Robust Fluorescence Collection Module for Wide-Bore Ion Cyclotron Resonance Mass Spectrometers.

Mass spectrometers are at the heart of the most powerful toolboxes available to scientists when studying molecular structure, conformation, and dynamics in controlled molecular environments. Improved molecular characterization brought about by the implementation of new orthogonal methods into mass spectrometry-enabled analyses opens deeper insight into the complex interplay of forces that underlie chemistry. Here, we detail how one can add fluorescence detection to commercial ultrahigh-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers without adverse effects to its preexisting analytical tools. This advance enables measurements based on fluorescence detection, such as Förster resonance energy transfer (FRET), to be used in conjunction with other MS/MS techniques to probe the conformation and dynamics of large biomolecules, such as proteins and their complexes, in the highly controlled environment of a Penning trap.

Self-localizing stabilized mega-pixel picoliter arrays with size-exclusion sorting capabilities.

We report on a liquid self-localizing process capable of producing Mega-pixel arrays of picoliter volumes on a 1 cm(2) area, within seconds, for high throughput sampling. The chip is based on principles of spatially varying wetting and stabilization. The key is to develop differential surface contact regions, which lead to both localization of the solution and increasing the surface adsorption energy to further pin the liquid to the surface, as highlighted by other studies. By exploiting surface roughness for enhanced wettability, we demonstrate wetting of wells with the aspect ratio of 100. The high precision of reactive ion etching (RIE) of silicon substrates allows for an extremely reproducible method of preparing the array of identical well structures and increased contact area to increase surface adsorption in the wells. "Dynamic wetting" is then readily achieved through inducing contact line instability by simply moving a drop of liquid on the top surface of the array. Liquid samples self-localize into the array pattern with the associated liquid flow leading to self-localization of suspended particles or analyte. The resulting picoliter volumes are both spatially ordered and stable for long periods of time, even for such small volumes, to permit selective measurements of the contents. This development will be particularly important in the assembly of the massive amounts of crystalline particles needed for atomically resolved structural dynamics using the latest advances in high number density electron and X-ray sources.

Fluorescence resonance energy transfer in gaseous, mass-selected polyproline peptides.

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.

Gas-phase fluorescence excitation and emission spectroscopy of mass-selected trapped molecular ions.

A flexible interface to perform optical spectroscopic measurements on gaseous ions stored in a modified commercial quadrupole ion trap (QIT) mass spectrometer is described. The modifications made to the mass spectrometer did not adversely affect its operating characteristics. Gas-phase ions are produced using electrospray ionization, mass isolated and stored in the trapping mass spectrometer. The ions are subsequently irradiated with visible light from a tunable laser and dispersed fluorescence spectra are recorded simultaneously. Mass spectra are recorded after the irradiation period. This set-up allows us to track a range of possible outcomes upon photoexcitation of selected ions including fluorescence, photofragmentation and photodetachment of electrons. The experimental set-up is characterized using rhodamine 590, which is a methyl ester variant of rhodamine 6G. Fluorescence excitation and emission spectra of gaseous rhodamine 590 are measured and compared with solution-phase spectra. Excitation and emission maxima for the gaseous ions are found to lie at higher energy than for the solvated rhodamine 590. In addition, the gas-phase Stokes shift is significantly smaller than the solution-phase Stokes shift. The effects of several experimental parameters on the observed fluorescence signal are investigated, including laser power, relative number of ions, q(z) trapping parameter and buffer gas pressure. In addition to its use for the photophysical characterization of the intrinsic properties of ionic chromophores, this set-up may be used to investigate the properties of mass-selected, dye-labeled biomolecules, both alone and in well-defined complexes and clusters.

Fluorescence imaging for visualization of the ion cloud in a quadrupole ion trap mass spectrometer.

Laser-induced fluorescence is used to visualize populations of gaseous ions stored in a quadrupole ion trap (QIT) mass spectrometer. Presented images include the first fluorescence image of molecular ions collected under conditions typically used in mass spectrometry experiments. Under these "normal" mass spectrometry conditions, the radial (r) and axial (z) full-width at half maxima (FWHM) of the detected ion cloud are 615 and 214 µm, respectively, corresponding to ~6% of r0 and ~3% of z0 for the QIT used. The effects on the shape and size of the ion cloud caused by varying the pressure of helium bath gas, the number of trapped ions, and the Mathieu parameter q z are visualized and discussed. When a "tickle voltage" is applied to the exit end-cap electrode, as is done in collisionally activated dissociation, a significant elongation in the axial, but not the radial, dimension of the ion cloud is apparent. Finally, using spectroscopically distinguishable fluorophores of two different m/z values, images are presented that illustrate stratification of the ion cloud; ions of lower m/z (higher qz) are located in the center of the trapping region, effectively excluding higher m/z (lower qz) ions, which form a surrounding layer. Fluorescence images such as those presented here provide a useful reference for better understanding the collective behavior of ions in radio frequency (rf) trapping devices and how phenomena such as collisions and space-charge affect ion distribution.

Carbohydrate amino acids: the intrinsic conformational preference for a beta-turn-type structure in a carbopeptoid building block.

Infrared ion-dip spectroscopy coupled with DFT and ab initio calculations are used to establish the intrinsic conformational preference of the basic structural unit of a peptide mimic, a cis-tetrahydrofuran-based "carbopeptoid" (amide-sugar-amide), isolated at low temperature in the gas phase. The carbopeptoid units form a beta-turn-type structure, stabilized by an intramolecular NH --> O=C hydrogen bond across the sugar ring, thus forming a 10-membered, C10 turn. Despite the clear preference for C10 beta-turn structures in the basic unit, however, the presence of multiple hydrogen-bond donating and accepting groups also generates a rich conformational landscape, and alternative structures may be populated in related molecules. Calculations on an extended, carbopeptoid dimer unit, which includes an alternating amide-sugar-amide-sugar-amide chain, identify conformers exhibiting alternative hydrogen-bonding arrangements that are somewhat more stable than the lowest-energy double beta-turn forming conformer.

Photodissociation spectroscopy of trapped protonated tryptophan.

We have coupled a quadrupole ion trap with a frequency doubled optical parametric oscillator laser. The photodissociation spectrum of the protonated tryptophan ion from 215 to 320 nm is reported. The yields of fragmentation on each mass channel as a function of the laser wavelength were obtained. We also report experiments involving multiple stages of laser induced dissociation and discuss possible structures for the fragmentation products.

Probing the glycosidic linkage: UV and IR ion-dip spectroscopy of a lactoside.

The beta(1-->4) glycosidic linkage found in lactose is a prevalent structural motif in many carbohydrates and glycoconjugates. Using UV and IR ion-dip spectroscopies to probe benzyl lactoside isolated in the gas phase, we find that the disaccharide unit adopts only a single, rigid structure. Its fully resolved infrared ion-dip spectrum is in excellent agreement with that of the global minimum structure computed ab initio. This has glycosidic torsion angles of phi(H) (H1-C1-O-C4') approximately 180 degrees and psi(H) (C1-O-C4'-H4') approximately 0 degrees which correspond to a rotation of approximately 150 degrees about the glycosidic bond compared to the accepted solution-phase conformation. We discuss the biological implications of this discovery and the generality of the strategies employed in making it.