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Welcome to

Frontiers in Mass Spectrometry @King’s

This two-day symposium will showcase how mass spectrometry (MS) supports and advances research across King's and with its partners. The aim of this symposium is to collate the portfolio of activity and to identify future avenues of exploration and collaboration. We have an agenda of exciting invited presentations including topics such as metabolomics, proteomics/protein analysis, lipidomics, analytical toxicology, chemical biology, pharmaceutical science, forensic science, environmental analysis and advanced data analysis & AI.

Chairperson’s welcome

On behalf of the organising committee, is my great pleasure to welcome you to Frontiers in Mass Spectrometry @King’s in the heart of London. We are currently reviewing how MS supports the research portfolio across King’s and this Symposium brings together most of its major users. Hopefully, the outcome of this meeting and the discussions held will inform us how to direct resources to support MS in the future. We are delighted to host you here at Bush House, King’s College London, the former

home of the BBC. Please find all the information you need about the event in this booklet including the scientific programme and the abstracts. Please make sure to visit the beautiful rooftop to see outstanding views of the London skyline while you are here. If you have any questions, please do not hesitate to contact either myself, Serena or Chris White during the day or afterwards.

Dr Leon Barron, King’s Forensics

Organising Committee Dr Christopher White; Dr Serena Mitchell; Prof. Frank Kelly; Prof. Neil Dalton; Dr Anthony Borysik; Dr Vincenzo Abbate; Dr Ciro Chiappini; Dr Ana Rodriguez-Mateos

Feel free to Tweet at #FrontiersMSKings

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4/2019

Shimadzu LCMS-9030 and MALDI-8020 time limited offer

Cover all bases with MALDI/LCMS combined

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AGENDA, 29th April Lecture Theatre 1 (BH (S) 1.01)

9.00 Registration Entrance Hall, Lecture Theatre 1

9.30 Welcome address Leon Barron

Session 1 in BH (S) 1.01 Chair: Leon Barron

9.45 Ana Rodriguez-Mateos Dept. Nutritional Sciences, KCL

You are what you excrete: using mass spectrometry to investigate the health benefits of plant foods and phytochemicals

10.15 Robin Mesnage Dept. Molecular Genetics, KCL

Combining omics data to identify health effects of pesticides

10.45 Leon Barron King’s Forensics, KCL

There’s something in the water: Using LC-MS to understand community-scale consumption and environmental impacts of drugs

11.15 Reza Razavi

Welcome from the Vice President & Vice-Principal (Research & Innovation) and Director of Research at King’s Health Partners, KCL

11.30 Coffee and Posters Breakout Area

Session 2 in BH (S) 1.01 Chair: Alan Brailsford

12.00 Manuel Müller Dept. Chemistry, KCL

Chemical Synthesis of Post-translationally Modified Proteins

12.30 Sufyan Pandor, Agilent Technologies LDA UK Ltd.

How developments/frontiers in metabolomic workflows is driving MS-based metabolome profiling

13.00 Lunch Breakout Area

Session 3 in BH (S) 1.01 Chair: Vincenzo Abbate

13.45 Simon Elliott Elliott Consulting Ltd., UK

Above and Beyond: Can KCL play a part in fixing clinical and forensic analytical toxicology?

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Max Hecht, Analytical Services International Ltd., UK Anca Frinculescu, Tictac Communications Ltd./Dept. Analytical, Environmental & Forensic Sciences, KCL

Good Golly, Miss ‘Molly’: Quantification and dissolution of ecstasy tablets seized 2001 – 2018

14.45 Mark Parkin Eurofins Forensic Services, UK

Mass spectrometry in the forensic toxicology laboratory – where are we now and where are we heading?

15.15 Coffee and Posters Breakout Area

Session 4 in BH (S) 1.01 Chair: Neil Dalton

15.30 Neil Dalton SpOtOn Clinical Diagnostics Limited, Evelina Children's Hospital

Targeted metabolomics: lessons learned

16.00 Varun Mehra Haematology, King's College Hospital

Invasive Fungal disease -Time for MS to step in

16.30 Charles Turner WellChild Laboratory, KCL and Evelina Children's Hospital

Peptide based analysis of proteins using electrospray MS/MS

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AGENDA, 30th April

Lecture Theatre 1 (BH (S) 1.01)

9.00 Registration Entrance Hall, Lecture Theatre 1

9.30 Welcome address Leon Barron

Session 5 in BH (S) 1.01 Chair: Theodora Stewart

9.45 Theodora Stewart, London Metallomics Facility, KCL

London Metallomics Facility: An emerging scientific platform for multidimensional metal analysis

10.15 Caitlyn Da Costa, Waters Corporation

Imaging mass spectrometry: visualization at the molecular level

10.45 Christopher Titman, Shimadzu UK Ltd.

Tools to help find components in complex mixtures by HRAM LC/MS/MS

11.15 Coffee and Posters Breakout Area

Session 6 in BH (S) 1.01 Chair: John Halket

11.30 Mariana Silva Dos Santos, The Francis Crick Institute

Metabolomics and lipidomics at the Crick: a case study in tuberculosis

12.00 Cagakan Ozbalci, The Randall, KCL Lipidomics in mammalian cells

12.30 Francesca Mazzacuva, Waterloo MS Facility, KCL

Bile acid profiling: research and clinical applications

13.00 Lunch and Posters Breakout Area

Session 7 in BH (S) 1.01 Chair: Ana Rodriguez-Mateos

13.45 Sean Burnap, Division of Vascular Biology, KCL

A multi-omics approach to understanding high-density lipoprotein functionality in cardiovascular disease

14.15 Louise Hesketh Dept. Cardiovascular Research, KCL

Examining the hypoxia-activated conversion of lidocaine N-oxide to lidocaine, an antiarrhythmic drug, in Langendorff-perfused isolated rat hearts

14.45 Yannis Paloyelis Dept. Neuroimaging, KCL

Social neuropeptides: basic physiology and measurement challenges

15.15 Erika Castrignanò, Drug Control Centre, KCL

Mass spectrometry as a “key success factor” in tackling doping

15.45 Drinks and Posters Breakout Area

16.00 Session 8 in BH (S) 1.01 Chair: Christopher White

ALL Panel discussion - Where do we go from here?

17.00 Close

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Abstracts

Oral Presentation Abstracts and Biographies

Please click any underlined names for KCL PURE research profiles for more details on latest outputs and research activity!

Dr Ana Rodriguez-Mateos You are what you excrete: using mass spectrometry to investigate the health benefits of plant foods and phytochemicals Diet has been recognized as one of the key modifiable risk factors for the prevention of chronic diseases, such as cardiovascular disease and diabetes. However, establishing relationships between diet and health is complex and there is a strong need to generate robust scientific evidence on the effects of specific diets, specific foods, and individual bioactive compounds within those foods that may be mediating health benefits. Mass spectrometry is increasingly becoming an essential tool in food and nutrition research. This presentation will cover some of the most important applications such as objective dietary assessment in epidemiological and clinical settings, identification of bioactive compounds in foods, interactions and synergisms between different food components when consumed together and elucidating inter-individual variability in response to foods. Ana is a Lecturer in Nutrition and leads the research group on Food Bioactives and Health within the Department of Nutritional Sciences at King’s College London. Her research aims to investigate the health benefits of plant foods and phytochemicals, with a strong focus on understanding the bioavailability, metabolism and cardiovascular health benefits of dietary polyphenols. More recent interests include the investigation of the role of the gut microbiome on the health benefits of phytochemicals, and the development of biomarkers of food intake using metabolomic approaches. Her expertise includes development and validation of analytical methods for the analysis of foods and biological samples using LC/GC-MS and performance of randomized controlled trials with cardiovascular outcomes. She is an Associate Editor of the Royal Society of Chemistry journal Food and Function and a member of the Editorial Board of Nutrition and Healthy Aging. Dr Robin Mesnage Combining omics data to identify health effects of pesticides Recent developments of molecular profiling (“omics”) tools have opened new avenues of investigation to understand the mechanisms of toxicity of chemical pollutants. Glyphosate is the world’s most used herbicide ingredient targeting aromatic amino acid biosynthesis through inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of the shikimate pathway in plants. Since EPSPS and the shikimate pathway also exists in some microorganisms, we conducted a toxicity study to investigate whether glyphosate has an effect on the serum and caecal metabolome in female rats. The results of ongoing analyses will be presented. Research Associate at King’s College London, Robin Mesnage uses high throughput biology techniques to understand the effects of chemical pollutants on human health. His research focus over the last 10 years has been on the safety evaluation of environmental contaminants, including pesticides. His current project aims at evaluating the effects of pesticides on the gut metagenome and metabolome in human populations and laboratory animals.

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Dr Leon Barron There’s something in the water: Using mass spectrometry to understand population-scale consumption and environmental impacts of drugs Pharmaceuticals, illicit drugs and their metabolites represent an emerging class of environmental contaminants of concern. This presentation overview advances in how liquid chromatography-mass spectrometry has evolved to characterise the occurrence, sources, fate and effects of these compounds. Particular attention will be placed on how mass spectrometry has enabled broad-scope targeted, untargeted and suspect screening workflows to be developed for hundreds of compounds in water, solids and biota. Lastly, our contribution to the European Monitoring Centre for Drugs and Drug Addiction wastewater analysis programme to determine the illicit drug consumption behaviour of over 38 million people across 85 cities from 27 countries from 2011-2019 will also be discussed. Leon is a Senior Lecturer in Forensic Science and leads the Environmental & Forensic Chemistry research group within the Dept. Analytical, Environmental & Forensic Sciences. His interests lie in the development of new, defensible strategies, technologies and methodologies to assist in the early discovery and response to pollution, the effects of new/emerging contaminants on biota and humans, wildlife crime, explosives and other security related activity. His core expertise lies in advanced sample preparation, separation science, mass spectrometry and machine learning. He has published >70 articles and book chapters in analytical, environmental and forensic science focused journals. He sits on the Editorial Board of Science & Justice and is also a committee member of the London Biological Mass Spectrometry Discussion Group. Dr Manuel Müller Chemical Synthesis of Post-translationally modified proteins Post-translational modifications (PTMs) play a central role in biological regulation. However, elucidating the role of specific PTMs is challenging due to the complexity of the systems involved. My lab focuses on developing and applying chemical biology tools – including synthesis of site-specifically modified proteins – to understand the role of individual PTMs in cell fate decisions, and how spontaneously occurring PTMs control protein structure and function. I studied biochemistry at ETH Zurich and stayed on to pursue a PhD with Prof. Don Hilvert on primordial enzymes. For my postdoctoral work, I joined Prof. Tom Muir’s lab in Princeton University where I developed and applied chemical biology tools to study chromatin modifying enzymes, and how these enzymes contribute to epigenetic phenomena and diseases. Since 2016, I am a Sir Henry Dale Fellow in the newly formed chemistry department at King’s College London. My research goals involve harnessing protein chemistry and engineering technologies to elucidate how an unusual class of post-translational modifications – occurring at the protein backbone – contribute to biological signalling. Sufyan Pandor How developments/frontiers in metabolomic workflows is driving MS-based metabolome profiling Metabolomics and lipidomics are powerful tools, with applications in pharmacology, systems biology, biochemistry, plant biotechnology, and human diseases. The complexity of samples, physicochemical differences and large data sets makes the analysis challenging - As a result, a range of chromatographic techniques are often required, together with powerful software platforms The talk will focus on our latest omic developments, such as:

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1. A new lipidomics workflow for identifying and profiling lipids on the QTOF and IM-QTOF

2. MassHunter Vistaflux software for automating and accelerating stable isotope labelling studies

3. Classification model building with the new MassHunter Classifier software.

As an LCMS Application Specialist at Agilent, I support our UK customers, focussing on life science applications. I have been with Agilent for over 7 years and started as an LCMS Engineer, before moving in to the Application Specialist role. I specialise in using our QTOFs and ion mobility-QTOF, for metabolomic and lipidomic studies. I am currently completing a part time PhD at Queens University Belfast, where I am developing new omic workflows to tackle issues of food safety and food security. Dr Mark C. Parkin Mass Spectrometry in the Forensic Toxicology Laboratory – Where Are We Now and Where Are We Heading? This presentation offers unique perspectives from a UK based commercial forensic toxicology laboratory where hyphenated chromatography mass spectrometry is used as the principal analytical tool. Current analytical challenges will be emphasised and thoughts on future directions discussed in order to highlight potential research themes and activities. Mark currently heads up the Toxicology laboratory at Eurofins Forensic Services (formally LGC Forensics) and is the current Chair of the London Toxicology Group. He holds a visiting lectureship at King’s Forensics and has over 15 years of experience using mass spectrometry for evidential purposes in both the commercial and academic setting. He has published extensively in the field of bioanalysis and analytical toxicology and frequently presents at national and international conferences. Mark’s research interests have focused on the development of highly sensitive hyphenated mass spectrometry methodologies for the detection of drugs and their metabolites in alternative matrices such as hair and saliva. Dr Simon Elliott Above and Beyond: Can KCL play a part in fixing clinical and forensic analytical toxicology? Analytical toxicology is at the centre of a changing landscape within clinical and forensic toxicology. The disciplines are facing challenges around the type of drugs available and the manner of their use as well as wider resource and funding issues. This presentation will look at such challenges and discuss the potential role King's College London could play in addressing these and assisting for the future. Dr. Simon Elliott has over 20 years’ experience in forensic toxicology and is a Consultant Forensic Toxicologist, independent Business Consultant and Director of Elliott Forensic Consulting Ltd. He is a Visiting Professor in Forensic Toxicology at King's College London. He was previously the Director of Global Forensics at Alere Inc (now part of Abbott) having also been the founder and Managing Director of Forensics Ltd (ROAR Forensics, subsequently Alere Forensics). Prof Elliott previously worked as a Clinical Scientist in the NHS at Birmingham City Hospital for over 10 years specifically involved in clinical and forensic toxicology as Section Head of Forensic Toxicology. He is the current Chair of the UK & Ireland Association of Forensic Toxicologists (UKIAFT) as well as being a Board member of The International Association of Forensic Toxicologists (TIAFT). As well as being an Associate Editor of the Journal of Analytical Toxicology and Drug Testing & Analysis and an Editor of Wiley’s WIREs Forensic Science (Toxicology) and Clarke’s Analysis of Drugs and Poisons, he is the author of over 70 scientific publications, articles and book chapters. Dr Elliott has presented at many national and international meetings (including invited speaker) as well as presenting expert evidence for many years in Coronial, Civil and Criminal Court. A member of the World Health Organisation (WHO) Expert Committee on Drug Dependence, he also advises the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and United Nations Office on Drugs and Crime (UNODC), especially regarding new psychoactive substances.

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Max Hecht & Anca Frinculescu Good Golly, Miss ‘Molly’: Quantification and Dissolution of Ecstasy Tablets Seized 2001 – 2018 This study evaluated the MDMA content of 412 tablets collected in the UK, 2001-2018 using a 2 min LC-MS method (WARP tapered bore column on a Waters Acquity™ UPLC-TQD). In addition, the variability in dissolution profiles of 247 tablets was analysed by a rapid-LC assay with only 36 seconds run time per sample on an Agilent 1290 UHPLC and Ultivo miniature triple quadrupole mass spectrometer. The data supports the concerns on the prevalence of high-content MDMA tablets in circulation. Drug identification is useful in non-laboratory settings but cannot fully-assess the risk to users due to fast- or slow-releasing MDMA tablets in circulation. Max Hecht is currently a PhD student in the field of analytical chemistry at the University of Tartu in Estonia and working as a trainee study director for Analytical Sciences International, London. The research focus is on direct ionization techniques and how to make them work in a clinical setup. He is also interested in developing hardware for mass spectrometer to be used with clinical samples. Anca Frinculescu is a registered pharmacist with an MSc in Forensics, a PhD student at Kings College London and working as a pharmaceutical analyst for TICTAC Communications Ltd. Anca oversees TICTAC’s laboratory and the “amnesty bin” analysis at music festivals which TICTAC pioneered since the late 90s, collaborating with a number of the UK’s largest festivals and clubs. Dr Varun Mehra Invasive Fungal disease -Time for MS to step in Invasive Aspergillosis (IA) accounts for a majority of Invasive Fungal diseases and is a potentially life-threatening condition with high mortality in immunocompromised patients with hematological malignancies and allogeneic hematopoietic stem cell transplants. However there is no gold standard diagnostic test for an early diagnosis of IA. Gliotoxin (GT) is an epipolythiodioxopiperazine (ETP) of molecular mass 326 Da, made only by fungi and appears to be a virulence factor associated with IA. We designed a prospective study focussing on improving our combined diagnostic strategies for Invasive fungal disease (IFD) with these novel fungal biomarkers by mass spectrometry & PCR methods on patients recruited as part of a single centre prospective Kings Invasive Aspergillosis Study II (KIASII), EudraCT number 2016-001223-31 with an aim to study its clinical performance compared to other diagnostic methods in all study patients. We discuss role of mass-spectrometry as a novel diagnostic tool with significant clinical application in diagnosis and effective stewardship of management of IA. Dr Varun Mehra is a UK trained Haematologist with a special interest in opportunistic infections in immunocompromised host and Graft versus Host disease in patients post allogeneic stem cell transplantation. Dr Mehra, who originally graduated from India, completed his specialist training in 2016 at one of UK’s reputed Haematology training program at Kings College Hospital London, where he has been working for past 8 years as a core member of the busy Stem Cell Transplant unit, while pursuing his MD (res) Degree with Kings College London in developing novel biomarkers in Fungal diagnostics for invasive aspergillosis in haemato-oncology patients. His core research interests have helped develop exciting research collaborations within Kings Health Partners with exciting bench to bed translation opportunities. Professor R Neil Dalton Targeted Metabolomics: Lessons Learned Targeted metabolomics, using electrospray tandem mass spectrometry (MSMS), for both rapid clinical diagnostics and biomarker validation requires a stable analytical platform. Sample preparation and MSMS analysis are the easy part of the analytical logistic pathway with sample

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collection, sample storage, assay standardisation, assay quality control, and data processing providing the real challenges and lessons learned. Neil is the Director of the WellChild Laboratory at the Evelina London Children’s Hospital, a founding director of SpOtOn Clinical Diagnostics, and Emeritus Professor of Paediatric Biochemistry at King’s College London. His current interests include the development of mass spectrometry based kits for newborn DBS screening and rapid inherited metabolic disease diagnosis, defining biomarkers of diabetic complications (AdDIT, SUMMIT, and JDRF biomarker consortia), and the measurement of renal function decline (eGFR-C study). Charles Turner Peptide based analysis of proteins using electrospray MSMS Electrospray MSMS using selected reaction monitoring, is a powerful analytical tool for the measurement of peptide biomarkers, either endogenous or generated by endopeptidase digestion. The technique offers a rapid, highly specific method to look at concentration and sequence variation of proteins and peptides in biological sample. Charles Turner is a Clinical Scientist & Deputy Director of the WellChild Laboratory at the Evelina London Children’s Hospital. He began his career at St Mary’s Hospital Medical School working on immunoassays for calcium regulating hormones (vitamin D metabolites and parathyroid hormone), and then came to work with Neil Dalton, then at Guy’s Hospital. The lab now concentrates on clinically based R&D, with an emphasis on biomarker analysis, validation, and discovery, in the areas of renal disease, diabetes, metabolic disease diagnosis and monitoring, and neonatal screening, mainly using electrospray MSMS. A spin-out company, SpOtOn Clinical Diagnostics, in which KCL & GSTT are majority shareholders was established in 2011 to exploit patented methods. Dr Theodora Stewart London Metallomics Facility: An Emerging Scientific Platform for Multidimensional Metal Analysis Metallomics takes an integrated approach to understand the metallobiochemistry of cells and organisms in health and disease, utilising analytical tools to decipher the biological roles of metals in both space and time. Identification of characteristic metal profiles within the life span of individual cells and tissues has significant value for fundamental scientific questions relating to determination of cell fate and health, trace element metabolism, and for translational science in diagnosis, prognosis, and therapy in a range of human diseases including cancer, neurodegeneration and metabolic diseases such as diabetes, Wilson’s disease, and thalassaemia. Simply put, metal ions are metabolites, and just as metabolomics is a rapidly growing field explored by molecular mass spectrometry, metallomics is an emerging field explored by elemental mass spectrometry. The London Metallomics Facility (LMF) is a newly created Wellcome Trust and King’s College London supported core facility, whose aim is to be a centralised hub for comprehensive multi-institutional integration of state-of-the-art metallomic analytics and correlative bioimaging, establishing direct interaction between leading research groups, commercial partners, and industry, whilst driving educational outreach and public engagement strategies. This presentation will focus on recent analytical developments within the LMF, highlighting bioimaging work within the context of metals in human health and disease. Dr Theodora Stewart is the lead scientist and manager of the new London Metallomics Facility (LMF) based at King's College London, focusing on developing correlative bioimaging workflows to advance our understanding of the roles of metals in biology. Her research interests lie at the interface of biology, chemistry and physics, with a specific focus on developing analytical techniques and workflows to quantify temporal dynamics of intracellular metal species, their subcellular localisation, and identification of key biomolecules involved in their binding and transport with minimal disruption to the

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system of study through the use of both elemental mass spectrometry and synchrotron X-ray spectroscopy based bioimaging techniques. Dr Caitlyn Da Costa Imaging Mass Spectrometry: Visualization at the molecular level The integration of mass spectrometry with imaging platforms, such as DESI and MALDI, provides a powerful tool to characterize surfaces at a molecular level. The approach can enable greater scientific insight into surface physiology and analyte distribution, lending itself to a range of diverse application areas. Caitlyn Da Costa received an honors degree in Biochemisty with Forensic Science from Queen Mary University of London before achieving both an MSc and PhD in Analytical Chemistry at Loughborough University under the supervision of Professor Colin Creaser. The PhD thesis titled “Applications of Desorption Electrospray Ionisation Mass Spectrometry and Ion Mobility Spectrometry to Petroleomic and Lubricant analysis” described the use of DESI-MS for the surface characterization of samples related to the chemical and environmental field. Upon completion of a PhD she took a Senior Application Scientist role at Waters Ltd, applying hyphenated MS technologies including imaging-MS to address a diverse range of analytical challenges. Dr Christopher Titman Tools to help find components in complex mixtures by HRAM LC/MS/MS Using the Find algorithm we show how to detect components in highly complex mixtures, and compare this with other platforms such as MS-DIAL. The talk will consider pesticide analysis and plasma metabolomics using HRAM sample analysis. It will highlight our passion for creative thinking and generating actionable data with greater confidence. Chris holds a degree in biochemistry from Imperial College, where his interest in protein science led to a PhD in biochemistry from the University of Cambridge. A change of direction led to a postdoctoral position in metabolomics which encouraged his interest in mass spectrometry. This led to Shimadzu, where he has been for over 11 years, providing support for mass spectrometry. Dr Mariana Silva Dos Santos Metabolomics and Lipidomics at The Crick: A case study in Tuberculosis The Metabolomics platform at the Crick works with over 100 researchers on a weekly basis, running 35,000 samples per year. Covering a wide range of biological questions - from Drosophila evolution and life span to organoid metabolism, and from fruit volatiles and their chemoattractant capabilities to intricate delineation of metabolic pathways in cancer and infectious disease - we use a multi-platform approach to both discovery metabolomics and bespoke, targeted studies. This presentation will give an overview of how we use different instrumentation and techniques to achieve answers to this broad range of biological research. In particular, recent in-house developments in lipidomics and metabolomics for investigating the metabolism of the bacterium Mycobacterium tuberculosis will be discussed Mariana received her B.Pharm from the Federal University of Minas Gerais (Brazil) where she completed her PhD in Pharmaceutical sciences in December 2014. In 2011 she joined the Centre of Metabolomics and Bioanalysis (CEMBIO), San Pablo, Spain) as part of her sandwich doctorate to investigate early biomarkers for canine leishmaniasis through metabolomic approaches. In 2015, she joined the Metabolomics STP at the Francis Crick Institute and is working on the development and application of LC-MS methods for metabolomics and lipidomics research. Her current project, in collaboration with Luiz Carvalho, aims to unveil protein function in M. tuberculosis through metabolomics and lipidomics approaches. She is also investigating the involvement of M. tuberculosis

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lipids in pathogenicity and virulence and how the TB lipidome could be used to identify new therapeutic targets. Dr Cagakan Ozbalci Lipidomics in mammalian cells We use LC-MS (liquid chromatography mass spectrometry) to investigate cellular lipidomes, coupled with cell biological analysis into their potential functions. While there are many promising advances in this field, there are still chemical, analytical and informatic challenges to be solved. Cagakan Ozbalci is a Research Associate in the Randall Centre for Cell & Molecular Biophysics at King’s College London. He received his BS and MSc from Istanbul University and Marmara University in Turkey and completed his PhD at Heidelberg University with Prof. Britta Bruegger, gaining experience in shotgun lipidomics analysis. He is now working in Prof. Ulrike Eggert’s group as a postdoctoral researcher developing non-targeted and targeted strategies in LC-MS based lipidomics to understand lipid functionality in cell division. As part of his project, he runs and maintains the QTOF- MS owned by the Eggert lab and teaches lipidomics workflow and data analysis to lab members and collaborators Dr Francesca Mazzacuva Bile acid profiling: research and clinical applications Bile acids are important end products of cholesterol catabolism. Recent findings suggest their relevance in clinical settings thus creating the need for high throughput screening analyses. In this context we have developed a selective and sensitive tandem mass spectrometry method now routinely employed in research and clinical diagnostics. Following a PhD in Pharmaceutical Chemistry and a post-doctoral specialisation in Clinical Applications of Mass Spectrometry, Dr Mazzacuva developed a research interest in the field of new biomarker discovery and translational medicine. She joined the mass spectrometry team at the Hospital of Florence, developing new diagnostic methods in endocrine diseases and then moved to London working as a research associate at Great Ormond Street-Institute of Child Health in the area of inborn errors of metabolism. Since moving to King’s College London in 2018, her research now focuses on the development of high throughput tandem mass spectrometry methods for biomedical applications. Sean Burnap A Multi-Omics Approach to Understanding High-Density Lipoprotein Functionality in Cardiovascular Disease New measurements to assess high-density lipoprotein (HDL) functionality are fundamental to further our understanding of the role HDL plays in the development of cardiovascular disease (CVD). This study combines proteomic and lipidomic technologies in the characterisation of HDL in the context of CVD in a large patient cohort. Sean Burnap is a recent graduate from King’s College London, where he studied Biomedical Sciences. An interest in the cardiovascular system built up throughout the course of his undergraduate degree, leading him to be accepted onto the 4-year MRes/PhD Cardiovascular Sciences programme at King’s College London, funded by the British Heart Foundation. The initial master’s year allowed him to be introduced to mass spectrometry and the Mayr lab, leading to the decision to remain in this lab to do his PhD.

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Louise Hesketh Examining the hypoxia-activated conversion of lidocaine N-oxide to lidocaine, an antiarrhythmic drug, in Langendorff-perfused isolated rat hearts Sudden cardiac death, resulting from fatal arrhythmias due to myocardial infarction, accounts for half of deaths from cardiovascular disease. Lidocaine N-oxide is a prodrug which, under hypoxic conditions, is converted to the antiarrhythmic drug lidocaine. The antiarrhythmic activity of the lidocaine produced during prodrug conversion was investigated in the isolated rat heart, and the conversion within myocardial tissue was quantified using a UPLC-MS/MS method to assess the potential of lidocaine N-oxide to be used as an antiarrhythmic drug. Louise Hesketh is a third year PhD student at King’s College London. She received a first-class degree in Biomedical Sciences from King’s College London in 2016 before beginning an MRC funded PhD researching into the development of a novel class of hypoxia-activated antiarrhythmic prodrugs, with collaborations at Imperial College London. In line with this, her scientific interests include pharmacology and cardiovascular physiology. Dr Yannis Paloyelis Social neuropeptides: basic physiology and measurement challenges Oxytocin (OT) and vasopressin (AVP) play diverse roles in the development and regulation of social behaviour and cognition (hence called “social” neuropeptides), but also in pain processing, energy intake, and even in the regulation of neuroinflammation. A key aspect of understanding the function of the OT and AVP systems requires the development of sensitive and precise ways to measure the concentration of these peptides in body fluids. In this talk I will focus on the central OT system and briefly describe its neurophysiology, patterns of OT release, functional significance and the challenges that we face in measuring OT in fluids. Yannis Paloyelis is Lecturer and Director of the BSc in Neuroscience and Psychology at the Institute of Psychiatry, Psychology and Neuroscience (IoPPN), King’s College London. His research focuses on the central oxytocin system and range from understanding the pharmacodynamics of oxytocin in the human brain, optimising methods of administration, to understanding the role of the oxytocin system in social behaviour and cognition and its involvement in neuropsychiatric disorders.

Dr Erika Castrignanò Mass spectrometry as a “key success factor” in tackling doping Tackling doping in sport is a continuous challenge due to its multifaceted nature, with potential doping agents varying from small molecules to large proteins. Hence, advanced analytical techniques need to keep up with the evolving dynamics of doping in sport detecting an ever greater number of analytes at increasing low detection limits. Mass spectrometry is a well-established “elite” technique capable of detecting, identifying and quantifying a number of diverse doping agents in athlete’s samples to a legally defensible standard. This presentation will provide the audience with an overview of the extended usage of mass spectrometry in the analysis of a wide range of compounds with the most updated approaches in the field of high resolution mass spectrometry and “omics” strategies applied to anti-doping testing.

Erika Castrignanò is a Senior Analyst at the Drug Control Centre within King's Forensics in the Department of Analytical, Environmental & Forensic Sciences. She has broad experience in analytical chemistry at both industrial and academic level and her research is currently aimed at advancing the boundaries of this discipline in three specific aspects: (i) anti-doping science, (ii) forensic toxicology for the bioanalysis of drugs of abuse and their enantiomeric profiling and (iii) environmental analysis (e.g. antimicrobial resistance and biormarkers) as an aid to understand human exposure to xenobiotics. She

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has been a Marie Skłodowska-Curie Researcher and she has published 25 peer-reviewed research articles in highly ranked international journals including Water Research, Scientific Reports and Analytica Chimica Acta.

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Poster Abstracts 1. NANOBIOPSY: Molecular Classification of Tumours by Spectroscopic Analysis of Tissue Replicas on Nanoneedles Davide A. Martella,1 MadsBergholt,1 Ciro Chiappini1 1. Centre for Craniofacial and Regenerative Biology, King’s College London, Faculty of Dentistry, Oral & Craniofacial Sciences, London, UK *Email: davide.martella@kcl.ac.uk Precision oncology aims to tailor patient treatment to the characteristics of individual malignancy. A key resource to tackle this challenge is molecular diagnostics, yet largely relying on biomarker detection from biopsies. Its diagnostic efficacy is negatively affected by labour-intensive protocols and limited information on the tumour overall molecular signature and heterogeneity. NANOBIOPSY develops a platform for accurate, minimally invasive, label-free mapping of the molecular profile of tumour. This map allows stratifying tumours based on their overall molecular composition and spatial distribution (heterogeneity). NANOBIOPSY uses chips patterned with arrays of porous-silicon nanoneedles pressed onto the tissue to generate a molecular replica. This reduces invasiveness and need for biopsies. The replica is analysed by Raman spectroscopy and mass spectrometry imaging, to combine high spatial and mass resolution. The stratification of clinical samples according to the shared molecular profile is performed through machine learning algorithms and the results are compared with histology. 2. Synthetic Cannabinoids/Alcohol Consumption – a New Metabolite Detected Orapan Apirakkan,1 Pierre Cheyanne,1 Lewis Couchman,2 Ivana Gavrilovic,1 David Cowan,1 Vincenzo Abbate1 1. King’ Forensics, Department of Analytical Environmental and Forensic Sciences, King’s College London 2. Analytical Services International, St. George's - University of London Cranmer Terrace London SW17 0RE Synthetic cannabinoids (SCs) constitute one of the most popular classes of new psychoactive substances (NPS). SCs are still posing a health threat to public due to their psychoactive but also peripheral toxic effects. Their toxicology and pharmacology are mostly unknown due to the dynamic nature of these recreational drugs. Metabolism of SCs is also relevant in clinical and forensic toxicology, as SCs are excreted in urine mostly as their metabolites. Thus, SCs' metabolites are widely used as biomarkers for identifying SCs intake. This research has identified a new metabolite, SC-ethanoate (ethyl ester), hereby proposed to be used as new biomarkers of SCs-alcohol consumption. This species is produced (in vitro using HLM systems) when SCs containing labile ester functional groups react with ethanol via enzymatic transesterification mediated esterase enzyme in the liver. Method validation for semi quantitative screening will be performed to detect and identify these new metabolites in biological matrices with an aim to finally investigate in vivo production of these novel markers in real cases using clinical and forensic samples

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3. Introducing the CEMS Proteomics Core Facility at King’s College London Steven Lynham1* 1, Proteomics Core Facility, Centre of Excellence for Mass Spectrometry, King’s College London. *Email: steven.lynham@kcl.ac.uk The CEMS Proteomics Core Facility is based in the James Black Centre on the Denmark Hill Campus. We provide a service and support to KCL and external researchers seeking to identify and quantify proteins and their modifications. We routinely analyse protein mixtures from gel slices, co-immunoprecipitations or enrichments and can identify several thousand proteins in complex samples such as cell lysates. Post-translational modification identification and mapping can also be performed whether it be to a specific single amino site or global identification. We also offer global quantitation of complex mixtures in multiplexed experiments which have been labelled with stable isotopes (SILAC), isobaric mass tags (TMT) or label-free. Support to help with scientific discussion, experimental design, sample preparation and analysis of mass spectrometry data is also offered to researchers wishing to undertake proteomic experiments. Please come and visit our poster to see how we can help your research. 4. Bioavailability of Aronia berry (poly)phenols Melanie Le Sayec,1 Geoffrey Istas,1 Eleanor Wood,1 Emilie Fromentin2 and Ana Rodriguez-Mateos1 1. Department of Nutritional Sciences, School of Life Course Sciences, Faculty of Life Sciences and Medicine, King’s College London, UK 2. R&D department, Naturex Inc., South Hackensack, New Jersey, US Aronia berries are a rich source of (poly)phenols with potential health benefits. We aimed to investigate the bioavailability of Aronia (poly)phenols after consumption of 500 mg of a (poly)phenols rich extract or a whole fruit powder for 12 weeks in 44 healthy men. Blood samples were collected at baseline and after 2 h post-consumption, on week 1 and week 12, and (poly)phenol metabolites were analyzed using LC-MS. A total of 63 phenolic metabolites were quantified in plasma, including derivatives of hippuric, benzoic, hydroxycinnamic, phenylacetic and propionic acids, benzaldehydes, catechols, pyrogallols, flavonols and valerolactones. Forty-eight and 22 compounds significantly increased at 2 h after consumption of the Aronia extract and Aronia whole fruit powder, respectively. After 12 weeks consumption, 22 and 14 compounds significantly increased compared to baseline in the extract and whole fruit groups, respectively. These findings demonstrate that Aronia berry (poly)phenols are more bioavailable than previously thought. 5. Structural Analysis of the Novel Fungal Peptide Toxin, Candidalysin Olivia Hepworth,1 Julian Naglik,2 Antoni Borysik1 1. Department of Chemistry, King’s College London 2. Centre for Host-Microbiome Interactions, Dental Institute, King’s College London Candidalysin is a cytolytic peptide toxin, produced by the fungus Candida albicans. It is the first described fungal toxin established as the hyphal moiety responsible for mucosal damage and epithelial cell activation in response to infection. [1] However, the peptide’s structure and dynamics are still poorly understood. In this study we use a range of mass spectrometry and biophysical techniques to characterize the membrane embedded peptide assemblies with a view to understanding the molecular basis of their toxicity. Using native mass spectrometry, we have

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identified that the peptide forms oligomers, and we aim to model the peptide’s binding affinity to the cell membrane in order to characterize the kinetics of association. To determine the thermodynamics of formation, we examined the sub-unit exchange between oligomers, and observed that the peptide forms stable structures in solution. The combination of these findings will hopefully provide significant insights into Candidalysin’s structure and its biological function. [1] Moyes, D. L., et al. (2016). Nature, 532, 64-68. 6. A Mass-Spectrometry-Based Modelling Workflow for Accurate Prediction of IgG Antibody Conformations in the Gas Phase Kjetil Hansen,1+ Andy M. Lau,1+ Kevin Giles,2 James M. McDonnell,3 Weston B. Struwe,4 Brian J. Sutton,3 and Argyris Politis1 1. Department of Chemistry, King's College London, 7 Trinity Street, London, SE1 1DB, UK. 2. Waters Corp., Stamford Road, Wilmslow, SK9 4AX, UK. 3. Randall Centre for Cell and Molecular Biophysics, King's College London, UK. 4. Department of Chemistry, University of Oxford, UK. + These authors contributed equally to this work Immunoglobulins are Y-shaped proteins which play an important role in the immune system. A unique property of immunoglobulins is their enhanced flexibility. This flexibility allows immunoglobulins to bind antigens and receptors, and allows proper function of the immune system. Here we have developed an integrative strategy combining ion mobility-mass spectrometry (IM-MS) with molecular modelling to interrogate the conformational dynamics of human immunoglobulin G (IgG). The collisional cross section (CCS) of IgG1-4 were measured in gas phase by IM-MS. Collapse of IgG was observed from the CCS data. Models of IgG1-4 were assembled and molecular dynamics successfully used to simulate this gas phase collapse. Our data show that this collapse occurs at the hinge region responsible for increased flexibility in IgG. The workflow presented here allows accurate structural representation of gas phase collapse of IgG molecules. 7. 3D-printed, multi-sorbent solid phase extraction and high-resolution trace chemical analysis for explosives in complex matrices Rachel Irlam,1 Leon Barron,1* Matthew Beardah,2 Michael O’Donnell,2 Mark Parkin,3 Dermot Brabazon4 1. Dept. Analytical, Environmental & Forensic Sciences, Faculty of Life Sciences and Medicine, King’s College London, London, UK 2. Forensic Explosives Laboratory (FEL), Dstl, Fort Halstead, Sevenoaks, Kent, UK 3. Eurofins Forensics, Teddington, Middlesex, UK 4. Advanced Processing Technology Research Centre & I-Form, School of Mechanical & Manufacturing Engineering, Dublin City University, Ireland *Email: leon.barron@kcl.ac.uk Complex samples often contain matrix interferences that challenge routine explosives detection. This work aimed to design a 3D-printed solid phase extraction (SPE) array to remove unwanted matrix from various sample types and efficiently extract target explosives. A fritless, 3D-printed SPE cartridge was designed and optimised (Fig. 1). Blocks could be ‘clicked together’ in series without sorbent bleed or leaking, enabling rapid assembly of sample-dependent, multi-sorbent SPE approaches. Recoveries from model solutions using these cartridges showed promising results, with values between 15 % and 69 % for 14 selected explosives analytes. The method was then applied to dried blood, cooking oil residues and soil. By removing additional matrix components, a

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combined-sorbent SPE approach enabled detection at the low picogram-on-column level. Additionally, the on-site sample preparation potential of miniaturised SPE is highly advantageous, minimising the chance of losing explosive analytes through evaporation or degradation and thus enhancing the likelihood of forensic detection.

Figure 1: CAD drawing of optimised sorbent block design (left) and the final 3D-printed part packed with selective extraction sorbent (right).

8. High resolution chemical analysis and machine learning for the identification of pharmaceutical and illicit drug compounds in water Helena Rapp-Wright,1,2 Fiona Regan,1 Blánaid White,1* Leon Barron2* 1. DCU Water Institute and School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland 2. Analytical & Environmental Sciences Division, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK *Email: Blanaid.white@dcu.ie; Leon.barron@kcl.ac.uk Contaminants of emerging concern (CECs) have been shown to occur in surface waters over the past decade (ng/L to µg/L) and their risks in the environment require further knowledge. A more flexible methodology combining liquid chromatography-high resolution mass spectrometry (LC-HRMS) and machine learning-based data analysis is presented for their prioritisation in water samples in the River Thames, UK. Daily samples were collected over one week from the central London and subjected to analyte enrichment with solid-phase extraction before analysis. A previously developed LC-HRMS method for >200 CECs was used to generate an artificial neural network-based retention time prediction tool [1]. Combining predicted retention times with accurate m/z (≤5 ppm) and theoretical isotope profile matching (≥80% fit) to a full-scan HRMS library of ~1500 compounds [2], suspect screening was successfully performed and subsequent formal confirmation using analytical standards of CECs. Overall, the use of chemical analysis and machine learning could enable more rapid shortlisting of CEC candidates for prioritised monitoring. Such approaches may also inform more efficient ecotoxicity testing strategies for potentially toxic compounds in the aquatic environment. [1] Munro, K., Miller, T.H., Martins, C.P.B., Edge, A.M., Cowan, D.A., Barron, L.P.; Artificial neural network modelling of pharmaceutical residue retention times in wastewater extracts using gradient liquid chromatography-high resolution mass spectrometry data (2015) Journal of Chromatography A, 1396, pp. 34-44. [2] Munro, K., Martins, C.P.B., Loewenthal, M., Comber, S., Cowan, D.A., Pereira, L., Barron, L.P.; Evaluation of combined sewer overflow impacts on short-term pharmaceutical and illicit drug occurrence in a heavily urbanised tidal river catchment (London, UK) (2019) Science of the Total Environment, 657, pp. 1099-1111.

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9. Emerging contaminant occurrence in a London urban river system measured using passive sampling and liquid chromatography-high resolution accurate mass spectrometry Alexandra K Richardson,1 Gary R Fones,2 Graham A Mills,2 David A Cowan,1 Anthony M Edge,3 David J Neep,3 Leon P Barron1* 1. Department of Analytical, Environmental & Forensic Sciences, School of Population Health & Environmental Sciences, Faculty of Life Sciences and Medicine, King's College London, 150 Stamford Street, London SE1 9NH, United Kingdom 2. School of Pharmacy and Biomedical Sciences, University of Portsmouth, White Swan Road, Portsmouth, PO1 2DT, United Kingdom 3. Agilent Technologies LDA UK Ltd., Essex Road, Church Stretton, Shropshire, United Kingdom *Email. leon.barron@kcl.ac.uk Targeted occurrence of 101 chemically diverse compounds was determined using two different types of passive sampler deployed for two weeks over the Christmas/New Year period in 2018/9 and analysed using reversed-phase chromatography coupled to high-resolution mass spectrometry (HRMS). Over 40 different compounds were found. The most frequent and highest intensity compounds included Clarithromycin, Diclofenac, Cocaine and Propanolol. Secondly, untargeted analysis was conducted using a previously developed machine learning model to predict retention times and using TraceFinder library of >1,500 compounds, mass spectra were evaluated based on accuracy <5 ppm and >80 % isotope ratio profile matching at 50,000 FWHM resolution. For retention prediction, a generalised regression neural network model achieved a prediction accuracy of <1.3 minutes for >75 % of all compounds. The model was then applied to a selection of potential suspects in river water extracts. 10. The Mass Spectrometry Facility – Franklin-Wilkins Building, King’s College London Anna Caldwell,1* Francesca Mazzacuva,1 Robert Gray,1 Andy Cakebread,1 Roger Tye,1 John Halket1

1. Mass Spectrometry Facility, Franklin-Wilkins Building, King’s College London, London SE1 9NH *Email: anna.caldwell@kcl.ac.uk; https://tinyurl.com/ycvcm3te The Mass Spectrometry Facility was established in 2001 to encourage the application of mass spectrometry in research. Staffed by highly experienced experts and well equipped (GC/MS, LC/MS/MS, HRMS, MALDI), it specialises in the qualitative and quantitative analysis of small organic molecules (up to approx. 3,000 Da) in forensic, biological, foodstuffs and environmental samples at ultratrace levels (~pg/mL, depending on the nature of the analyte and matrix). The capability of the Facility is briefly described and illustrated by several examples including biomarker discovery, metabolic profiling (metabolomics) as well as targeted and quantitative analyses of endogenous and drug-related molecules in a variety of samples.

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11. Charge derivatization to enhance the analysis of acidic metabolites and glucuronide conjugates by liquid chromatography/mass spectrometry Francesca Mazzacuva,1 Anna Caldwell,1 Robert Gray,1 David Cowan,2 John M. Halket1* 1. Mass Spectrometry Facility 2. Drug Control Centre, Franklin-Wilkins Building, King’s College London, London SE1 9NH * Email: john.halket@kcl.ac.uk Chemical derivatization procedures, particularly trimethylsilylation, are commonly employed to alter the physicochemical properties of metabolites and aid their analysis by gas chromatography/mass spectrometry (GC/MS). In LC/MS, derivatization techniques are also being introduced to enhance sensitivity for problematic analytes. The current work describes the application of pre-charged trimethoxyphenylphosphonium (TMPP) derivatives [1,2] to improve detection of acidic metabolites such as short-chain fatty acids, organic acids and glucuronide conjugates. High mass parent and fragment ions are obtained reducing the significance of chemical noise and excellent separations are obtained on reverse-phase LC:

A library of highly reproducible tandem mass spectra (ion trap) of organic acid derivatives is described. The analysis of anabolic steroid glucuronide conjugates by LC/MS/MS is complicated by the apparent different proton affinities of these compounds [3.4]. Depending on the steroid structure, ammonium adducts may be formed preferentially. The TMPP method helps to address this problem by the formation of positively charged derivatives and signal/noise enhancements in urine can be 1000-fold [5]. [1] Sadagopan, N, Watson, JT, J. Am. Soc. Mass Spectrom., 11, 107-119, 2000. [2] Leavens, WJ, Lane, SJ, Carr, RM, Lockie, AM, Waterhouse, I, Rapid Commun. Mass Spectrom., 16, 433-441, 2002. [3] Bowers, LD, Sanaullah, N, J. Chromatogr. B 687, 61-68, 1996. [4] Kuuranne, T, Vahermo, M. Leinonen, A, Kostiainen, R, J. Am. Soc. Mass Spectrom. 11, 722-730, 2000. [5] Turfus, SC, Halket, JM, Parkin, MC, Cowan, DA, Braithwaite, RA, Kicman, AT, (2014). Drug Testing and Analysis, (October 2014), https://doi.org/10.1002/dta.1661

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12. GC-NICI-MS determination of short chain fatty acids in serum using pentafluorobenzyl derivatives Robert Gray,1* Amrita Vijay,2 Anna Caldwell,1 Francesca Mazzacuva,1 John Halket1 1. Mass Spectrometry Facility, Franklin-Wilkins Building, King’s College London, London SE1 9NH 2. TwinsUK, Department of Twins Research, King’s College London, St Thomas’ Hospital Campus, Westminster Bridge Road, London SE1 7EH, *Email: robert.gray@kcl.ac.uk Short chain fatty acids (SCFAs) are an important subclass of fatty acids produced by the gut microbiota during the fermentation of partially and nondigestible polysaccharides. An abundance of evidence suggests their role in the maintenance of health and the development of disease. Despite the need for the accurate measurement of SCFAs in serum, technical difficulties in analysis often arise due to their volatility and hydrophilicity. Here we present a highly sensitive and selective method for the profiling of SCFAs in human serum using the Agilent 5973 inert GC/MSD equipped with CI capability. To circumvent the aforementioned issues, we employed in situ derivatization with pentafluorobenzyl bromide followed by hexane extraction. Using this approach, an NICI based method for C2 to C5 (Acetic to Valeric) acids was developed, with all species in this range successfully identified and quantified. 13. Exploring the “Dark Matter” of the Metabolome: Curation and quality assurance of mass spectral libraries of unknowns John M. Halket,1* Anna Caldwell,1 Robert Gray,1 Francesca Mazzacuva,1 W. Gary Mallard,2 Stephen E. Stein2 1. Mass Spectrometry Facility, Franklin-Wilkins Building, King’s College London, London SE1 9NH 2. Mass Spectrometry Data Center, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA *Email: john.halket@kcl.ac.uk A major bottleneck in metabolite profiling is the very large number of unidentified metabolites. As a contribution to solving this major problem, libraries of confirmed recurring unknown spectra in biological materials are created. The eventual web-based sharing [1] of such libraries in the metabolomics research community will assist the annotation of such unknowns. During a study of >6,000 metabolic profiles of paediatric urine [2], it was found that the use of two chromatographic columns of slightly differing polarities together with mass spectral deconvolution had utility for verifying the integrity of the unknown spectra encountered, i.e. the detection of “false” spectra formed by the exact superposition of two or more spectra. A resulting mass spectral library is available for download. This work has been extended to incorporate a third chromatographic dimension. The power of the new method is illustrated by examples from the profiling of over 60 plant-based food materials. [1] Askenazi, M, Stein, SE, The Fragmentarium: A Universal Query Service Enabling Partial Matching of Unidentified Spectra across the Full Gamut NIST MS Spectral Libraries, Proc. 64th ASMS Conf., San Antonio, TX, 2016 [2] Mallard WG, Andriamaharavo NR, Mirokhin YA, Halket JM, Stein SE. Anal. Chem. 86, 10231–10238, 2014.

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14. An isotope-dilution GC/MS method for the quantitation of 25-hydroxy-vitamin D3 in

human serum

Anna Caldwell,1 Graham Carter,2 Julia Jones,2 Robert Gray,1 Francesca Mazzacuva,1 John M. Halket1,2*

1. Mass Spectrometry Facility, Franklin-Wilkins Building, King’s College London, London SE1 9NH 2. Imperial College Healthcare NHS Trust, London W6 8RF *Email: john.halket@kcl.ac.uk

The level of 25-hydroxy-vitamin D3 in serum is currently the most used indicator of vitamin D status, a parameter of increasing importance in many aspects of health. Although GC/MS methods have been developed [1,2], they have been considered too slow for routine use. Immunoassay, HPLC and LC/MS/MS methods are widespread but modern GC/MS has superior chromatographic resolution. In the current work, a simplified procedure as been developed with potential as a standard reference method available to external quality assurance schemes such as DEQAS.

Calibrators, blank, spiked serum and patient serum samples (1 ml spiked with d6-25-hydroxy-vitamin D3 as internal standard) are deproteinized with acetonitrile and purified by solid phase extraction. Dry residues are trimethylsilylated and analysed by GC/MS in SIM mode.

Validation data are presented together with results of spiking experiments as well as data from a range of DEQAS samples analysed.

[1] I Björkhem, I Holmberg, Clin. Chim. Acta, 68, 1976, 215-221.

[2] RD Coldwell, DJH Trafford, MJ Varley, HLJ Makin, DN Kirk, Biol. Mass Spectrom, 16, 1988, 81-85.

15. Good Golly, Miss ‘Molly’: Quantification and Dissolution of Ecstasy Tablets Seized 2001 - 2018 Anca Frinculescu,1,5 Max Hecht,2,4 Catarina C. Sobreira,1,3 Karin Kipper,2,4 Trevor Shine,1 John Ramsey,1 Atholl Johnston,2,3 David W. Holt,2 Lewis Couchman2,5

1. TICTAC Communications Ltd., Cramer Terrace, London, SW 17 0RE, United Kingdom 2. Analytical Services International Ltd., Cramer Terrace, London, SW 17 0RE, United Kingdom 3. Barts and The London Queen Mary’s School of Medicine and Dentistry, Mile End Road London E1 4NS, United Kingdom 4. University of Tartu, Institute of Chemistry, 14a Ravila Street, 50411 Tartu, Estonia 5. King’s College London, Strand, London WC2R 2LS, United Kingdom MDMA (‘ecstasy’) tablets are recreationally used and known to vary in their appearance and MDMA content. This project studied the variation in dose and dissolution profiles for samples collected between 2001- 2018.

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Figure 1: Variation in MDMA content (red) and total tablet mass (blue) (A) and dissolution rate (B) for tablets collected between 2001 and 2018. For quantification, approximately 10 mg of crushed tablet was accurately weighed and dissolved in a methanol/water solution. For dissolution, 247 whole tablets were dissolved in an HCl solution (37 °C) with samples collected at 11 time points over a 3 h period. All measurements were conducted using LC-MS/MS. The median MDMA content (N=412) decreased from 2001 to 2009 and increased after 2010. The proportion of slow-releasing tablets decreased over the period studied, but there was still significant between-tablet variability in dissolution. A B Our data supports the concerns on the prevalence of high-content MDMA tablets in circulation. Drug identification is useful in non-laboratory settings but cannot fully-assess the risk to users due to fast- or slow releasing MDMA tablets in circulation. 16. Protein Atlas of the Human Vascular Extracellular Matrix

Ferheen Baig,1* Javier Barallobre-Barreiro,1 Marika Fava,1 Konstantinos Theofilatos,1 Marc Lynch,1 Marieke Rienks,1 Elizaveta Ermolaeva,1 Marjan Jahangiri,2 Manuel Mayr1*

1. King’s British Heart Foundation Centre, King’s College London, London, UK 2. St George’s University of London, NHS Trust, United Kingdom *Email: ferheen.baig@kcl.ac.uk, manuel.mayr@kcl.ac.uk

Extracellular matrix (ECM) remodeling is a hallmark of vascular disease. We used proteomics to quantify ECM composition of human vessels. Aortas, internal mammary arteries, radial arteries and saphenous veins (n=10 each) were characterised using untargeted proteomics. 364 ECM and ECM-

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associated proteins were identified. Veins displayed increased basement membrane proteins and collagen VI. Hyalectin proteoglycans, essential for viscoelasticity were abundant in aortas, and small leucine rich proteoglycans were reduced in internal mammary arteries, used for bypass surgery. We created a panel of 125 ECM proteins for multiple reaction monitoring (MRM) quantification. When applied to type 2 diabetes, aortas of diabetic patients displayed higher levels of laminins β2, α4 and

α5, and collagen α3(VI), supporting the role of basement membrane proteins in diabetic vascular

pathologies. We provide the first comprehensive characterisation of the vascular ECM revealing distinct ECM signatures. MRM allowed detection of ECM changes before manifestation of macroscopically evident vascular disease.

17. Glycoproteomic analysis of the aortic extracellular matrix in patients with Marfan syndrome X Yin,1* S Wanga,2 A Fellows,1 J Barallobre-Barreiro,1 R Lu,1 R Franken,3 M Fava,1 P Skroblin1, Q Xing,1 DR Koolbergen,4 M Groenink,3,5 AH Zwinderman,6 R Balm,7 CJM de Vries,2 BJM Mulder,3,8 R Viner,9 M Jahangiri,10 V de Waard,2 M Mayr1* 1. King’s British Heart Foundation Centre, King’s College London, London, UK 2. Department of Medical Biochemistry, Amsterdam UMC, Academic Medical Center, Amsterdam, The Netherlands 3. Department of Cardiology, Amsterdam UMC, Academic Medical Center, Amsterdam, The Netherlands 4. Department of Cardiothoracic Surgery, Amsterdam UMC, Academic Medical Center, Amsterdam, The Netherlands 5. Department of Radiology, Amsterdam UMC, Academic Medical Center, Amsterdam, The Netherlands 6. Department of Clinical Epidemiology, Biostatistics & Bioinformatics, Amsterdam UMC, Academic Medical Center, Amsterdam, The Netherlands 7. Department of Surgery, Amsterdam UMC, Academic Medical Center, Amsterdam, The Netherlands 8. Netherlands Heart Institute, Utrecht, The Netherlands 9. Thermo Fisher Scientific, San Jose, USA. 10 St George’s, University of London, London, UK *Email: xiaoke.yin@kcl.ac.uk, manuel.mayr@kcl.ac.uk Marfan syndrome (MFS) is a genetic disorder caused by mutations in fibrillin-1, an extracellular matrix (ECM) component which is regulated by glycosylation. ECM extracts from aneurysmal ascending aorta from patients with and without MFS were enriched for glycopeptides and 141 N-glycoforms from 35 glycoproteins were directly identified by LC-MS/MS. Notably, microfibril-associated glycoprotein 4 (MFAP4) showed increased and more diverse N-glycosylation in MFS group. MFAP4 transcript levels were higher in MFS tissue and protein levels were increased at the predilection site for aneurysm formation in BAV patients. In hSMCs, MFAP4 mRNA expression was induced by TGFβ whereas knockdown of MFAP4 upregulated elastin but downregulated fibrillin-1 expression. Higher plasma MFAP4 in MFS patients were associated with a larger aortic root diameter, lower distensibility, and greater incidence of dissection. In conclusion, glycoproteomic analysis revealed enhanced MFAP4 glycosylation in aortic ECM of MFS patients and plasma MFAP4 level associated with higher risk of aortic dissection in MFS patients

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