Enhanced Declustering Enables Native Top-Down Analysis of Membrane Protein Complexes using Ion-Mobility Time-Aligned Fragmentation.

Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries.

Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid-Liquid Phase Separation.

Liquid-liquid phase separation (LLPS) is a process by which biomacromolecules, particularly proteins, condense into a dense phase that resembles liquid droplets. Dysregulation of LLPS is implicated in disease, yet the relationship between protein conformational changes and LLPS remains difficult to discern. This is due to the high flexibility and disordered nature of many proteins that phase separate under physiological conditions and their tendency to oligomerize.

Energy-Resolved Ion Mobility Spectrometry: Composite Breakdown Curves for Distinguishing Isomeric Product Ions.

Identification of lipopeptides (AA) synthesized from bacteria involves the study of structural characterization. Twenty AA have been characterized using commercial tandem high-resolution mass spectrometers in negative electrospray, employing nonresonant excitation in "RF only" collision cells and generally behave identically. However, [AA-H] (AA = Asp or Glu) shows surprising fragmentation pathways, yielding a complementary fatty acid carboxylate and dehydrated amino acid fragment anions.

Protein Unfolding in Freeze Frames: Intermediate States are Revealed by Variable-Temperature Ion Mobility-Mass Spectrometry.

The gas phase is an idealized laboratory for the study of protein structure, from which it is possible to examine stable and transient forms of mass-selected ions in the absence of bulk solvent. With ion mobility-mass spectrometry (IM-MS) apparatus built to operate at both cryogenic and elevated temperatures, we have examined conformational transitions that occur to the monomeric proteins: ubiquitin, lysozyme, and α-synuclein as a function of temperature and in source activation. We rationalize the experimental observations with a temperature-dependent framework model and comparison to known conformers.

Mapping Isomeric Peptides Derived from Biopharmaceuticals Using High-Resolution Ion Mobility Mass Spectrometry.

The identification and localization of isomeric peptide modifications is a critical requirement of the biopharmaceutical industry. Despite the ability of liquid chromatography-mass spectrometry to identify many of the common post translational modifications, the identification of isobaric or racemized peptides is confounded by modern mass spectrometry-based techniques. Here, we present a novel approach combining liquid chromatography with a high-resolution ion mobility mass spectrometry system to differentiate peptide and peptide fragments based upon their mobility and mass.

Investigations into pesticide charge site isomers using conventional IM and cIM systems.

A growing number of pesticides are being used around the world necessitating strict regulatory policies to guarantee consumer safety. Liquid Chromatography - Mass Spectrometry (LC-MS) is a highly sensitive method for pesticide screening, which provides retention time, mass/charge ratios and the relative abundances of characteristic product ions. Variability in the latter necessitates relatively large tolerances (±30%, SANCO/12682/2019, current EU regulation).

Cyclic Ion Mobility-Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase.

Ion mobility coupled to mass spectrometry (IM-MS) is widely used to study protein dynamics and structure in the gas phase. Increasing the energy with which the protein ions are introduced to the IM cell can induce them to unfold, providing information on the comparative energetics of unfolding between different proteoforms. Recently, a high-resolution cyclic IM-mass spectrometer (cIM-MS) was introduced, allowing multiple, consecutive tandem IM experiments (IM) to be carried out.

LESA Cyclic Ion Mobility Mass Spectrometry of Intact Proteins from Thin Tissue Sections.

Liquid extraction surface analysis (LESA) is an ambient surface sampling technique that allows the analysis of intact proteins directly from tissue samples via mass spectrometry. Integration of ion mobility separation to LESA mass spectrometry workflows has shown significant improvements in the signal-to-noise ratios of the resulting protein mass spectra and hence the number of proteins detected. Here, we report the use of a quadrupole-cyclic ion mobility-time-of-flight mass spectrometer (Q-cIM-ToF) for the analysis of proteins from mouse brain and rat kidney tissues sampled via LESA.

Collision Cross Sections of Charge-Reduced Proteins and Protein Complexes: A Database for Collision Cross Section Calibration.

The use of charge-reducing reagents to generate lower-charge ions has gained popularity in the field of native mass spectrometry (MS) and ion mobility mass spectrometry (IM-MS). This is because the lower number of charged sites decreases the propensity for Coulombic repulsions and unfolding/restructuring, helping to preserve the native-like structure. Furthermore, lowering the charge state consequently increases the mass-to-charge values (/), effectively increasing spacing between signals originating from small mass differences, such as different proteoforms or protein-drug complexes.

High-resolution ion mobility spectrometry-mass spectrometry of isomeric/isobaric ribonucleotide variants.

In this report, we explored the benefits of cyclic ion mobility (cIM) mass spectrometry in the analysis of isomeric post-transcriptional modifications of RNA. Standard methyl-cytidine samples were initially utilized to test the ability to correctly distinguish different structures sharing the same elemental composition and thus molecular mass. Analyzed individually, the analytes displayed characteristic arrival times (t ) determined by the different positions of the modifying methyl groups onto the common cytidine scaffold.

Carbohydrate isomer resolution via multi-site derivatization cyclic ion mobility-mass spectrometry.

Oligosaccharides serve many roles in extant life and may have had a significant role in prebiotic chemistry on the early Earth. In both these contexts, the structural and isomeric diversity among carbohydrates presents analytical challenges necessitating improved separations. Here, we showcase a chemical derivatization approach, where 3-carboxy-5-nitrophenylboronic acid (3C5NBA) is used to label vicinal hydroxyl groups, amplifying the structural difference between isomers.

Isolation of Crude Oil Peaks Differing by / ∼0.1 via Tandem Mass Spectrometry Using a Cyclic Ion Mobility-Mass Spectrometer.

Mass spectrometry is widely used in studying the structures of compounds present in crude oil. In this study, a novel mass spectrometer incorporating a cyclic ion mobility separator was used to obtain tandem mass spectra of crude oil compounds in a narrow mass-to-charge ratio ( window. Isolation of specific peaks was performed by combining quadrupole and ion mobility separation.

Structure Determination of Large Isomeric Oligosaccharides of Natural Origin through Multipass and Multistage Cyclic Traveling-Wave Ion Mobility Mass Spectrometry.

Carbohydrate isomers with identical atomic composition cannot be distinguished by mass spectrometry. By separating the ions according to their conformation in the gas phase, ion mobility (IM) coupled to mass spectrometry is an attractive approach to overcome this issue and extend the limits of mass spectrometry in structural glycosciences. Recent technological developments have significantly increased the resolving power of ion mobility separators.

A Cyclic Ion Mobility-Mass Spectrometry System.

Improvements in the performance and availability of commercial instrumentation have made ion mobility-mass spectrometry (IM-MS) an increasingly popular approach for the structural analysis of ionic species as well as for separation of complex mixtures. Here, a new research instrument is presented which enables complex experiments, extending the current scope of IM technology. The instrument is based on a Waters SYNAPT G2-S i IM-MS platform, with the IM separation region modified to accept a cyclic ion mobility (cIM) device.

Gas Phase Stability of Protein Ions in a Cyclic Ion Mobility Spectrometry Traveling Wave Device.

Ion mobility mass spectrometry (IM-MS) allows separation of native protein ions into "conformational families". Increasing the IM resolving power should allow finer structural information to be obtained and can be achieved by increasing the length of the IM separator. This, however, increases the time that protein ions spend in the gas phase and previous experiments have shown that the initial conformations of small proteins can be lost within tens of milliseconds.

Cyclic Ion Mobility Mass Spectrometry Distinguishes Anomers and Open-Ring Forms of Pentasaccharides.

There is increasing biopharmaceutical interest in oligosaccharides and glycosylation. A key requirement for these sample types is the ability to characterize the chain length, branching, type of monomers, and importantly stereochemistry and anomeric configuration. Herein, we showcase the multi-function capability of a cyclic ion mobility (cIM) separator embedded in a quadrupole/time-of-flight mass spectrometer (Q-ToF MS).

Initial Steps of Amyloidogenic Peptide Assembly Revealed by Cold-Ion Spectroscopy.

The early stages of fibril formation are difficult to capture in solution. We use cold-ion spectroscopy to examine an 11-residue peptide derived from the protein transthyretin and clusters of this fibre-forming peptide containing up to five units in the gas phase. For each oligomer, the UV spectra exhibit distinct changes in the electronic environment of aromatic residues in this peptide compared to that of the monomer and in the bulk solution.

Structurally Flexible and Solution Stable [LnTM(OH)(L)(OCR)(MeOH)](ClO): A Playground for Magnetic Refrigeration.

The family of compounds of general formula [LnTM(OH)(L)(OCR)(MeOH)](ClO) {[GdZn(OH)(hmp)(OCPr)](ClO) (1a); [YZn(OH)(hmp)(OCPr)](ClO) (1b); [GdCu(OH)(hmp)(OCPr)](ClO) (2a); [YCu(OH)(hmp)(OCPr)](ClO) (2b); [GdCu(OH)(hep)(OCPr)](ClO) (3a); [GdCu(OH)(Hpdm)(OCBu)](ClO) (4a); [GdCu(OH)(ea)(OCMe)](ClO) (5a); [GdNi(OH)(hmp)(OCEt)(MeOH)](ClO) (6a); [YNi(OH)(hmp)(OCEt)(MeOH)](ClO) (6b); [GdCo(OH)(hmp)(OCEt)(MeOH)](ClO) (7a); [YCo(OH)(hmp)(OCEt)(MeOH)](ClO) (7b)} can be formed very simply and in high yields from the reaction of Ln(NO)·6HO and TM(ClO)·6HO and the appropriate ligand blend in a mixture of CHCl and MeOH in the presence of a suitable base. Remarkably, almost all the constituent parts, namely the lanthanide (or rare earth) ions Ln (here Ln = Gd or Y), the transition metal ions TM (here TM = Zn, Cu, Ni, Co), the bridging ligand L (Hhmp = 2-(hydroxymethyl)pyridine; Hhep = 2-(hydroxyethyl)pyridine; Hpdm = pyridine-2,6-dimethanol; Hea = 2-ethanolamine), and the carboxylates can be exchanged while maintaining the structural integrity of the molecule. NMR spectroscopy of diamagnetic complex 1b reveals the complex to be fully intact in solution with all signals from the hydroxide, ligand L, and the carboxylates equivalent on the NMR time scale, suggesting the complex possesses greater symmetry in solution than in the solid state.

New High Resolution Ion Mobility Mass Spectrometer Capable of Measurements of Collision Cross Sections from 150 to 520 K.

We present a new variable temperature (VT), high resolution ion mobility (IM) drift tube coupled to a commercial mass spectrometer (MS). Ions are generated in an electrospray ion source with a sampling cone interface and two stacked ring RF guides which transfer ions into the mobility analyzer located prior to a quadrupole time-of-flight mass spectrometer. The drift cell can be operated over a pressure range of 0.

Sizing and Discovery of Nanosized Polyoxometalate Clusters by Mass Spectrometry.

Ion mobility-mass spectrometry (IM-MS) is a powerful technique for structural characterization, e.g., sizing and conformation, particularly when combined with quantitative modeling and comparison to theoretical values.

Making hybrid [n]-rotaxanes as supramolecular arrays of molecular electron spin qubits.

Quantum information processing (QIP) would require that the individual units involved--qubits--communicate to other qubits while retaining their identity. In many ways this resembles the way supramolecular chemistry brings together individual molecules into interlocked structures, where the assembly has one identity but where the individual components are still recognizable. Here a fully modular supramolecular strategy has been to link hybrid organic-inorganic [2]- and [3]-rotaxanes into still larger [4]-, [5]- and [7]-rotaxanes.

UV photodissociation of trapped ions following ion mobility separation in a Q-ToF mass spectrometer.

An ion mobility mass spectrometer has been modified to allow optical interrogation of ions with different mass-to-charge (m/z) ratios and/or mobilities (K). An ion gating and trapping procedure has been developed which allows us to store ions for several seconds enabling UV photodissociation (UVPD).

Luminescent, enantiopure, phenylatopyridine iridium-based coordination capsules.

The first molecular capsule based on an [Ir(ppy)(2)](+) unit (ppy = 2-phenylatopyridine) has been prepared. Following the development of a method to resolve rac-[(Ir(ppy)(2)Cl)(2)] into its enantiopure forms, homochiral Ir(6)L(4) octahedra where obtained with the tritopic 1,3,5-tricyanobenzene. Solution studies and X-ray diffraction show that these capsules encapsulate four of the six associated counteranions and that these can be exchanged for other anionic guests.

Shapes of supramolecular cages by ion mobility mass spectrometry.

Mass spectrometry and drift tube ion mobility mass spectrometry have been used to analyse several isobaric, multicomponent cages yielding information on three dimensional structure, interactions and dynamics of assembly in the gas phase.