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Understanding the fundamental reactivity of polymetallic complexes is challenging due to the complexity of their structures with many possible bond breaking and forming processes. Here, we apply ion mobility mass spectrometry coupled with density functional theory to investigate the disassembly mechanisms and energetics of a family of heterometallic rings and rotaxanes with the general formula [NH

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and identification of robust biomarkers to complement clinical diagnosis will accelerate treatment options. Here we demonstrate the use of direct infusion of sebum from skin swabs using paper spray ionisation coupled with ion mobility mass spectrometry (PS-IM-MS) to determine the regulation of molecular classes of lipids in sebum that are diagnostic of PD. A PS-IM-MS method for sebum samples that takes three minutes per swab was developed and optimised. The method was applied to skin swabs collected from 150 people and elucidates ~ 4200 features from each subject which were independently analysed. The data included high molecular weight lipids (>600 Da.) that differ significantly in the sebum of people with PD. Putative metabolite annotations of several lipid classes, predominantly triglycerides and larger acyl glycerides, were obtained using accurate mass, tandem mass spectrometry and collision cross section measurements.

Understanding the fundamental reactivity of polymetallic complexes is challenging due to the complexity of their structures with many possible bond breaking and forming processes. Here we apply ion mobility mass spectrometry (IM-MS) coupled with density functional theory (DFT) to investigate the disassembly mechanisms and energetics of a family of heterometallic rings and rotaxanes with the general formula [NH2RR’][Cr7MF8(O2CtBu)16] with M = MnII, FeII, CoII, NiII, CuII, ZnII, CdII. Our results show that their stability can be tuned both by altering the d-metal composition in the macrocycle and by the end groups of the secondary ammonium cation [NH2RR’]+. Ion mobility probes the conformational landscape of the disassembly process from intact complex to structurally distinct isobaric fragments, providing unique insights to how a given divalent metal tunes the structural dynamics.

The effect of temperature on the stability of proteins is well explored above 298 K, but harder to track experimentally below 273 K. Variable-temperature ion mobility mass spectrometry (VT IM-MS) allows us to measure the structure of molecules at sub-ambient temperatures. Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies (mAbs) on cooling by measuring their collision cross sections (CCS) at discrete drift gas temperatures from 295 to 160 K. The CCS at 250 K is larger than predicted from collisional theory and experimental data at 295 K. This restructure is attributed to change in the strength of stabilizing intermolecular interactions. Below 250 K the CCS of the mAbs increases in line with prediction implying no rearrangement. Comparing data from isotypes suggest disulfide bridging influences thermal structural rearrangement. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the native fold and VT IM-MS measurements at sub ambient temperatures provide new insights to the phenomenon of cold denaturation.

Ultraviolet photodissociation (UVPD) has emerged as a useful technique for characterizing peptide, protein, and protein complex primary and secondary structure. 193 nm UVPD, specifically, enables extensive covalent fragmentation of the peptide backbone without the requirement of a specific side chain chromophore and with no precursor charge state dependence. We have modified a commercial quadrupole-ion mobility-time-of-flight (Q-IM-TOF) mass spectrometer to include 193 nm UVPD following ion mobility. Ion mobility (IM) is a gas-phase separation technique that enables separation of ions by their size, shape, and charge, providing an orthogonal dimension of separation to mass analysis. Following instrument modifications, we characterized the performance of, and information that could be generated from, this new setup using the model peptides substance P, melittin, and insulin chain B. These experiments show extensive fragmentation across the peptide backbone and a variety of ion types as expected from 193 nm UVPD. Additionally, y-2 ions (along with complementary a+2 and b+2 ions) N-terminal to proline were observed. Combining the IM separation and mobility gating capabilities with UVPD, we demonstrate the ability to accomplish both mass- and mobility-selection of bradykinin des-Arg9 and des-Arg1 peptides followed by complete sequence characterization by UVPD. The new capabilities of this modified instrument demonstrate the utility of combining IM with UVPD because isobaric species cannot be independently selected with a traditional quadrupole alone.

Careful transfer of ions into the gas-phase permits the measurement of protein structures, with ion mobility-mass spectrometry, which provides shape and stoichiometry information. Collision cross sections (CCS) can be obtained from measurements made of the ions mobility through a given gas, and such structural information once obtained should also permit interlaboratory comparisons. However, until recently, there was not a recommended standard form for the reporting of such measurements. In this study, we explore the use of collision cross section distributions to allow comparisons of IM-MS data for commonly analyzed proteins. We present measurements from seven proteins across three IM-MS configurations, namely, an Agilent 6560 IMQToF, a Waters Synapt G2 possessing a TWIMS cell and a modified Synapt G2 possessing an RF confining linear field drift cell. Mobility measurements were taken using He and N2 as the drift gases. To aid comparability across instruments and best assess the corresponding gas-phase conformational landscapes of the protein "standards", we present the data in the form of averaged CCS distributions. For experiments carried out in N2, CCS values for the most compact ion conformations have an interinstrument variability of ≤3%, and the total CCS distributions are generally similar across platforms. For experiments carried out in He, we observe the total CCS distributions to follow the same trend as observed in N2, while CCS for the most compact ion conformations sampled on the 6560 are systematically smaller by up to 10% than those observed on the G2. The calibration procedure (for TWIMS) yields TWCCS for native-like proteins which are largely similar to those obtained on DTIMS instruments. We collate previously reported values of CCS for these proteins in the form of histograms which bear a remarkable similarity to the CCS distributions, reflecting the conformational heterogeneity of proteins and also how conformer populations can be altered on transfer from solution to the detector. This gives concern for some caution when calibrating sample protein drift times simply with single numeric CCS values.

Bio-based production of industrial chemicals using synthetic biology can provide alternative green routes from renewable resources, allowing for cleaner production processes. To efficiently produce chemicals on-demand through microbial strain engineering, biomanufacturing foundries have developed automated pipelines that are largely compound agnostic in their time to delivery. Here we benchmark the capabilities of a biomanufacturing pipeline to enable rapid prototyping of microbial cell factories for the production of chemically diverse industrially relevant material building blocks. Over 85 days the pipeline was able to produce 17 potential material monomers and key intermediates by combining 160 genetic parts into 115 unique biosynthetic pathways. To explore the scale-up potential of our prototype production strains, we optimized the enantioselective production of mandelic acid and hydroxymandelic acid, achieving gram-scale production in fed-batch fermenters. The high success rate in the rapid design and prototyping of microbially-produced material building blocks reveals the potential role of biofoundries in leading the transition to sustainable materials production., Graphical abstract Image 1, Highlights • An automated biomanufacturing pipeline is benchmarked for the production of material monomers. • Over 85 days, 160 genes were screened for activity, assembled into 115 unique pathways and tested for in vivo production. • E. coli production strains were successfully constructed to produce 17 target compounds at competitive titers. • Scale-up potential is demonstrated through enantioselective production of mandelic acid targets at gram-scale in bioreactors. • The high success rate demonstrates the capabilities of biofoundries to rapidly prototype microbial production strains.

Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising strategy. However, high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance.Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which blocks both p53/MDM2 and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA and protoporphyrin IX (PpIX). Ion-mobility mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions.Contribution to the fieldGiven the immense importance of the p53 tumor suppressor for cancer, efforts have been made to identify p53 activators, which sterically inhibit MDM2. Because high-affinity MDM2 inhibitors are facing problems with considerable side effects, other approaches are needed to reactivate p53 for improved cancer therapy. The allosteric mechanism of action of p53 activator RITA, which we discovered, and its dependence on the oncogenic switch, is an unexpected turn in the p53 story. Our findings provide a basis for the development of p53 activators with a similar mode of functioning, either through the classical drug discovery route or through the drug repurposing approach. Allosteric modulators might have great potential as single agents, or in combination with the standard of care. Further, p53 modulators could serve as invaluable tools to better understand its biology.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, which is characterised by degeneration of distinct neuronal populations, including dopaminergic neurons of the substantia nigra. Here, we use a metabolomics profiling approach to identify changes to lipids in PD observed in sebum, a non-invasively available biofluid. We used liquid chromatography-mass spectrometry (LC-MS) to analyse 274 samples from participants (80 drug naïve PD, 138 medicated PD and 56 well matched control subjects) and detected metabolites that could predict PD phenotype. Pathway enrichment analysis shows alterations in lipid metabolism related to the carnitine shuttle, sphingolipid metabolism, arachidonic acid metabolism and fatty acid biosynthesis. This study shows sebum can be used to identify potential biomarkers for PD., Studies of metabolites in neurodegeneration have not yet used sebum as a source fluid. Here the authors demonstrate the potential of metabolomics of sebum samples from individuals with Parkinson’s disease and controls.

Supramolecular chemistry has grown rapidly over the past three decades, yet synthetic supramolecular chemists still face several challenges when it comes to characterising their compounds. In this review, we present an introduction to structural characterisation techniques commonly used for non-crystalline supramolecular molecules, e.g. nuclear magnetic and electron paramagnetic resonance spectroscopy (NMR and EPR), mass spectrometry (MS), ion mobility mass spectrometry (IM-MS), small-angle neutron and X-ray scattering (SANS and SAXS) as well as cryogenic transmission electron microscopy (cryo-TEM). We provide an overview of their fundamental concepts based on case studies from different fields of supramolecular chemistry, e.g. interlocked structures, molecular self-assembly and host-guest chemistry, while focussing on particular strengths and weaknesses of the discussed methods. Additionally, three multi-technique case studies are examined in detail to illustrate the benefits of using complementary techniques simultaneously.

The effect of temperature on the stability of proteins is well explored for high temperatures, but harder to track below the freezing point of water. This challenge is met with the use of variable temperature ion mobility mass spectrometry (VT IM-MS), which allows the structure of isolated, solvent free molecules to be measured at sub ambient temperatures in the form of their collision cross section (CCS). Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies over a temperature range from 295 to 165 K. For each we observe a large increase in the magnitude of the CCS at 250K (-20 °C) substantially above that predicted. This loss of structure in the absence of bulk solvent is attributed to a change in the strength of stabilizing intermolecular interactions, causing rearrangement. At 190 K (-80 °C) the CCS distribution narrows which we attribute to better resolution. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the gas phase native fold supporting this practice in vitro. Comparing the data for each isotype suggests that the disulfide bridging influences thermal structural rearrangement and taken together we show that this method provides unique insights to the phenomenon of cold denaturation.

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 the conformational transitions of the monomeric proteins: ubiquitin, lysozyme and alpha-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. Data from ubiquitin shows unfolding transitions that proceed through diverse and highly elongated intermediate states, which converge to more compact structures. These findings contrast with data obtained from lysozyme – a protein where (un)-folding plasticity is restricted by four disulfide linkages, although this is alleviated in its reduced form. For structured proteins, collision activation of the protein ions in- source, enables subsequent “freezing” or thermal annealing of unfolding intermediates whereas disordered proteins restructure substantially at 250 K even without activation, indicating that cold denaturation can occur without solvent. These data are presented in the context of a toy model framework which describes the relative occupancy of the available conformational space.

BackgroundThe COVID-19 pandemic is likely to represent an ongoing global health issue given the potential for vaccine escape and the low likelihood of eliminating all reservoirs of the disease. Whilst diagnostic testing has progressed at pace, there is an unmet clinical need to develop tests that are prognostic, to triage the high volumes of patients arriving in hospital settings. Recent research has shown that serum metabolomics has potential for prognosis of disease progression. 1 In a hospital setting, collection of saliva samples is more convenient for both staff and patients, and therefore offers an alternative sampling matrix to serum. We demonstrate here for the first time that saliva metabolomics can reveal COVID-19 severity.Methods88 saliva samples were collected from hospitalised patients with clinical suspicion of COVID-19, alongside clinical metadata. COVID-19 diagnosis was confirmed using RT-PCR testing. COVID severity was classified using clinical descriptors first proposed by SR Knight et al. Metabolites were extracted from saliva samples and analysed using liquid chromatography mass spectrometry.ResultsIn this work, positive percent agreement of 1.00 between a PLS-DA metabolomics model and the clinical diagnosis of COVID severity was achieved. The negative percent agreement with the clinical severity diagnosis was also 1.00, for overall percent agreement of 1.00.ConclusionsThis research demonstrates that liquid chromatography-mass spectrometry can identify salivary biomarkers capable of separating high severity COVID-19 patients from low severity COVID-19 patients in a small cohort study.

Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B12-dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B12 to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B12 photochemistry results in tetramer dissociation, releasing DNA for transcription. Although the details of the response of CarH to light are beginning to emerge, the biophysical mechanism of B12-binding in the dark and how this drives domain assembly is poorly understood. Here – using a combination of molecular dynamics simulations, native ion mobility mass spectrometry and time-resolved spectroscopy – we reveal a complex picture that varies depending on the availability of B12. When B12 is in excess, its binding drives structural changes in CarH monomers that result in the formation of head-to-tail dimers. The structural changes that accompany these steps mean that they are rate-limiting. The dimers then rapidly combine to form tetramers. Strikingly, when B12 is scarcer, as is likely in nature, tetramers with native-like structures can form without a B12 complement to each monomer, with only one apparently required per head-to-tail dimer. We thus show how a bulky chromophore such as B12 shapes protein/protein interactions and in turn function, and how a protein can adapt to a sub-optimal availability of resources. This nuanced picture should help guide the engineering of B12-dependent photoreceptors as light-activated tools for biomedical applications., The function of the bacterial photoreceptor protein, CarH, is regulated by changes to its oligomeric state. Camacho et al. detail how binding of vitamin B12 in the dark drives assembly of the protein tetramer that in turn blocks transcription.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate. ispartof: ANALYTICAL CHEMISTRY vol:93 issue:26 pages:9041-9048 ispartof: location:United States status: published

Activated ion mobility measurements provide insights to the stability of tertiary and quaternary structures of proteins and when coupled with dissociation strategies can delineate how the fold is disrupted. In this work, we use 213 nm photodissociation coupled with ion mobility mass spectrometry and collisional activation to probe the conformational landscape of model proteins. UVPD experiments are performed on proteins following in source activation and on collisionally activated photoproducts post ion mobility separation. For all observed conformations of cytochrome c, there is a significant increase in the UVPD fragmentation yield with the addition of collisional activation post mobility. Similar strategies are deployed with the multimeric proteins, concanavalin a, and haemoglobin. For these complexes’ CID results in ‘classical’ asymmetric charge distribution in subunit products, which when preceded by UV irradiation, yields fragments from within sub-units. Combining these strategies provides complex multidimensional datasets, rich in information, which here we mine with multivariate analysis (MVA). This approach readily determines differences in UVPD and CID fragmentation patterns as a function of conformation and reveals diagnostic information about the precursor native structure without the limitations of current methods that only assign backbone cleavage sites.

Pyridine[4]arenes have previously been considered as anion binding hosts due to the electron-poor nature of the pyridine ring. Herein, we demonstrate the encapsulation of Me4N+ cations inside a dimeric hydrogen-bonded pyridine[4]arene capsule, which contradicts with earlier assumptions. The complexation of a cationic guest inside the pyridine[4]arene dimer has been detected and studied by multiple gas-phase techniques, ESI-QTOF-MS, IRMPD, and DT-IMMS experiments, as well as DFT calculations. The comparison of classical resorcinarenes with pyridinearenes by MS and NMR experiments reveals clear differences in their host–guest chemistry and implies that cation encapsulation in pyridine[4]arene is an anion-driven process.

[Image: see text] The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27(Kip1). We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, κ14 and κ56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase molecular dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.

Intrinsically disordered proteins have been reported to undergo ‘disorder to order’ transitions upon binding to their partners in the cell. The extent of the ordering on binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behaviour for function, particularly in the C-terminal half of the protein, positioning it as an exemplary system to probe conformational diversity. Here we employ native ion mobility with mass spectrometry (IM-MS) to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. This stepwise approach reveals the conformational distributions of the constituent proteins and how they are restructured on complex formation; the trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity cf. Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N and C termini of p27 interact with Cdk2/cyclin A in multiple closely associated states.

The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s per sample. The heat map output allows rapid selection of active enzymes within 96-well plates facilitating identification of industrially relevant biocatalysts. This DiBT-MS screening workflow has been applied to the directed evolution of a phenylalanine ammonia lyase (PAL) as a case study, enhancing its activity toward electron-rich cinnamic acid derivatives which are relevant to lignocellulosic biomass degradation. Additional benefits of the screening platform include the discovery of biocatalysts (kinases, imine reductases) with novel activities and the incorporation of ion mobility technology for the identification of product hits with increased confidence.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that does not currently have a robust clinical diagnostic test. Nonmotor symptoms such as skin disorders have long since been associated with the disease, and more recently a characteristic odor emanating from the skin of people with Parkinson’s has been identified. Here, dynamic head space (DHS) thermal desorption (TD) gas chromatography–mass spectrometry (GC-MS) is implemented to directly measure the volatile components of sebum on swabs sampled from people with Parkinson’s—both drug naïve and those on PD medications (n = 100) and control subjects (n = 29). Supervised multivariate analyses of data showed 84.4% correct classification of PD cases using all detected volatile compounds. Variable importance in projection (VIP) scores were generated from these data, which revealed eight features with VIP > 1 and p < 0.05 which all presented a downregulation within the control cohorts. Purified standards based on previously annotated analytes of interest eicosane and octadecanal did not match to patient sample data, although multiple metabolite features are annotated with these compounds all with high spectral matches indicating the presence of a series of similar structured species. DHS-TD-GC-MS analysis of a range of lipid standards has revealed the presence of common hydrocarbon species rather than differentiated intact compounds which are hypothesized to be breakdown products of lipids. This replication study validates that a differential volatile profile between control and PD cohorts can be measured using an analytical method that measures volatile compounds directly from skin swabs., We validate our previously published findings (cohort of 64) that distinct metabolites in sebum differentiate a person with Parkinson’s from matched controls with an improved (84−86%) correct classification rate.

Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B12-dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B12 to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B12 photochemistry results in tetramer dissociation, releasing DNA for transcription. Although the details of the response of CarH to light are beginning to emerge, the biophysical mechanism of B12-binding in the dark and how this drives domain assembly is poorly understood. Here - using a combination of molecular dynamics simulations, native ion mobility mass spectrometry and time-resolved spectroscopy - we reveal a complex picture that varies depending on the availability of B12. When B12 is in excess, its binding drives structural changes in CarH monomers that result in the formation of head-to-tail dimers. The structural changes that accompany these steps mean that they are rate-limiting. The dimers then rapidly combine to form tetramers. Strikingly, when B12 is scarcer, as is likely in nature, tetramers with native-like structures can form without a B12 complement to each monomer, with only one apparently required per head-to-tail dimer. We thus show how a bulky chromophore such as B12 shapes protein/protein interactions and in turn function, and how a protein can adapt to a sub-optimal availability of resources. This nuanced picture should help guide the engineering of B12-dependent photoreceptors as light-activated tools for biomedical applications.

Activated ion mobility measurements provide Insights to the stability of tertiary and quaternary structures of proteins and pairing such approaches with fragmentation can delineate which part(s) of the primary sequence are disrupted from a folded structure. In this work we use 213 nm photodissociation coupled with ion mobility mass spectrometry and collisional activation to determine the conformational landscape of model proteins. UVPD experiments are performed on proteins following in source activation as well as on collisionally activated photoproducts post ion mobility separation. For cytochrome c, there is a significant increase in the fragmentation yield with collisional activation post mobility, for all conformational states. Similar strategies are deployed with the model multimeric proteins, concanavalin a, and haemoglobin. For these complexes’ CID leads to classic asymmetric charge distribution in subunit products, which when preceded by UV irradiation yields fragments from within the sub-unit that can be mapped to the quaternary fold. Data driven, multivariate analysis (MVA) was used to determine the significant differences in UVPD and CID fragmentation pattern following in source activation. This data driven approach reveals diagnostic fragments without a priori assignments limited to predicated backbone cleavage and provides a new approach to map conformation landscapes that may have wider utility.

The COVID-19 pandemic has led to an urgent and unprecedented demand for testing – both for diagnosis and prognosis. Here we explore the potential for using sebum, collected via swabbing of a patient’s skin, as a novel sampling matrix to fulfil these requirements. In this pilot study, sebum samples were collected from 67 hospitalised patients (30 PCR positive and 37 PCR negative). Lipidomics analysis was carried out using liquid chromatography mass spectrometry. Total fatty acid derivative levels were found to be depressed in COVID-19 positive participants, indicative of dyslipidemia. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) modelling showed promising separation of COVID-19 positive and negative participants when comorbidities and medication were controlled for. Given that sebum sampling is rapid and non-invasive, this work may offer the potential for diagnostic and prognostic testing for COVID-19.

High- and ultrahigh-throughput label-free sample analysis is required by many applications, extending from environmental monitoring to drug discovery and industrial biotechnology. HTS methods predominantly are based on a targeted workflow, which can limit their scope. Mass spectrometry readily provides chemical identity and abundance for complex mixtures, and here, we use microdroplet generation microfluidics to supply picoliter aliquots for analysis at rates up to and including 33 Hz. This is demonstrated for small molecules, peptides, and proteins up to 66 kDa on three commercially available mass spectrometers from salty solutions to mimic cellular environments. Designs for chip-based interfaces that permit this coupling are presented, and the merits and challenges of these interfaces are discussed. On an Orbitrap platform droplet infusion rates of 6 Hz are used for analysis of cytochrome c, on a DTIMS Q-TOF similar rates were obtained, and on a TWIMS Q-TOF utilizing IM-MS software rates up to 33 Hz are demonstrated. The potential of this approach is demonstrated with proof of concept experiments on crude mixtures including egg white, unpurified recombinant protein, and a biotransformation supernatant.

The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.

Ultraviolet photodissociation (UVPD) has emerged as a useful technique for characterizing peptide, protein, and protein complex primary and secondary structure. We present here the modification of a commercial ion mobility (IM) mass spectrometer to incorporate 193 nm UVPD following IM separation, enabling IM-UVPD to fragment species selected by both their m/z (via quadrupole selection) and conformation (mobility-selection).

Louis Pasteur has particular resonance in the midst of the coronavirus disease 2019 (COVID-19) pandemic, given his work on the germ theory of disease that led to the development of vaccines for rabies and anthrax. Some years before these better-known discoveries, Pasteur had already made a great contribution to science with his observation of molecular chirality in crystals ([ 1 ][1]). Chirality is a fundamental property of molecular asymmetry, which in turn dictates the secondary structure of proteins and gives rise to handedness in DNA helices. Some 170 years later, on page 1465 of this issue, Daly et al. ([ 2 ][2]) present a new method, mass-resolved electronic circular dichroism (CD) ion spectroscopy, that allows the chirality of secondary structures—in this case, isolated guanine-rich DNA quadruplexes—to be unambiguously defined. In the 1830s, the acceptance of the concept of chemical structure was some decades away, but developments in optics were revealing regularity in the ordering of crystalline materials at a submicrometer level. Light could be filtered through polarizing optics, such as Nicol prisms. Although not understood at the time, this process causes the oscillating electric field of the light to move in only one direction. Using a polarized light source, Jean-Baptiste Biot ([ 3 ][3]) observed that a saturated solution of tartaric acid would rotate the polarized light clockwise or anticlockwise, but he presented no explanation for this phenomenon. Some 20 years later, in 1848, Pasteur crystallized paratartaric acid, produced from overboiled wine, and noticed that it produced two types of crystals, one that looked like tartaric acid crystals and one that did not. He repeated Biot's experiment with a 50-50 mixture of these crystals and found that this solution would not rotate the polarized light. From these observations, Pasteur concluded that each crystal had an intrinsic property, and that the material in each crystal was the mirror image of the other, like a left and a right hand. Pasteur named this phenomenon chirality (from kheir , the Greek word for hand). It soon became evident that this is a property of fundamental importance to chemistry, where different substituents on tetravalent carbon create a chiral center. ![Figure][4] An exciting way to measure handedness Molecular handedness (chirality) is usually measured with circular dichroism, in which the direction of polarized light is rotated by absorption of light by molecules in solution. Daly et al. determined such spectra for individual molecules in the gas phase. GRAPHIC: V. ALTOUNIAN/ SCIENCE Naturally occurring biopolymers such as proteins and DNA are made of monomeric units with such chiral centers. When these repeat at regular intervals, they favor repeating noncovalent interactions that give rise to distinctive secondary structural motifs with characteristic optical signatures. Circular dichroism, the modern version of the experiments of Biot and Pasteur, measures the absorbance of polarized light and can verify, for example, that secondary structures of proteins are correctly folded. However, CD measures the aggregate effect of a mixture and can be insensitive to the presence of an enantiomer at lower concentrations, or to the particular form of the molecule that is causing the optical rotation. To address this limitation, Daly et al. built an experimental apparatus that can make precise measurements of chirality in biomolecules (see the figure). Unlike an optical absorption experiment in solution, the optical source (laser excitation) releases characteristic photoelectrons that lead to charge-reduced ions, so this method is an action spectroscopy. Electrospray ionization introduces guanine-rich DNA quadruplex molecules of different chirality into the vacuum chamber of a mass spectrometer. This transfer is performed carefully under conditions that aim to leave the solvated structure unperturbed. A few thousand molecular ions are trapped and irradiated with polarized laser light. The masses of the left- and right-handed forms of these DNA complexes are identical, so mass spectrometry alone cannot determine chirality. Further, unlike solution experiments where the concentration of molecules is high enough that the absorbance of light can be readily measured, the gas-phase DNA ions are dilute. Instead, Daly et al. use the mass spectrometer to measure the action of the polarized light on the DNA molecule. Facile release of a photoelectron upon irradiation gives a signal caused by the formation of the charge-reduced anion. The laser light pulses are polarized, so the efficiency of photoelectron removal depends on the helix handedness. By monitoring the intensity of the distinctive charge-reduced species as a function of the polarization, they are able to determine the chirality of the molecule. This type of experiment can isolate any given molecular complex because each analyte is unequivocally defined by its mass-to-charge ratio. Prior work by this group revealed coexisting structures formed by oligonucleotides in the presence of cations or organic molecules ([ 4 ][5]); this work was aimed at understanding the balance of forces that guide folding and self-assembly. Stoichiometry and quantitative measurements of nucleic acid complexes were readily made by combining mass and ion-mobility spectrometries, but this approach could not determine chirality or details of secondary structure. Mass-resolved electronic CD ion spectroscopy complements other structural mass spectrometry methods because it can provide secondary structure information in addition to molecular identity. Daly et al. also studied complexes of DNA with ammonium and potassium counterions and obtained a tantalizing glimpse of the effects of individual molecule solvation. The wavelength dependence of the action of polarized light followed the same trend as data obtained in solution, albeit with differences in magnitude, indicating that structures are preserved and that these gas-phase results are relevant to molecules in solution. They extended their measurement on human telomeric DNA sequences to determine enantiomeric ratios of mixtures of G-quadruplex topologies. Intriguingly, the difference in electron-detachment efficiency for the left-handed molecule and the right-handed molecule under a given circularly polarized light is equivalent to the slight enantiomeric excess in the products—up to 1% at some wavelengths—thereby demonstrating the high sensitivity as well as the potential of this new method. 1. [↵][6]1. L. Pasteur , C. R. Seances Acad. Sci. 26, 535 (1848). [OpenUrl][7] 2. [↵][8]1. S. Daly, 2. F. Rosu, 3. V. Gabelica , Science 368, 1465 (2020). [OpenUrl][9][Abstract/FREE Full Text][10] 3. [↵][11]1. J.-B. Biot , Mem. Acad. Sci. Inst. Fr. 15, 93 (1836). [OpenUrl][12] 4. [↵][13]1. M. Porrini et al ., ACS Cent. Sci. 3, 454 (2017). [OpenUrl][14] [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: pending:yes [5]: #ref-4 [6]: #xref-ref-1-1 "View reference 1 in text" [7]: {openurl}?query=rft.jtitle%253DC.%2BR.%2BS%25E9ances%2BAcad.%2BSci.%26rft.volume%253D26%26rft.spage%253D535%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [8]: #xref-ref-2-1 "View reference 2 in text" [9]: {openurl}?query=rft.jtitle%253DScience%26rft.stitle%253DScience%26rft.aulast%253DDaly%26rft.auinit1%253DS.%26rft.volume%253D368%26rft.issue%253D6498%26rft.spage%253D1465%26rft.epage%253D1468%26rft.atitle%253DMass-resolved%2Belectronic%2Bcircular%2Bdichroism%2Bion%2Bspectroscopy%26rft_id%253Dinfo%253Adoi%252F10.1126%252Fscience.abb1822%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [10]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjEzOiIzNjgvNjQ5OC8xNDY1IjtzOjQ6ImF0b20iO3M6MjM6Ii9zY2kvMzY4LzY0OTgvMTQyNi5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30= [11]: #xref-ref-3-1 "View reference 3 in text" [12]: {openurl}?query=rft.jtitle%253DMem.%2BAcad.%2BSci.%2BInst.%2BFr.%26rft.volume%253D15%26rft.spage%253D93%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [13]: #xref-ref-4-1 "View reference 4 in text" [14]: {openurl}?query=rft.jtitle%253DACS%2BCent.%2BSci.%26rft.volume%253D3%26rft.spage%253D454%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx

Parkinson’s disease (PD) is the second most common neurodegenerative disorder for which identification of robust biomarkers to complement clinical PD diagnosis would accelerate treatment options and help to stratify disease progression. Here we demonstrate the use of paper spray ionisation coupled with ion mobility mass spectrometry (PSI IM-MS) to determine diagnostic molecular features of PD in sebum. PSI IM-MS was performed directly from skin swabs, collected from 34 people with PD and 30 matched control subjects as a training set and a further 91 samples from 5 different collection sites as a validation set. PSI IM-MS elucidates ~ 4200 features from each individual and we report two classes of lipids (namely phosphatidylcholine and cardiolipin) that differ significantly in the sebum of people with PD. Putative metabolite annotations are obtained using tandem mass spectrometry experiments combined with accurate mass measurements. Sample preparation and PSI IM-MS analysis and diagnosis can be performed ~5 minutes per sample offering a new route to for rapid and inexpensive confirmatory diagnosis of this disease.

Biotin protein ligase (BPL) is an essential enzyme in all kingdoms of life, making it a potential target for novel anti-infective agents. Whilst bacteria and archaea have simple BPL structures (class I and II), the homologues from certain eukaryotes such as mammals, insects and yeast (class III) have evolved a more complex structure with a large extension on the N-terminus of the protein in addition to the conserved catalytic domain. The absence of atomic resolution structures of any class III BPL hinders structural and functional analysis of these enzymes. Here, two new class III BPLs from agriculturally important moulds Botrytis cinerea and Zymoseptoria tritici were characterised alongside the homologue from the prototypical yeast Saccharomyces cerevisiae. Circular dichroism and ion mobility-mass spectrometry analysis revealed conservation of the overall tertiary and secondary structures of all three BPLs, corresponding with the high sequence similarity. Subtle structural differences were implied by the different thermal stabilities of the enzymes and their varied Michaelis constants for their interactions with ligands biotin, MgATP, and biotin-accepting substrates from different species. The three BPLs displayed different preferences for fungal versus bacterial protein substrates, providing further evidence that class III BPLs have a ‘substrate validation’ activity for selecting only appropriate proteins for biotinylation. Selective, potent inhibition of these three BPLs was demonstrated despite sequence and structural homology. This highlights the potential for targeting BPL for novel, selective antifungal therapies against B. cinerea, Z. tritici and other fungal species Refereed/Peer-reviewed

Glycation is a protein modification prevalent in the progression of diseases such as Diabetes and Alzheimer’s, as well as a byproduct of therapeutic protein expression, notably for monoclonal antibodies (mAbs). Quantification of glycated protein is thus advantageous in both assessing the advancement of disease diagnosis and for quality control of protein therapeutics. Vibrational spectroscopy has been highlighted as a technique that can easily be modified for rapid analysis of the glycation state of proteins, and requires minimal sample preparation. Glycated samples of lysozyme and albumin were synthesised by incubation with 0.5 M glucose for 30 days. Here we show that both FTIR-ATR and Raman spectroscopy are able to distinguish between glycated and non-glycated proteins. Principal component analysis (PCA) was used to show separation between control and glycated samples. Loadings plots found specific peaks that accounted for the variation - notably a peak at 1027 cm-1 for FTIR-ATR. In Raman spectroscopy, PCA emphasised peaks at 1040 cm-1 and 1121 cm-1. Therefore, both FTIR-ATR and Raman spectroscopy found changes in peak intensities and wavenumbers within the sugar C-O/C-C/C-N region (1200-800 cm-1). For quantification of the level of glycation of lysozyme, partial least squares regression (PLSR), with statistical validation, was employed to analyse Raman spectra from solution samples containing 0 – 100% glycated lysozyme, generating a robust model with R2 of 0.99. We therefore show the scope and potential of Raman spectroscopy as a high throughput quantification method for glycated proteins in solution that could be applied in disease diagnostics, as well as therapeutic protein quality control.

As experimentalists, we normally rely on assessing observables. However sometimes, the most fascinating phenomena are not noticeable directly. An example of such is our data and the corresponding interpretation presented in this manuscript. We have designed and constructed a ion mobility mass spectrometer (acs.analchem.6b01812) capable of taking ion mobility measurements over a temperature range from 150-500K. We chose to benchmark this new instrument, using the small proteins Ubiquitin and Lysozyme extensively studied as a “model proteins” in many in-silico, -solution and -vacuo studies focusing on conformational dynamics.In this work, we activate and subsequently thermally equilibrate the protein ions at several temperatures prior to collision cross section measurement. For Ubiquitin at 300K and above, the protein unfolds in a “step-wise” fashion as previously reported (by David Clemmer) and for other proteins including lysozyme, and cytochrome c by us and also by Martin Jarrold. However, to our surprise, activation and equilibration of ubiquitin at 150K yields a plethora of highly extended forms of the protein. We attribute these as kinetically trapped unfolding intermediates. Since the activation process is the same at both temperatures we infer that the unfolding must always proceed via these extended intermediate forms, which then converge to commonly reported conformations. Intriguingly, this “convergence” appears to occur mostly below the temperature of irreversible conformational thermal transition of Ubiquitin reported in many solution phase studies. For Lysozyme the same experiment is performed and similar results are obtained although we cannot activate the gaseous ensemble too far from the native fold and the activation barrier to refolding is low enough to allow it to be re-accessed on the experimental timescale we use.

High and ultra-high-throughput label-free sample analysis is required by many applications, extending from environ-mental monitoring to drug discovery and industrial biotechnology. HTS methods predominantly are based on a targeted workflow, which can limit their scope. Mass spectrometry readily provides chemical identity and abundance for complex mixtures and here, we use microdroplet generation microfluidics to supply picolitre aliquots for analysis at rates up to and including 33 Hz. This is demon-strated for small molecules, peptides and proteins up to 66 kDa on three commercially available mass spectrometers from salty solutions to mimic cellular environments. Designs for chip-based interfaces that permit this coupling are presented and the merits and challenges of these interfaces are discussed. On an Orbitrap platform droplet infusion rates of 6 Hz are used for the analysis of cytochrome c, on a DTIMS Q-TOF similar rates were obtained and on a TWIMS Q-TOF utilizing IM-MS software rates up to 33 Hz are demonstrated. The potential of this approach is demonstrated with proof of concept experiments on crude mixtures including egg white, unpurified recombinant protein and a biotransformation supernatant.

In this chapter, we highlight recent developments using laser irradiation to probe ions in mass spectrometers. We will primarily cover the structural characterisation of biomolecules. This chapter will introduce the principles behind laser irradiation and describe some of the common instrument configurations, with an emphasis on benchtop laser systems coupled to instruments in laboratories. We have ordered the narrative in this chapter according to what has been achieved with three different irradiation wavelengths namely UV, visible and infrared. Instrumentation developed to permit laser irradiation along with mass spectrometry is described in each section along with some examples of recent applications primarily focused on the analysis of peptides and proteins.

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88–99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.

Rapid and comprehensive genetic sequencing has shed light on the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and allowed timely implementation of PCR tests to determine the presence of viral RNA. PCR tests for SARS-CoV-2 are some way from being reliably qualitative and will never indicate how the disease might progress in an individual. As COVID-19 becomes endemic, there is a concomitant need for accurate serological assays to detect antibodies to SARS-CoV-2 antigens and ultimately tests for prognostic markers to target treatment options.1,2 With this considerable genetic insight, and the emerging structural information, comes associated questions regarding the molecular descriptors that contribute to disease progression, especially when we consider spread across different populations. The power of mass spectrometry to generate rapid, precise, and reproducible diagnostic information that complements genomic information and accelerates our understanding of the disease, is now becoming a reality.

The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKAc) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKAc- and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKAc-regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κB DNA. Graphical Abstractᅟ Electronic supplementary material The online version of this article (10.1007/s13361-018-1984-0) contains supplementary material, which is available to authorized users.

Entire functional proteins as well as large regions of proteins lack structural elements which are resolvable via crystallography or NMR. These intrinsically disordered proteins (IDPs) or regions (IDRs) are often involved in cell regulation processes, for example in signalling hubs and as a result aberrant behaviour can cause or be representative of disease. As a consequence there is a pressing need to develop alternative structural methods for IDPs and the interactions that they may form with other proteins and/or with potential inhibitors of binding. One such method is ion mobility mass spectrometry (IM-MS) coupled with careful application of electrospray ionisation, which shows great promise as a technique that does not 'care' if a protein is structured or not. We highlight recent work which has employed IM-MS to study conformational heterogeneity in disordered proteins, and discuss the opportunities, as well as the challenges of this approach.

Chromatography-based mass spectrometry approaches (xC-MS) are commonly used in untargeted metabolomics, providing retention time, m/z values and metabolite-specific fragments, all of which are used to identify and validate an unknown analyte. Ion mobility-mass spectrometry (IM-MS) is emerging as an enhancement to classic xC-MS strategies, by offering additional ion separation as well as collision cross section (CCS) determination. In order to apply such an approach to a metabolomics workflow, verified data from metabolite standards is necessary. In this work we present experimental DTCCSN2 values for a range of metabolites in positive and negative ionisation modes using drift tube-ion mobility-mass spectrometry (DT-IM-MS) with nitrogen as the buffer gas. The value of DTCCSN2 measurements for application in metabolite identification relies on a robust technique that acquires measurements of high reproducibility. We report that the CCS values found for 86% of metabolites measured in replicate have a relative standard deviation lower than 0.2%. Examples of metabolites with near identical mass are demonstrated to be separated by ion mobility with over 4% difference in DTCCSN2 values. We conclude that the integration of ion mobility into current LC-MS workflows can aid in small molecule identification for both targeted and untargeted metabolite screening.