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

Structural isomers of -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 -glycans and their conformers. A series of IM-MS experiments were able to separate the low abundance -glycans and their linkage-based isomers (α1-3 and α1-6 for A2G1).

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.

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.

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.

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.

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.

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.

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.

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.