both steady state spectra and lifetime measurements are used to monitor energy transfer in a series of dye-labeled polyproline-based peptides. FRET efficiency is explored as a function of peptide chain length and charge state. For the peptide with eight proline repeats, virtually complete energy transfer is observed. For the peptide with 14 proline repeats, energy transfer decreases as the charge state increases, consistent with Coulomb repulsion induced elongation of the peptide backbone. FRET measurements of the longest peptide examined, which has 20 proline repeats, indicates that the peptide adopts a bent configuration. Evidence for multiple conformations present within the ensemble of trapped ions is provided by fluorescence lifetime measurements. Gas-phase FRET measurements promise to be a new route to probe the conformations of large gaseous ions.

An Electrostatic Charge Partitioning Model for the Dissociation of Protein Complexes in the Gas Phase

“…This correlation greatly expands the scope of gas phase structural investigations such as ion mobility measurements [22][23][24], gas phase hydrogen exchange [25], and optical spectroscopy [26]. In addition, a host of ion activation methods are available for the dissociation of protein assemblies [27][28][29][30][31][32].…”

Action-FRET: Probing the Molecular Conformation of Mass-Selected Gas-Phase Peptides with Förster Resonance Energy Transfer Detected by Acceptor-Specific Fragmentation

“…38 The donor chromophore is 5-carboxyrhodamine 575 N-succinimidyl ester (rh575) (Life Technologies), absorbing at 550 nm and fluorescing at 575 nm in solution. Rh575 was also used as the donor chromophore by Talbot et al 18 , and both the fluorescence excitation and emission spectra along with photofragmentation mass spectra have been previously studied in detail. 39 Both chromophores are designed to be functionalized onto primary amines, and thus peptides containing two primary amine groups only were chosen.…”

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

“…In the future, free electron lasers may provide atomically resolved images of solvent-free biomolecules [11], but at present other approaches have to be used. These include spectroscopic tools [12][13][14], dissociation experiments [5,6,8], gas phase H/D exchange [15,16], soft landing/EM [4,17], and computer simulations [10,[18][19][20]. The technique that is currently most widely used for characterizing biomolecular conformations in the gas phase is ion mobility spectrometry (IMS) [21][22][23][24][25][26][27].…”