Peter B. O’ Connor

Two-dimensional mass spectrometry (2DMS) is a powerful data-independent analysis (DIA) technique that allows mixtures to be analyzed without chromatographic separation of the components. Until now, high-resolution 2DMS has only been possible using Fourier Transform Ion Cyclotron Resonance (FTICR) instruments, which are impractical for most laboratories. Herein, we present the first experimental demonstration of 2DMS using a quadrupole time-of-flight (QToF) mass spectrometer. These instruments are commonplace, but offer high resolution and the ability to assign peaks in the 2D spectrum based on accurate mass. The key development is the application of stored waveform ion radius modulation (SWIM) to ions trapped in the quadrupole prior to mass analysis by the ToF analyzer. SWIM is an encoding technique that uses broadband dipolar excitation and a radius-dependent fragmentation method to modulate precursor and product ion signals. We show how each component of a peptide mixture can be individually, yet simultaneously, sequenced using collision-induced dissociation (CID) and/or ultraviolet photodissociation (UVPD).

Cyclic peptide-polymer conjugates offer a unique biocompatible system with many advantages but come at the cost of being analytically challenging. Developing further analytical techniques of complex polymer-conjugate systems is key to understanding synthetic and medicinal properties. In this contribution, a synthetic cyclic peptide-polymer conjugate is analyzed using electron capture dissociation (ECD), infrared multiphoton absorption dissociation (IRMPD), and 193 nm ultraviolet photodissociation (UVPD) on the same mass spectrometry system. IRMPD and UVPD were shown to effectively characterize unconjugated cyclic peptide species. ECD was less informative during cyclic peptide analysis due to the production of multiple sequence scrambling fragments and radical side chain losses. ECD was shown to produce extensive fragmentation and enable the characterization of conjugated side chains of cyclic species. ECD and IRMPD thus provided complementary data, enabling the target analysis of conjugated systems. UVPD effectively characterized both the cyclic peptide and the conjugating polymer in one experiment, being able to produce complete cyclic peptide fragmentation via b/y fragment pathways and polymer fragmentation via a/x poly(2-ethyl-2-oxazoline) fragment pathways.

The analysis of complex biological mixtures remains a significant challenge in mass spectrometry (MS), particularly when using conventional direct infusion MS/MS approaches due to inherent limitations in resolving power and spectral complexity. Here, we demonstrate the integration of trapped ion mobility spectrometry (TIMS) with two-dimensional mass spectrometry (2DMS) to enable high-resolution TIMS-MS/2DMS experiments for detailed protein characterization within mixtures. TIMS provides separation based on the ion's size-to-charge ratio, effectively reducing the occurrence of chimeric tandem mass spectra containing fragments from more than one precursor ion. This coupling allows for an improved peak capacity and reduced ambiguity in tandem spectral interpretation. When applied to a model protein mixture, the TIMS-MS/2DMS method allows resolution of near m/z species, including isomeric and isonucleonic species, and it was possible to assign secondary fragmentation with greater confidence.

Two-dimensional mass spectrometry (2DMS) is a truly data-independent acquisition technique used in the analysis of complex mixtures; however, the nature of the noise within these spectra is not well understood. In this work, 2DMS is tested for conformity with the Fellgett principle: (signal/noise) proportional to root (no. of data points). Since 2DMS functions through the modulation of ions through a fragmentation region across many scans, the individual scans are considered data points in this experiment. Random noise was shown to be prevalent as the main source of noise in this experiment with minor systematic noise. This means that the minimum size for a 2DMS spectrum that displays a target fragment ion can be determined using a fast-2D equation detailed herein. The effects of existing denoising algorithms were also found to change the relationship between the signal-to-noise ratio and the scan numbers to be of a quasi-linear nature rather than the square root trend observed before denoising.

Multimodal tandem mass spectrometry sequencing of oligonucleotides using CID, EDD, IRMPD, and UVPD as fragmentation methods, and using 2-dimensional mass spectrometry with signal averaging to differentiate fragments from closely spaced precursors.

The accumulation of amyloid plaques and increased brain redox burdens are neuropathological hallmarks of Alzheimer's disease. Altered metabolism of essential biometals is another feature of Alzheimer's, with amyloid plaques representing sites of disturbed metal homeostasis. Despite these observations, metal-targeting disease treatments have not been therapeutically effective to date. A better understanding of amyloid plaque composition and the role of the metals associated with them is critical. To establish this knowledge, the ability to resolve chemical variations at nanometer length scales relevant to biology is essential. Here, we present a methodology for the label-free, nanoscale chemical characterization of amyloid plaques within human Alzheimer's disease tissue using synchrotron X-ray spectromicroscopy. Our approach exploits a C-H carbon absorption feature, consistent with the presence of lipids, to visualize amyloid plaques selectively against the tissue background, allowing chemical analysis to be performed without the addition of amyloid dyes that alter the native sample chemistry. Using this approach, we show that amyloid plaques contain elevated levels of calcium, carbonates, and iron compared to the surrounding brain tissue. Chemical analysis of iron within plaques revealed the presence of chemically reduced, low-oxidation-state phases, including ferromagnetic metallic iron. The zero-oxidation state of ferromagnetic iron determines its high chemical reactivity and so may contribute to the redox burden in the Alzheimer's brain and thus drive neurodegeneration. Ferromagnetic metallic iron has no established physiological function in the brain and may represent a target for therapies designed to lower redox burdens in Alzheimer's disease. Additionally, ferromagnetic metallic iron has magnetic properties that are distinct from the iron oxide forms predominant in tissue, which might be exploitable for the in vivo detection of amyloid pathologies using magnetically sensitive imaging. We anticipate that this label-free X-ray imaging approach will provide further insights into the chemical composition of amyloid plaques, facilitating better understanding of how plaques influence the course of Alzheimer's disease.

Proteolysis targeting chimeras (PROTACs) are specialized molecules that bind to a target protein and a ubiquitin ligase to facilitate protein degradation. Despite their significance, native PROTACs have not undergone tandem mass spectrometry (MS) analysis. To address this gap, we conducted a pioneering investigation on the fragmentation patterns of two PROTACs in development, dBET1 and VZ185. Employing diverse cations (sodium, lithium, and silver) and multiple tandem-MS techniques, we enhanced their structural characterization. Notably, lithium cations facilitated comprehensive positive-mode coverage for dBET1, while negative polarity mode offered richer insights. Employing de novo structure determination on 2DMS data from degradation studies yielded crucial insights. In the case of VZ185, various charge states were observed, with [M + 2H]²⁺ revealing fewer moieties than [M + H]⁺ due to charge-related factors. Augmenting structural details through silver adducts suggested both charge-directed and charge-remote fragmentation. This comprehensive investigation identifies frequently dissociated bonds across multiple fragmentation techniques, pinpointing optimal approaches for elucidating PROTAC structures. The findings contribute to advancing our understanding of PROTACs, pivotal for their continued development as promising therapeutic agents.

Sample preparation of complex, natural mixtures such as lignin prior to mass spectrometry analysis, however minimal, is a critical step in ensuring accurate and interference-free results. Modern shotgun-MS techniques, where samples are directly injected into a high-resolution mass spectrometer (HRMS) with no prior separation, usually still require basic sample pretreatment such as filtration and appropriate solvents for full dissolution and compatibility with atmospheric pressure ionization interfaces. In this study, sample preparation protocols have been established for a unique sample set consisting of a wide variety of degraded lignin samples from numerous sources and treatment processes. The samples were analyzed via electrospray (ESI)-HRMS in negative and positive ionization modes. The resulting information-rich HRMS datasets were then transformed into the mass defect space with custom R scripts as well as the open-source Constellation software as an effective way to visualize changes between the samples due to the sample preparation and ionization conditions as well as a starting point for comprehensive characterization of these varied sample sets. Optimized conditions for the four investigated lignins are proposed for ESI-HRMS analysis for the first time, giving an excellent starting point for future studies seeking to better characterize and understand these complex mixtures. Graphical Abstract

Two-dimensional mass spectrometry (2DMS) allows for the analysis of complex mixtures of all kinds at high speed and resolution without data loss from isolation or biased acquisition, effectively generating tandem mass spectrometry information for all ions at once. Currently, this technique is limited to instruments utilizing an ion trap such as the Fourier transform ion cyclotron resonance or linear ion traps. To overcome this limitation, new fragmentation waveforms were used in either a temporal or spatial configuration, allowing for the application of 2DMS on a much wider array of instruments. A simulated example of a time-of-flight-based instrument is shown with the new waveforms, which allowed for the correlation of fragment ions to their respective precursors through the processing of the modulation of fragmentation intensity with a Fourier transform. This application indicated that 2D modulation and Fourier precursor/fragment intensity correlation are possible in any case where separation, either temporally or spatially, can be achieved, allowing 2DMS to be applied to almost every type of mass spectrometry instrument.

Ultraviolet photodissociation is a fast, photon-mediated fragmentation method that yields high sequence coverage and informative cleavages of biomolecules. In this work, 193 nm UVPD was coupled with a 12 Tesla FT-ICR mass spectrometer and 10.6 μm infrared multi-photon dissociation to provide gentle slow-heating of UV-irradiated ions. No internal instrument hardware modifications were required. Adjusting the timing of laser pulses to the ion motion within the ICR cell provided consistent fragmentation yield shot-to-shot and may also be used to monitor ion positions within the ICR cell. Single-pulse UVPD of the native-like 5+ charge state of ubiquitin resulted in 86.6% cleavage coverage. Additionally, IR activation post UVPD doubled the overall fragmentation yield and boosted the intensity of UVPD-specific x-type fragments up to 4-fold. This increased yield effect was also observed for the 6+ charge state of ubiquitin, albeit less pronounced. This indicates that gentle slow-heating serves to sever tethered fragments originating from non-covalently linked compact structures and makes activation post UVPD an attractive option to boost fragmentation efficiency for top-down studies. Lastly, UVPD was implemented and optimized as a fragmentation method for 2DMS, a data-independent acquisition method. UVPD-2DMS was demonstrated to be a viable method using BSA digest peptides as a model system.