Advancing PROTAC Characterization: Structural Insights through Adducts and Multimodal Tandem-MS Strategies.

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.

Characterizing lignins from various sources and treatment processes after optimized sample preparation techniques and analysis via ESI-HRMS and custom mass defect software tools.

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.

Chemoselective derivatisation and ultrahigh resolution mass spectrometry for the determination of hydroxyl functional groups within complex bio-oils.

Bio-oils are a renewable alternative resource for the production of fine chemicals and fuels. Bio-oils are characterised by a high content of oxygenated compounds with a diverse array of different chemical functionalities. Here, we performed a chemical reaction to transform the hydroxyl group of the various components in a bio-oil prior to characterisation with ultrahigh resolution mass spectrometry (UHRMS). The derivatisations were first evaluated using twenty lignin-representative standards with different structural features. Our results indicate a highly chemoselective transformation of the hydroxyl group despite the presence of other functional groups. Mono- and di-acetate products were observed in acetone-acetic anhydride (acetone-Ac2O) mixtures for non-sterically hindered phenols, catechols and benzene diols. Dimethyl sulfoxide-Ac2O (DMSO-Ac2O) reactions favoured the oxidation of primary and secondary alcohols and the formation of methylthiomethyl (MTM) products of phenols. The derivatisations were then performed in a complex bio-oil sample to gain insights into the hydroxyl group profile of the bio-oil. Our results indicate that the bio-oil before derivatisation is composed of 4500 elemental compositions containing 1-12 oxygen atoms. After the derivatisation in DMSO-Ac2O mixtures, the total number of compositions increased approximately five-fold. The reaction was indicative of the variety of hydroxyl group profiles within the sample in particular the presence of phenols that were ortho and para substituted, non-hindered phenols (about 34%), aromatic alcohols (including benzylic and other non-phenolic alcohols) (25%), and aliphatic alcohols (6.3%) could be inferred. Phenolic compositions are known as coke precursors in catalytic pyrolysis and upgrading processes. Thus, the combination of chemoselective derivatisations in conjunction with UHRMS can be a valuable resource to outline the hydroxyl group profile in elemental chemical compositions in complex mixtures.

Analytical chemistry solutions to hazard evaluation of petroleum refining products.

Products of petroleum refining are substances that are both complex and variable. These substances are produced and distributed in high volumes; therefore, they are heavily scrutinized in terms of their potential hazards and risks. Because of inherent compositional complexity and variability, unique challenges exist in terms of their registration and evaluation. Continued dialogue between the industry and the decision-makers has revolved around the most appropriate approach to fill data gaps and ensure safe use of these substances. One of the challenging topics has been the extent of chemical compositional characterization of products of petroleum refining that may be necessary for substance identification and hazard evaluation. There are several novel analytical methods that can be used for comprehensive characterization of petroleum substances and identification of most abundant constituents. However, translation of the advances in analytical chemistry to regulatory decision-making has not been as evident. Therefore, the goal of this review is to bridge the divide between the science of chemical characterization of petroleum and the needs and expectations of the decision-makers. Collectively, mutual appreciation of the regulatory guidance and the realities of what information these new methods can deliver should facilitate the path forward in ensuring safety of the products of petroleum refining.

Characterization of Mineral and Synthetic Base Oils by Gas Chromatography-Mass Spectrometry and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Base oil is a main component of engine oil that enables smooth operation of an internal combustion engine. There are two types of base oils, such as mineral oil and synthetic oil. In this study, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and gas chromatography-mass spectrometry (GC-MS) were used to characterize the base oils. One difficulty in analyzing base oils using MS is that the ionization of alkanes can be problematic due to low ionization efficiencies and the predominance of fragmentation. Despite these limitations, the combination of GC-MS and FT-ICR MS data can provide qualitative insights into the composition differences for these various sample types. The distinctive total ion chromatogram obtained by GC-MS of the different base oils allowed the classification of mineral oil from synthetic oil. The additional structural characteristics of paraffinic compounds were also inferred by GC-MS. FT-ICR MS coupled to two different ionization methods, atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI), was tested for the analysis of base oils. It was determined that APPI was suitable for the analysis of aliphatic hydrocarbon compounds, where APPI minimizes the decomposition of hydrocarbon compounds compared to atmospheric pressure chemical ionization. Using APPI FT-ICR MS, the components of the oils were characterized, including not only paraffinic compounds but also cyclic compounds. In addition, the alpha olefin monomer of the synthetic oil was determined, and the homogeneity of the branched compound of the synthetic base oil was confirmed using GC-MS and FT-ICR MS results.

Enhancing Biomolecule Analysis and 2DMS Experiments by Implementation of (Activated Ion) 193 nm UVPD on a FT-ICR Mass Spectrometer.

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.

Use of QuEChERS as a manual and automated high-throughput protocol for investigating environmental matrices.

Environmental pollution has strong links to adverse human health outcomes with risks of pollution through production, use, ineffective wastewater (WW) remediation, and/or leachate from landfill. 'Fit-for-purpose' monitoring approaches are critical for better pollution control and mitigation of harm, with current sample preparation methods for complex environmental matrices typically time-consuming and labour intensive, unsuitable for high-throughput screening. This study has shown that a modified 'Quick Easy Cheap Effective Rugged and Safe' (QuEChERS) sample preparation is a viable alternative for selected environmental matrices required for pollution monitoring (e.g. WW effluent, treated sludge cake and homogenised biota tissue). As a manual approach, reduced extraction times (hours to ∼20 min/sample) with largely reproducible (albeit lower) recoveries of a range of pharmaceuticals and biocidal surfactants have been reported. Its application has shown clear differentiation of matrices via chemometrics, and the measurement of pollutants of interest to the UK WW industry at concentrations significantly above suggested instrument detection limits (IDL) for sludge, indicating insufficient removal and/or bioaccumulation during WW treatment. Furthermore, new pollutant candidates of emerging concern were identified - these included detergents, polymers and pharmaceuticals, with quaternary ammonium compound (QAC) biocides observed at 2.3-70.4 mg/kg, and above levels associated with priority substances for environmental quality regulation (EQSD). Finally, the QuEChERS protocol was adapted to function as a fully automated workflow, further reducing the resource to complete both the preparation and analysis to <40 min. This operated with improved recovery for soil and biota (>62%), and when applied to a largely un-investigated clay matrix, acceptable recovery (88.0-131.1%) and precision (≤10.3% RSD) for the tested pharmaceuticals and biocides was maintained. Therefore, this preliminary study has shown the successful application of a high-throughput QuEChERS protocol across a range of environmental solids for potential deployment in a regulated laboratory.

Investigating the Influence of n-Heptane versus n-Nonane upon the Extraction of Asphaltenes.

The composition of asphaltenes is of interest due to the challenges they pose for industry and their high complexity, encompassing a range of heteroatom contents, molecular weights, double bond equivalents (DBEs), and structural motifs. They are well-known for aggregating above critical concentrations, hindering the upstream and downstream processes. Asphaltenes are defined by solubility, as they are insoluble in light paraffins such as n-heptane and soluble in aromatic solvents such as toluene. Today, enormous efforts are being invested into the characterization of asphaltenes to shed light into their structural profiles to benefit the petroleum industry and environmental sustainability. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides molecular level analysis with unparalleled mass resolving power and mass accuracy, which is vital for the characterization of inherently complex crude oils and their asphaltene fractions. The aim of this research is to elucidate and compare the compositional profiles of asphaltene fractions of two petroleum samples, fractioned through two approaches: using n-heptane, as is typical practice, and n-nonane, for the purpose of testing extraction using higher molecular weight alkanes. The results highlight that the choice of solvents does indeed influence the accessibility of different species and therefore changes the observed molecular profiles of the extracted asphaltenes. n-Heptane afforded broader contributions of different heteroatomic classes and greater carbon number ranges of the observed components; the DBE distribution vs carbon number profiles were different, where the extracts produced using n-nonane displayed a greater prevalence of lower DBE species.

Stochasticity of poly(2-oxazoline) oligomer hydrolysis determined by tandem mass spectrometry.

Understanding modification of synthetic polymer structures is necessary for their accurate synthesis and potential applications. In this contribution, a series of partially hydrolyzed poly(2-oxazoline) species were produced forming poly[(2-polyoxazoline)-co-(ethylenimine)] (P(EtOx-co-EI)) copolymers; EI being the hydrolyzed product of Ox. Bulk mass spectrometry (MS) measurements accurately measured the EI content. Tandem mass spectrometry analysis of the EI content in the copolymer samples determined the distribution of each monomer within the copolymer and corresponded to a theoretically modelled random distribution. The EI distribution across the polymers was shown to be effected by the choice of terminus, with a permanent hydrolysis event observed at an OH terminus. A neighbouring group effect wasn't observed at the polymer length analysed (approximately 25-mer species), suggesting that previously observed neighbouring group effects require a larger polymer chain. Although clearly useful for random polymer distribution this approach may be applied to many systems containing non-specific modifications to determine if they are directed or random locations across peptides, proteins, polymers, and nucleic acids.

Fine Structure in Isotopic Peak Distributions Measured Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: A Comparison between an Infinity ICR Cell and a Dynamically Harmonized ICR Cell.

The fine structure of isotopic peak distributions of glutathione in mass spectra is measured using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) at 12 and 15 T magnetic field, with an infinity cell and a dynamically harmonized cell (DHC) respectively. The resolved peaks in the fine structure of glutathione consist of 2H, 13C, 15N, 17O, 18O, 33S, 34S, 36S, and combinations of them. The positions of the measured fine structure peaks agree with the simulated isotopic distributions with the mass error less than 250 ppb in broadband mode for the infinity cell and no more than 125 ppb with the DHC after internal calibration. The 15 T FT-ICR MS with DHC cell also resolved around 30 isotopic peaks in broadband with a resolving power (RP) of 2 M. In narrowband (m/z 307-313), our current highest RP of 13.9 M in magnitude mode was observed with a 36 s transient length by the 15 T FT-ICR MS with the DHC and 2ω detection on the 15 T offers slightly higher RP (14.8 M) in only 18 s. For the 12 T FT-ICR MS with the infinity cell, the highest RP achieved was 15.6 M in magnitude mode with a transient length of 45 s. Peak decay was observed for low abundance peaks, which could be due to the suppression effects from the most abundant peak, as result of ion cloud Coulombic interactions (space-charge).

Multimodal Tandem Mass Spectrometry Techniques for the Analysis of Phosphopeptides.

Collisionally activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), electron capture dissociation and electron detachment dissociation (EDD) experiments were conducted on a set of phosphopeptides, in a Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation patterns were compared and varied according to the fragmentation mechanisms and the composition of the peptides. CAD and IRMPD produced similar fragmentation profiles of the phosphopeptides, while UVPD produced a large number of complementary fragments. Electron-based dissociation techniques displayed lower fragmentation efficiencies, despite retaining the labile phosphate group, and drastically different fragmentation profiles. EDD produced complex spectra whose interpretation proved challenging.

Revealing the Reactivity of Individual Chemical Entities in Complex Mixtures: the Chemistry Behind Bio-Oil Upgrading.

Bio-oils are precursors for biofuels but are highly corrosive necessitating further upgrading. Furthermore, bio-oil samples are highly complex and represent a broad range of chemistries. They are complex mixtures not simply because of the large number of poly-oxygenated compounds but because each composition can comprise many isomers with multiple functional groups. The use of hyphenated ultrahigh-resolution mass spectrometry affords the ability to separate isomeric species of complex mixtures. Here, we present for the first time, the use of this powerful analytical technique combined with chemical reactivity to gain greater insights into the reactivity of the individual isomeric species of bio-oils. A pyrolysis bio-oils and its esterified bio-oil were analyzed using gas chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry, and in-house software (KairosMS) was used for fast comparison of the hyphenated data sets. The data revealed a total of 10,368 isomers in the pyrolysis bio-oil and an increase to 18,827 isomers after esterification conditions. Furthermore, the comparison of the isomeric distribution before and after esterification provide new light on the reactivities within these complex mixtures; these reactivities would be expected to correspond with carboxylic acid, aldehyde, and ketone functional groups. Using this approach, it was possible to reveal the increased chemical complexity of bio-oils after upgrading and target detection of valuable compounds within the bio-oils. The combination of chemical reactions alongside with in-depth molecular characterization opens a new window for the understanding of the chemistry and reactivity of complex mixtures.

Solvent and Flow Rate Effects on the Observed Compositional Profiles and the Relative Intensities of Radical and Protonated Species in Atmospheric Pressure Photoionization Mass Spectrometry.

Sample preparation and instrument parameters have regularly been demonstrated to impact upon the observed results in atmospheric pressure photoionization, mass spectrometry (MS), and analytical techniques in general but may be overlooked when such methods are applied to the characterization of real-world samples. An initial investigation into different solvent systems demonstrated that the inclusion of ethyl acetate inverted the ratio of relative intensities of radical and protonated species (R/P). Design of experiments was performed and indicated that the injection flow rate is also a significant factor. The impact of the solvent system and flow rate on signal intensity, the observed compositional profile, and R/P of selected molecular groups is demonstrated further. An inversion of R/P is observed at higher flow rates in solvent systems commonly used in petroleomics studies, effecting a loss of molecular speciation. The findings presented reiterate the critical importance in considering experimental parameters when interpreting the results of analytical procedures.

Voice Behavior in Healthcare: A Scoping Review of the Study of Voice Behavior in Healthcare Workers.

BACKGROUND: Patient safety and quality care require learning organizations. For organizations to pivot and progress they need a workforce skilled and confident in voicing ideas and errors. Expressing issues related to patient safety and quality improvement can be defined as voice behavior. METHODS: This scoping review aimed to answer two questions: 1) What is the scope of the study of voice behavior in healthcare? 2) How do new graduates in allied health use their voice? The Joanna Briggs guidelines were used to identify 76 articles that were explored using a descriptive and evaluative approach to map the depth and breadth of this topic and identify research gaps. FINDINGS: The review revealed that nursing and medicine feature in voice behavior studies, but few studies focus on allied health. An exploration of the concepts emerging from these studies revealed eight topics: power, risk, tenure, leadership, intervention, self, climate, and strategies. CONCLUSION: Synthesis of the reviewed studies highlights that we know little about allied health new graduates' voice behavior in healthcare. Exploration of voice behaviors with this group will inform education and workplace settings about ways to grow professionals who are confident and competent in speaking up for patient safety and quality improvement.

Characterization Across a Dispersity: Polymer Mass Spectrometry in the Second Dimension.

Due to the natural dispersity that is present in synthetic polymers, an added complexity is always present in the analysis of polymeric species. Tandem mass spectrometry analysis requires the isolation of individual precursors before a fragmentation event to allow the unambiguous characterization of these species and is not viable at certain levels of complexity due to achievable isolation widths. Two-dimensional mass spectrometry (2DMS) fragments ions and correlates fragments with their corresponding precursors without the need for isolation. In this study, 2DMS electron capture dissociation (ECD) fragmentation of a polyoxazoline and polyacrylamide species was carried out, resulting in the analysis of byproducts and individual polymer species without the use of chromatographic techniques. This study shows that 2DMS ECD is a powerful tool for the analysis of polyacrylamide and polyoxazoline species and offers a new dimension in the characterization of polymers.

Combining Ultraviolet Photodissociation and Two-Dimensional Mass Spectrometry: A Contemporary Approach for Characterizing Singly Charged Agrochemicals.

Ultraviolet photodissociation (UVPD) has been shown to produce extensive structurally informative data for a variety of chemically diverse compounds. Herein, we demonstrate the performance of the 193 nm UVPD fragmentation technique on structural/moiety characterization of 14 singly charged agrochemicals. Two-dimensional mass spectrometry (2DMS) using infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID) have previously been applied to a select range of singly charged pesticides. The ≥80% moiety coverage achieved for the majority of the species by the UVPD and 2D-UVPD methods was on par with and, in some cases, superior to the data obtained by other fragmentation techniques in previous studies, demonstrating that UVPD is viable for these types of species. A three-dimensional (3D) peak picking method was implemented to extract the data from the 2DMS spectrum, overcoming the limitations of the line extraction method used in previous studies, successfully separating precursor specific fragments with milli-Dalton accuracy. Whole spectrum internal calibration combined with 3D peak picking obtained sub-part-per-million (ppm) to part-per-billion (ppb) mass accuracies across the entire 2DMS spectrum.

Two-Dimensional Mass Spectrometry Analysis of IgG1 Antibodies.

Two-dimensional mass spectrometry (2DMS) is a new, and theoretically ideal, data-independent analysis tool, which allows the characterization of a complex mixture and was used in the bottom-up analysis of IgG1 for the identification of post-translational modifications. The new peak picking algorithm allows the distinction between chimeric peaks in proteomics. In this application, the processing of 2DMS data correlates fragments to their corresponding precursors, with fragments from precursors which are <0.1 m/z at m/z 840 easily resolved, without the need for quadrupole or chromatographic separation.

Making their way as academics: A qualitative study examining how nurse academics understand and (re)construct academic identity.

OBJECTIVES: This study aims to explore the way in which nurse academics understand and (re)construct their academic identity. DESIGN: An exploratory qualitative approach was adopted. METHODS: Individual semi-structured interviews were conducted with 15 nurse academics who were employed in positions requiring both research and teaching in two New Zealand university nursing schools. Participants' demographic information and critical career incidents were also collected. Interview data were analysed thematically. RESULTS: Nurse academics understand their academic identity as research-related, relational and dynamic. Although their academic identity can be in conflict with their clinical one, the former is (re)constructed as an extension of the latter. To navigate each university's multiple expectations of academics, nurse academics in this study strive to maintain their engagement with teaching, research and clinical practice, although in some cases, clinical practice is reconceptualised in relation to teaching and researching rather than direct contact with patients. CONCLUSION: Academic identity development is fluid and interdependent on that of clinical identity in this young and vocationally-based discipline. There is an interrelated, dual process of identity (re)construction - while (re)constructing academic identity, nurse academics also reshape and redefine their clinical identity. The trajectory of their identity development challenges institutions to consider the needs of nurse academics (and others in similar disciplines) by recognising and supporting the maintenance of a form of clinical currency in order to strengthen the discipline within the academy.

Electron Capture Dissociation of Trithiocarbonate-Terminated Acrylamide Homo- and Copolymers: A Terminus-Directed Mechanism?

The structure and sequence elucidation of complex homo- and copolymers is key for further understanding polymers, polymer synthesis, and polymer interactions in biological processes. In this contribution, poly(dimethylacrylamide) homo- and dimethylacrylamide/4-acryloylmorpholine block copolymers were synthesized and analyzed by electron capture dissociation (ECD) and Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry. Double-resonance experiments were carried out, providing a better understanding of the fragmentation process. A novel radical dissociation process is presented, and electron capture caused a specific cleavage at the terminal butyl-trithiocarbonate group, which initiated a free radical dissociation process.