Mass spectrometry-based studies for the investigation of protein structure and dynamics

The use of mass spectrometry to study of proteins and their non-covalent complexes has grown during the last few decades thanks to the introduction of soft ionisation techniques capable of preserving the weak molecular bonds present in higher order protein structure. The integration of the shape selective technique ion mobility with mass spectrometry has allowed the study of conformation and dynamics of native proteins. The work presented in this thesis focuses on the use of ion mobility mass spectrometry to investigate both protein conformation, and conformational differences induced by mutation. Prolyl oligopeptidase family enzymes are proteases characterised by their unique function; these enzymes are only capable of digesting short amino acid sections of no more than thirty amino acids. These proteins have been implicated in a number of neurological disorders and have been targeted as potential drug candidates. PREP is an 80.7 kDa monomeric protein that has been targeted as a potential drug candidate. PREP has been crystallised in two distinct conformations, open and closed, and it has been suggested that the protein exists in equilibrium between the two states in solution, with only the open conformation allowing substrate entry via a domain separation mechanism. Ion mobility mass spectrometry has been used to confirm the presence of both conformations, and to investigate the effect of ligand binding on conformation. It was found that, at lower energy states, PREP was only capable of adopting a single conformation, and that more extended conformations were only present following activation of the protein. Binding of ligand appeared to increase the relative stability of the protein. DPP IV, is a larger, dimeric, protein from the same family. Unlike PREP, DPP IV has only been crystallised in a single conformation and it was proposed that small loop movements, rather than domain separation, allowed substrate entry. Ion mobility measurements show only a single conformation of DPP IV, consistent with no large conformational changes, supporting this hypothesis. Haemoglobin is the main protein involved in gas transport in mammalian systems, taking oxygen from the lungs to the tissues of the body. Disorders of haemoglobin represent the most common of all inherited disorders, with an estimated 7% of the global population being carriers for a haemoglobin disorder. A previous study by Scarff et al used ion mobility mass spectrometry to identify conformational differences between normal HbA and HbS, the haemoglobin mutant responsible for sickle cell disease. Recent experimental improvements in sample preparation, data collection and data processing have been used in this research to provide improved experimental information. HbA, HbS and HbC, a third haemoglobin variant known to form crystals within erythrocytes, were investigated using ion mobility mass spectrometry. Calibration of ion mobility data using native protein standards indicated that the structural differences between HbA and HbS were smaller than previously reported, and that the CCS measurements of the two proteins were similar for the native charge states. The HbC molecule does however adopt a smaller conformation. All three proteins unfold as a factor of increased protonation, with HbS and HbC showing evidence of adopting a more extended conformation at lower charge states when compared to HbA, suggesting possible differences in protein stability. These stability differences were investigated using collision-induced activation of the protein, the results suggesting that HbA is more resistant to unfolding that either HbS or HbC. Monoclonal antibodies represent a new generation of biotherapeutic capable of high specificity and selectivity, with diminished risk of inducing a host immune response. Antibodies therapeutics have an added benefit of interacting with host cellular systems, promoting host immune response. Engineering of antibodies has become well established to improve or diminish antibody-receptor binding in order to increase or abolish this interaction. Four antibody Fc variants have been studied using a combination of ion mobility mass spectrometry and hydrogen deuterium exchange mass spectrometry; a wild type, a TM mutant, a YTE mutant and a TMYTE double mutant. Previous studies have shown that the introduction of both TM and YTE mutations leads to a decrease in the thermal stability of the protein, and it was an aim of the study to provide structural information to explain this thermal destabilisation. Ion mobility mass spectrometry measurements suggest that there is little change in global conformation between the four variants. HDX results show that mutation introduces changes in local conformation across the protein, with increases in deuterium uptake observed at sites distant to the mutation sites. One region, located between the TM and YTE mutation sites, showed a greater than additive increase in deuterium uptake in TMYTE mutant compared with either TM or YTE mutants, indicating a region that hade become destabilised in the double mutant. These changes could be responsible for the observed loss of thermal stability

Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/anie.20150722

Structural Plasticity of the Semliki Forest Virus Glycome upon Interspecies Transmission

Cross-species viral transmission subjects parent and progeny alphaviruses to differential post-translational processing of viral envelope glycoproteins. Alphavirus biogenesis has been extensively studied, and the Semliki Forest virus E1 and E2 glycoproteins have been shown to exhibit differing degrees of processing of N-linked glycans. However the composition of these glycans, including that arising from different host cells, has not been determined. Here we determined the chemical composition of the glycans from the prototypic alphavirus, Semliki Forest virus, propagated in both arthropod and rodent cell lines, by using ion-mobility mass spectrometry and collision-induced dissociation analysis. We observe that both the membrane-proximal E1 fusion glycoprotein and the protruding E2 attachment glycoprotein display heterogeneous glycosylation that contains N-linked glycans exhibiting both limited and extensive processing. However, E1 contained predominantly highly processed glycans dependent on the host cell, with rodent and mosquito-derived E1 exhibiting complex-type and paucimannose-type glycosylation, respectively. In contrast, the protruding E2 attachment glycoprotein primarily contained conserved under-processed oligomannose-type structures when produced in both rodent and mosquito cell lines. It is likely that glycan processing of E2 is structurally restricted by steric-hindrance imposed by local viral protein structure. This contrasts E1, which presents glycans characteristic of the host cell and is accessible to enzymes. We integrated our findings with previous cryo-electron microscopy and crystallographic analyses to produce a detailed model of the glycosylated mature virion surface. Taken together, these data reveal the degree to which virally encoded protein structure and cellular processing enzymes shape the virion glycome during interspecies transmission of Semliki Forest virus

The Anthropocene is best understood as an ongoing, intensifying, diachronous event

Current debate on the status and character of the Anthropocene is focussed on whether this interval of geological time should be designated as a formal unit of epoch/series rank in the International Chronostratigraphic Chart/Geological Time Scale, or whether it is more appropriate for it to be considered as an informal ‘event’ comparable in significance with other major transformative events in deeper geological time. The case for formalizing the Anthropocene as a chronostratigraphical unit with a base at approximately 1950 CE is being developed by the Anthropocene Working Group of the Subcommission on Quaternary Stratigraphy. Here we outline the alternative position and explain why the time-transgressive nature of human impact on global environmental systems that is reflected in the recent stratigraphical record means that the Anthropocene is better seen not as a series/epoch with a fixed lower boundary, but rather as an unfolding, transforming and intensifying geological event

A practical solution: the Anthropocene is a geological event, not a formal epoch

The Anthropocene has yet to be defined in a way that is functional both to the international geological community and to the broader fields of environmental and social sciences. Formally defining the Anthropocene as a chronostratigraphical series and geochronological epoch with a precise global start date would drastically reduce the Anthropocene’s utility across disciplines. Instead, we propose the Anthropocene be defined as a geological event, thereby facilitating a robust geological definition linked with a scholarly framework more useful to and congruent with the many disciplines engaging with human-environment interactions. Unlike formal epochal definitions, geological events can recognize the spatial and temporal heterogeneity and diverse social and environmental processes that interact to produce anthropogenic global environmental changes. Consequently, an Anthropocene Event would incorporate a far broader range of transformative human cultural practices and would be more readily applicable across academic fields than an Anthropocene Epoch, while still enabling a robust stratigraphic characterization

The Anthropocene is best understood as an ongoing, intensifying, diachronous event

Current debate on the status and character of the Anthropocene is focussed on whether this interval of geological time should be designated as a formal unit of epoch/series rank in the International Chronostratigraphic Chart/Geological Time Scale, or whether it is more appropriate for it to be considered as an informal ‘event’ comparable in significance with other major transformative events in deeper geological time. The case for formalizing the Anthropocene as a chronostratigraphical unit with a base at approximately 1950 CE is being developed by the Anthropocene Working Group of the Subcommission on Quaternary Stratigraphy. Here we outline the alternative position and explain why the time‐transgressive nature of human impact on global environmental systems that is reflected in the recent stratigraphical record means that the Anthropocene is better seen not as a series/epoch with a fixed lower boundary, but rather as an unfolding, transforming and intensifying geological event

The management of Staphylococcus aureus bacteremia in the United Kingdom and Vietnam: a multi-centre evaluation.

Background: Staphylococcus aureus bacteremia is a common and serious infection worldwide and although treatment guidelines exist, there is little consensus on optimal management. In this study we assessed the variation in management and adherence to treatment guidelines of S. aureus bacteremia. Methodology/Principal Findings: We prospectively recorded baseline clinical characteristics, management, and in-hospital outcome of all adults with S. aureus bacteremia treated consecutively over one year in eight centres in the United Kingdom, three in Vietnam and one in Nepal. 630 adults were treated for S. aureus bacteremia: 549 in the UK (21% methicillin-resistant), 80 in Vietnam (19% methicillin-resistant) and 1 in Nepal. In the UK, 41% had a removable infection focus (50% intravenous catheter-related), compared to 12% in Vietnam. Significantly (p50% of treatment with oral antibiotics alone (25% versus 4%). UK centres varied significantly (p50% of treatment (range 12-40%), in those treated for longer than 28 days (range 13-54%), and in those given combination therapy (range 14-94%). 24% died during admission: older age, time in hospital before bacteremia, and an unidentified infection focus were independent predictors of in-hospital death (p<0.001). Conclusions/Significance: The management of S. aureus bacteremia varies widely between the UK and Vietnam and between centres in the UK with little adherence to published guidelines. Controlled trials defining optimal therapy are urgently required

All clinically-relevant blood components transmit prion disease following a single blood transfusion: a sheep model of vCJD

Variant CJD (vCJD) is an incurable, infectious human disease, likely arising from the consumption of BSE-contaminated meat products. Whilst the epidemic appears to be waning, there is much concern that vCJD infection may be perpetuated in humans by the transfusion of contaminated blood products. Since 2004, several cases of transfusion-associated vCJD transmission have been reported and linked to blood collected from pre-clinically affected donors. Using an animal model in which the disease manifested resembles that of humans affected with vCJD, we examined which blood components used in human medicine are likely to pose the greatest risk of transmitting vCJD via transfusion. We collected two full units of blood from BSE-infected donor animals during the pre-clinical phase of infection. Using methods employed by transfusion services we prepared red cell concentrates, plasma and platelets units (including leucoreduced equivalents). Following transfusion, we showed that all components contain sufficient levels of infectivity to cause disease following only a single transfusion and also that leucoreduction did not prevent disease transmission. These data suggest that all blood components are vectors for prion disease transmission, and highlight the importance of multiple control measures to minimise the risk of human to human transmission of vCJD by blood transfusion

Adjunctive rifampicin to reduce early mortality from Staphylococcus aureus bacteraemia (ARREST): study protocol for a randomised controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common and serious infection, with an associated mortality of ~25%. Once in the blood, S. aureus can disseminate to infect almost any organ, but bones, joints and heart valves are most frequently affected. Despite the infection's severity, the evidence guiding optimal antibiotic therapy is weak: fewer than 1,500 patients have been included in 16 randomised controlled trials investigating S. aureus bacteraemia treatment. It is uncertain which antibiotics are most effective, their route of administration and duration, and whether antibiotic combinations are better than single agents. We hypothesise that adjunctive rifampicin, given in combination with a standard first-line antibiotic, will enhance killing of S. aureus early in the treatment course, sterilise infected foci and blood faster, and thereby reduce the risk of dissemination, metastatic infection and death. Our aim is to determine whether adjunctive rifampicin reduces all-cause mortality within 14 days and bacteriological failure or death within 12 weeks from randomisation. METHODS: We will perform a parallel group, randomised (1:1), blinded, placebo-controlled trial in NHS hospitals across the UK. Adults (≥ 18 years) with S. aureus (meticillin-susceptible or resistant) grown from at least one blood culture who have received ≤ 96 h of active antibiotic therapy for the current infection and do not have contraindications to the use of rifampicin will be eligible for inclusion. Participants will be randomised to adjunctive rifampicin (600-900 mg/day; orally or intravenously) or placebo for the first 14 days of therapy in combination with standard single-agent antibiotic therapy. The co-primary outcome measures will be all-cause mortality up to 14 days from randomisation and bacteriological failure/death (all-cause) up to 12 weeks from randomisation. 940 patients will be recruited, providing >80% power to detect 45% and 30% reductions in the two co-primary endpoints of death by 14 days and bacteriological failure/death by 12 weeks respectively. DISCUSSION: This pragmatic trial addresses the long-standing hypothesis that adjunctive rifampicin improves outcome from S. aureus bacteraemia through enhanced early bacterial killing. If proven correct, it will provide a paradigm through which further improvements in outcome from S. aureus bacteraemia can be explored.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Association of cardiometabolic microRNAs with COVID-19 severity and mortality

AIMS: Coronavirus disease 2019 (COVID-19) can lead to multiorgan damage. MicroRNAs (miRNAs) in blood reflect cell activation and tissue injury. We aimed to determine the association of circulating miRNAs with COVID-19 severity and 28 day intensive care unit (ICU) mortality. METHODS AND RESULTS: We performed RNA-Seq in plasma of healthy controls (n = 11), non-severe (n = 18), and severe (n = 18) COVID-19 patients and selected 14 miRNAs according to cell- and tissue origin for measurement by reverse transcription quantitative polymerase chain reaction (RT–qPCR) in a separate cohort of mild (n = 6), moderate (n = 39), and severe (n = 16) patients. Candidates were then measured by RT–qPCR in longitudinal samples of ICU COVID-19 patients (n = 240 samples from n = 65 patients). A total of 60 miRNAs, including platelet-, endothelial-, hepatocyte-, and cardiomyocyte-derived miRNAs, were differentially expressed depending on severity, with increased miR-133a and reduced miR-122 also being associated with 28 day mortality. We leveraged mass spectrometry-based proteomics data for corresponding protein trajectories. Myocyte-derived (myomiR) miR-133a was inversely associated with neutrophil counts and positively with proteins related to neutrophil degranulation, such as myeloperoxidase. In contrast, levels of hepatocyte-derived miR-122 correlated to liver parameters and to liver-derived positive (inverse association) and negative acute phase proteins (positive association). Finally, we compared miRNAs to established markers of COVID-19 severity and outcome, i.e. SARS-CoV-2 RNAemia, age, BMI, D-dimer, and troponin. Whilst RNAemia, age and troponin were better predictors of mortality, miR-133a and miR-122 showed superior classification performance for severity. In binary and triplet combinations, miRNAs improved classification performance of established markers for severity and mortality. CONCLUSION: Circulating miRNAs of different tissue origin, including several known cardiometabolic biomarkers, rise with COVID-19 severity. MyomiR miR-133a and liver-derived miR-122 also relate to 28 day mortality. MiR-133a reflects inflammation-induced myocyte damage, whilst miR-122 reflects the hepatic acute phase response