Exploring Gas-Phase MS Methodologies for Structural Elucidation of Branched N-Glycan Isomers.

Structural isomers of N-glycans that are identical in mass and atomic composition provide a great challenge to conventional mass spectrometry (MS). This study employs additional dimensions of structural elucidation including ion mobility (IM) spectroscopy coupled to hydrogen/deuterium exchange (HDX) and electron capture dissociation (ECD) to characterize three main A2 N-glycans and their conformers. A series of IM-MS experiments were able to separate the low abundance N-glycans and their linkage-based isomers (α1-3 and α1-6 for A2G1). HDX-IM-MS data indicated the presence of multiple gas-phase structures for each N-glycan including the isomers of A2G1. Identification of A2G1 isomers by their collision cross section was complicated due to the preferential collapse of sugars in the gas phase, but it was possible by further ECD fragmentation. The cyclic IM-ECD approach was capable of assigning and identifying each isomer to its IM peak. Two unique cross-ring fragments were identified for each isomer: m/z = 624.21 for α1-6 and m/z = 462.16 for α1-3. Based on these key fragments, the first IM peak, indicating a more compact conformation, was assigned to α1-3 and the second IM peak, a more extended conformer, was assigned to α1-6.

Fluorescence-Based Detection of the Desolvation Process of Protein Ions Generated in an Aqueous Electrospray Plume.

A new experimental setup to study laser-induced fluorescence from analytes at different locations in an electrospray plume has been developed. The high fluorescence collection efficiency (∼2%) of the setup, along with a sensitive charge coupled device (CCD) detector, enabled the study of low ion concentrations (down to ∼fM) in the plume. The use of small electrospray tip inner diameters (<1 µm) facilitated the fast desolvation of gaseous protein ions in an aqueous electrospray plume. Fluorescence spectra were acquired from specific locations along the plume axis in different aqueous electrospray plumes with three different analytes: a rhodamine dye and two proteins (ubiquitin and apomyoglobin) labeled with rhodamine dyes. To confirm the presence of gaseous ions, pure gas-phase fluorescence spectra were acquired in the vacuum of a modified ion trap mass spectrometer. These spectra were used to fit to confirm the presence of gaseous species in the corresponding spectra obtained from the electrospray plume. This study shows that with small inner diameter spray capillaries, gaseous protein ions generated at atmospheric pressure in an electrospray plume can be detected with fluorescence-based techniques. Fluorescence measurements can be used to study their structure in the electrospray plume, and the dynamics as they transition from solution to the gas phase and in the early stages after desolvation from charged droplets. Other techniques can also be applied to further study gaseous biomolecular structures under ambient conditions immediately after desolvation.

Breaking the Brightness Barrier: Design and Characterization of a Selected-Ion Fluorescence Measurement Setup with High Optical Detection Efficiency.

A quadrupole ion trap (QIT) mass spectrometer has been modified and coupled with tunable laser excitation and highly sensitive fluorescence detection systems to perform fluorescence studies on mass-selected ions. Gaseous ions, generated using nanoelectrospray ionization (nano-ESI), are trapped in the QIT that allows optical access for laser irradiation. The emitted fluorescence is collected from a 5.0 mm diameter hole drilled into the ring electrode of the QIT and is directed toward the detection setup. Due to the small inner diameter (7.07 mm) of the ring electrode and a relatively large opening for fluorescence collection, a fluorescence collection efficiency of 2.3% is achieved. After some losses in transmission, around 1.8% of the emitted fluorescence reaches the detectors, more than any other similar instrument reported in the literature. This improved fluorescence collection translates to a much shorter measurement time for a fluorescence signal. Another key feature of this setup is the ability to perform a variety of fluorescence experiments on trapped ions including excitation and emission spectroscopy, lifetime measurement, and ion imaging. The capabilities of the instrument are demonstrated by measuring fluorescence spectra of dyes and biomolecules labeled with dyes in a range of different excitation and emission wavelengths, quantum yields, m/z, and different polarities. A fluorescence lifetime measurement and ion image of trapped rhodamine 6G cations are also shown. With a wide array of functionality and high fluorescence detection performance, this setup provides an opportunity to study biomolecular structures and photophysics of fluorophores in well-controlled environments.

Studying biomolecular folding and binding using temperature-jump mass spectrometry.

Characterizing folding and complex formation of biomolecules provides a view into their thermodynamics, kinetics and folding pathways. Deciphering kinetic intermediates is particularly important because they can often be targeted by drugs. The key advantage of native mass spectrometry over conventional methods that monitor a single observable is its ability to identify and quantify coexisting species. Here, we show the design of a temperature-jump electrospray source for mass spectrometry that allows one to perform fast kinetics experiments (0.16-32 s) at different temperatures (10-90 °C). The setup allows recording of both folding and unfolding kinetics by using temperature jumps from high to low, and low to high, temperatures. Six biological systems, ranging from peptides to proteins to DNA complexes, exemplify the use of this device. Using temperature-dependent experiments, the folding and unfolding of a DNA triplex are studied, providing detailed information on its thermodynamics and kinetics.

Linear and Kinked Oligo(phenyleneethynylene)s as Ideal Molecular Calibrants for Förster Resonance Energy Transfer.

We show that oligo(phenyleneethynylene)s (oligoPEs) are ideal spacers for calibrating dye pairs used for Förster resonance energy transfer (FRET). Ensemble FRET measurements on linear and kinked diads with such spacers show the expected distance and orientation dependence of FRET. Measured FRET efficiencies match excellently with those predicted using a harmonic segmented chain model, which was validated by end-to-end distance distributions obtained from pulsed electron paramagnetic resonance measurements on spin-labeled oligoPEs with comparable label distances.

Mechanistic Studies on Cationization in MALDI-MS Employing a Split Sample Plate Set-up.

In the analysis of polymers by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), a commonly observed ionization pathway is cation-adduct formation, as polymers often lack easily ionizable (basic/acidic) functional groups. The mechanism of this process has been hypothesized to involve gas-phase cation attachment. In previous experiments, a split sample plate set-up has been introduced, enabling separate deposition of the components on individual MALDI plates. The plates are divided by a small gap of a few micrometers, allowing simultaneous laser irradiation from both plates, while precluding the possibility of any other interactions prior to ablation. Here, we extend on these studies by using different polymer-salt combinations to test the generalizability of a gas-phase ionization process. Clear evidence for in-plume ionization is presented for the model polymers poly (methyl methacrylate) and polystyrene. Furthermore, the contribution of in-plume processes to the overall ion formation by cationization is gauged, providing a first estimate for the importance of this pathway.

A Modified Traveling Wave Ion Mobility Mass Spectrometer as a Versatile Platform for Gas-Phase Ion-Molecule Reactions.

Taken individually, chemical labeling and mass spectrometry are two well-established tools for the structural characterization of biomolecular complexes. A way to combine their respective advantages is to perform gas-phase ion-molecule reactions (IMRs) inside the mass spectrometer. This is, however, not so well developed because of the limited range of usable chemicals and the lack of commercially available IMR devices. Here, we modified a traveling wave ion mobility mass spectrometer to enable IMRs in the trapping region of the instrument. Only one minor hardware modification is needed to allow vapors of a variety of liquid reagents to be leaked into the trap traveling wave ion guide of the instrument. A diverse set of IMRs can then readily be performed without any loss in instrument performance. We demonstrate the advantages of implementing IMR capabilities in general, and to this quadrupole-ion mobility-time-of-flight (Q-IM-TOF) mass spectrometer in particular, by exploiting the full functionality of the instrument, including mass selection, ion mobility separation, and post-mobility fragmentation. The potential to carry out gas-phase IMR kinetics experiments is also illustrated. We demonstrate the versatility of the setup using gas-phase IMRs of established utility for biological mass spectrometry, including hydrogen-deuterium exchange, ion-molecule proton transfer reactions, and covalent modification of DNA anions using trimethylsilyl chloride.

Sensitive probes of protein structure and dynamics in well-controlled environments: combining mass spectrometry with fluorescence spectroscopy.

Combining the selectivity of mass spectrometry (MS) with laser-induced fluorescence (LIF) presents a promising route to probe the intrinsic conformation, stability and dynamics of biological macromolecules. However, applications to proteins are in their infancy. Recent advances include the realization of Förster (fluorescence) resonance energy transfer (FRET) to provide nm-range distance constraints in de-solvated proteins, and measurement of dynamic fluorescence quenching rates to assess shorter-range interactions in peptides and Trp-cage. Temperature-dependent experiments employing FRET and dynamic quenching as conformational probes enable determination of enthalpy and entropy of conformational change in de-solvated biomolecules. These developments show the feasibility of using MS-LIF to dissect complex molecular interactions. For example, MS-LIF of protein-ligand complexes and partially hydrated proteins will better elucidate the energetics of specific binding interactions and the role of the solvent in protein structure and folding.

Gas-Phase FRET Efficiency Measurements To Probe the Conformation of Mass-Selected Proteins.

Electrospray ionization and mass spectrometry have revolutionized the chemical analysis of biological molecules, including proteins. However, the correspondence between a protein's native structure and its structure in the mass spectrometer (where it is gaseous) remains unclear. Here, we show that fluorescence (Förster) resonance energy transfer (FRET) measurements combined with mass spectrometry provides intramolecular distance constraints in gaseous, ionized proteins. Using an experimental setup which combines trapping mass spectrometry and laser-induced fluorescence spectroscopy, the structure of a fluorescently labeled mutant variant of the protein GB1 was probed as a function of charge state. Steady-state fluorescence emission spectra and time-resolved donor fluorescence measurements of mass-selected GB1 show a marked decrease in the FRET efficiency with increasing number of charges on the gaseous protein, which suggests a Coulombically driven unfolding and expansion of its structure. This lies in stark contrast to the pH stability of GB1 in solution. Comparison with solution-phase single-molecule FRET measurements show lower FRET efficiency for all charge states of the gaseous protein examined, indicating that the ensemble of conformations present in the gas phase is, on average, more expanded than the native form. These results represent the first FRET measurements on a mass-selected protein and illustrate the utility of FRET for obtaining a new kind of structural information for large, desolvated biomolecules.

Understanding photophysical effects of cucurbituril encapsulation: a model study with acridine orange in the gas phase.

Encapsulation of dyes by cucurbituril macrocycles has proven profitable as a strategy to alter fluorescence characteristics in useful ways. Encapsulation generally results in longer fluorescence lifetimes, enhanced brightness, and solvatochromic effects not normally seen in the condensed phase. These effects have been attributed variously to both the removal of interactions with solvent molecules and to the confined environment of extremely low polarizability provided by the cucurbituril interior. It is difficult to disentangle these effects in solution. Here, we present results from gas-phase experiments designed to separate these effects, using cucurbit[7]uril (CB7), and the cationic dye acridine orange (AOH(+)) as a probe. Fluorescence properties of gaseous AOH(+) are compared with those of the gaseous AOH(+)-CB7 complex and with the properties of the dye and complex in aqueous solution. The dependence on the local environment of several spectroscopic properties is discussed, including the fluorescence excitation and emission maxima, the size of the Stokes shift, fluorescence lifetime and relative brightness. An understanding of the modulation of fluorescence properties by the local environment, such as that promoted by this work, will aid in the rational design of improved fluorophores and fluorescent sensors.