Probing the Stability of a ß-Hairpin Scaffold after Desolvation.

Probing the structural characteristics of biomolecular ions in the gas phase following native mass spectrometry (nMS) is of great interest, because noncovalent interactions, and thus native fold features, are believed to be largely retained upon desolvation. However, the conformation usually depends heavily on the charge state of the species investigated. In this study, we combine transition metal ion Förster resonance energy transfer (tmFRET) and ion mobility-mass spectrometry (IM-MS) with molecular dynamics (MD) simulations to interrogate the ß-hairpin structure of GB1p in vacuo. Fluorescence lifetime values and collisional cross sections suggest an unfolding of the ß-hairpin motif for higher charge states. MD simulations are consistent with experimental constraints, yet intriguingly provide an alternative structural interpretation: preservation of the ß-hairpin is not only predicted for 2+ but also for 4+ charged species, which is unexpected given the substantial Coulomb repulsion for small secondary structure scaffolds.

Transition Metal Ion FRET-Based Probe to Study Cu(II)-Mediated Amyloid-ß Ligand Binding.

Recent therapeutic strategies suggest that small peptides can act as aggregation inhibitors of monomeric amyloid-ß (Αß) by inducing structural rearrangements upon complexation. However, characterizing the binding events in such dynamic and transient noncovalent complexes, especially in the presence of natively occurring metal ions, remains a challenge. Here, we deploy a combined transition metal ion Förster resonance energy transfer (tmFRET) and native ion mobility-mass spectrometry (IM-MS) approach to characterize the structure of mass- and charge-selected Aß complexes with Cu(II) ions (a quencher) and a potential aggregation inhibitor, a small neuropeptide named leucine enkephalin (LE). We show conformational changes of monomeric Αß species upon Cu(II)-binding, indicating an uncoiled N-terminus and a close interaction between the C-terminus and the central hydrophobic region. Furthermore, we introduce LE labeled at the N-terminus with a metal-chelating agent, nitrilotriacetic acid (NTA). This allows us to employ tmFRET to probe the binding even in low-abundance and transient Aß-inhibitor-metal ion complexes. Complementary intramolecular distance and global shape information from tmFRET and native IM-MS, respectively, confirmed Cu(II) displacement toward the N-terminus of Αß, which discloses the binding region and the inhibitor's orientation.

Influence of the Fluorophore Mobility on Distance Measurements by Gas-Phase FRET.

Gas-phase Förster resonance energy transfer (FRET) combines mass spectrometry and fluorescence spectroscopy for the conformational analysis of mass-selected biomolecular ions. In FRET, fluorophore pairs are typically covalently attached to a biomolecule using short linkers, which affect the mobility of the dye and the relative orientation of the transition dipole moments of the donor and acceptor. Intramolecular interactions may further influence the range of motion. Yet, little is known about this factor, despite the importance of intramolecular interactions in the absence of a solvent. In this study, we applied transition metal ion FRET (tmFRET) to probe the mobility of a single chromophore pair (Rhodamine 110 and Cu2+) as a function of linker lengths to assess the relevance of intramolecular interactions. Increasing FRET efficiencies were observed with increasing linker length, ranging from 5% (2 atoms) to 28% (13 atoms). To rationalize this trend, we profiled the conformational landscape of each model system using molecular dynamics (MD) simulations. We captured intramolecular interactions that promote a population shift toward smaller donor-acceptor separation for longer linker lengths and induce a significant increase in the acceptor's transition dipole moment. The presented methodology is a first step toward the explicit consideration of a fluorophore's range of motion in the interpretation of gas-phase FRET experiments.

Determining the gas-phase structures of α-helical peptides from shape, microsolvation, and intramolecular distance data.

Mass spectrometry is a powerful technique for the structural and functional characterization of biomolecules. However, it remains challenging to accurately gauge the gas-phase structure of biomolecular ions and assess to what extent native-like structures are maintained. Here we propose a synergistic approach which utilizes Förster resonance energy transfer and two types of ion mobility spectrometry (i.e., traveling wave and differential) to provide multiple constraints (i.e., shape and intramolecular distance) for structure-refinement of gas-phase ions. We add microsolvation calculations to assess the interaction sites and energies between the biomolecular ions and gaseous additives. This combined strategy is employed to distinguish conformers and understand the gas-phase structures of two isomeric α-helical peptides that might differ in helicity. Our work allows more stringent structural characterization of biologically relevant molecules (e.g., peptide drugs) and large biomolecular ions than using only a single structural methodology in the gas phase.

Structural Studies of a Stapled Peptide with Native Ion Mobility-Mass Spectrometry and Transition Metal Ion Förster Resonance Energy Transfer in the Gas Phase.

Native mass spectrometry has emerged as an important tool for gas-phase structural biology. However, the conformations that a biomolecular ion adopts in the gas phase can differ from those found in solution. Herein, we report a synergistic, native ion mobility-mass spectrometry (IM-MS) and transition metal ion Förster resonance energy transfer (tmFRET)-based approach to probe the gas-phase ion structures of a nonstapled peptide (nsp; Ac-CAARAAHAAAHARARA-NH2) and a stapled peptide (sp; Ac-CXARAXHAAAHARARA-NH2). The stapled peptide contains a single hydrocarbon chain connecting the peptide backbone in the i and i + 4 positions via a Grubbs ring-closure metathesis. Fluorescence lifetime measurements indicated that the Cu-bound complexes of carboxyrhodamine 6g (crh6g)-labeled stapled peptide (sp-crh6g) had a shorter donor-acceptor distance (rDA) than the labeled nonstapled peptide (nsp-crh6g). Experimental collision cross-section (CCS) values were then determined by native IM-MS, which could separate the conformations of Cu-bound complexes of nsp-crh6g and sp-crh6g. Finally, the experimental CCS (i.e., shape) and rDA (i.e., distance) values were used as constraints for computational studies, which unambiguously revealed how a staple reduces the elongation of the peptide ions in the gas phase. This study demonstrates the superiority of combining native IM-MS, tmFRET, and computational studies to investigate the structure of biomolecular ions.

Adapting a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer for Gas-Phase Fluorescence Spectroscopy Measurement of Trapped Biomolecular Ions.

Gas-phase fluorescence spectroscopy is still in its infancy, which demands further instrumental developments. In this study, a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), equipped with a lab-developed data acquisition system, was coupled to a tunable femtosecond laser and a state-of-the-art optical system for fluorescence studies of mass-selected ions. For excitation, a laser beam was focused (beam size < 1.0 mm) into the cylindrical ICR cell. A wire mesh replaced the back trapping plate, allowing ∼10% of the fluorescence emitted from trapped ions to be collected by a lens installed beside the wire mesh. The collected fluorescence light was then transmitted outside of the mass spectrometer via fiber optics. A novel accumulation during detection (ADD) scheme was developed to increase the duty cycle of gas-phase fluorescence spectroscopy experiments. With ADD, >90% duty cycle for mass spectrometry and fluorescence experiments could be achieved. This instrument was able to perform fluorescence experiments on various ions, from simple rhodamine dyes to large biomolecules (i.e., peptides and proteins) labeled with dyes of various optical properties. A fluorescence lifetime measurement of trapped rhodamine 6G cations was also performed, yielding a value of 5.97 ± 0.23 ns. This setup has a broad mass range and decent fluorescence spectroscopy performance (i.e., the emission spectrum of rhodamine 6G can be acquired with good S/N in a minute). Finally, this setup also allows more challenging gas-phase fluorescence spectroscopy experiments, for example, of low quantum yield fluorophores and large biomolecules in their native state that appear at high m/z, which may not be doable with quadrupole ion traps (QIT).

Transition Metal Ion FRET in the Gas Phase: A 10-40 Å Range Molecular Ruler for Mass-Selected Biomolecular Ions.

Structural studies of mass-selected biomolecules in the gas phase can reveal their intrinsic properties and provide useful benchmarks for biomolecular modeling. Here, we report the first evidence of transition metal ion FRET (tmFRET) in the gas phase and its application to measure short (10-40 Å) biomolecular backbone distances. The measured FRET efficiencies in rhodamine dye (donor) labeled helical peptides complexed with Cu2+ ions (acceptor) decreased with increasing donor - acceptor distances, confirming the occurrence of tmFRET. The distances estimated for similar peptide sequences from the FRET efficiencies were consistently longer in the gas phase compared to those reported in solution, indicating an expanded structure and a possible loss of helicity.

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.

How Peptides Dissociate in Plasmonic Hot Spots.

Plasmon-induced hot carriers enable dissociation of strong chemical bonds by visible light. This unusual chemistry has been demonstrated for several diatomic and small organic molecules. Here, the scope of plasmon-driven photochemistry is extended to biomolecules and the reactivity of proteins and peptides in plasmonic hot spots is described. Tip-enhanced Raman spectroscopy (TERS) is used to both drive the reactions and to monitor their products. Peptide backbone bonds are found to dissociate in the hot spot, which is reflected in the disappearance of the amide I band in the TER spectra. The observed fragmentation pathway involves nonthermal activation, presumably by dissociative capture of a plasmon-induced hot electron. This fragmentation pathway is known from electron transfer dissociation (ETD) of peptides in gas-phase mass spectrometry (MS), which suggests a general similarity between plasmon-induced photochemistry and nonergodic reactions triggered by electron capture. This analogy may serve as a design principle for plasmon-induced reactions of biomolecules.

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.