Accelerating the Implementation of Mass Spectrometry in the Clinical Lab

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EUROPE 2019

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Plenary & Keynote Lecture Series


Distinguished Contribution Award Lecture


On the Award

>> Wednesday 16:30 in Mozart 1-5
15 years of Ambient Mass Spectrometry: From Amino Acid Clusters to Surgical Robotics
Zoltan Takats
Imperial College London


Plenary Lectures


>> Tuesday 14:15 in Mozart 1-5
Inflammatory Stories in Time and Space: Using Mass Spectrometry Imaging, Ion Mobility and High Throughput Lipidomics to Understand Human Disease
Jules Griffin
Computational and Systems Medicine, Surgery and Cancer, Imperial College London, London and Department of Biochemistry, University of Cambridge, Cambridge, UK

A central aspect of the development of many of the pathologies associated with the metabolic syndrome is a chronic progression of inflammation in the affected tissues. This is in part driven by lipid remodelling in the cell membrane and the production of pro-inflammatory lipid mediators produced from polyunsaturated fatty acids such as arachidonic, docosahexaenoic and eicosapentaenoic acids. To explore lipid remodelling during the development of non-alcoholic fatty liver disease we have applied MALDI-based mass spectrometry imaging (MSI) to examine both human tissue and animal models of the disease progression. Using a combination of high fat feeding and genetic modification (the ob/ob mouse which lacks leptin) to cause hepatic steatosis with and without inflammation, MSI shows that one of the events associated with disease progression is a lipid remodelling of phosphatidylcholines (PCs), and in particular, a reduction in arachidonic acid containing PCs. We have also developed a ultra-high performance liquid chromatography ion mobility mass spectrometry-based method to profile known and novel lipid mediators, using a KNIME workflow to process the data and annotate the detected lipids, in part relying on collision cross-section values for these species to aid assignments. This will be illustrated in following the time course of lipid changes in thrombin activated human platelets.
>> Thursday 17:15 in Mozart 4-5
Donor-derived cell-free DNA as a biomarker in organ transplantation
Michael Oellerich
Institute for Clinical Pharmacology, University Medicine Göttingen, Germany

Molecular biomarkers have attracted special attention in solid organ transplantation because of unresolved problems that limit long-term outcome. There is a lack of reliable noninvasive markers. Immunosuppressive drug monitoring mainly indicates potential toxicity, but is a poor biomarker of graft damage. In kidney transplant patients, for example, an increase of plasma creatinine may be also be due to exsiccation, the use of ACE inhibitors, or immunosuppressive drug toxicity. By the time a rejection-related increase in plasma creatinine is evident, a significant degree of tissue damage has already occurred within the kidney. A further limitation of the current standard of care is that rejection episodes can only be confirmed by biopsies. Biomarkers are needed to achieve personalized immunosuppression to reduce premature graft loss. Against this background, a particularly promising new approach for the early detection of acute or chronic rejection or asymptomatic graft injury leading to irreversible damage is based on the determination of donor-derived circulating cell-free DNA (dd-cfDNA). Data on clinical validity have been documented in more than 48 independent studies which have shown that dd-cfDNA detects rejection episodes early, at an actionable stage, and is a more reliable marker of graft injury, compared to conventional tests. dd-cfDNA may also be useful to guide changes in immunosuppression, to monitor immunosuppression minimization (e.g. during tapering), and to prevent immune activation. The high negative predictive value of dd-cfDNA is the reason why this test can be helpful to avoid unnecessary biopsies. It could be shown that dd-cfDNA can be useful to detect subclinical (e.g. clinically unsuspected) graft damage as a result of immune activation triggered by under-immunosuppression. Early diagnosis of subclinical antibody-mediated rejection may improve outcomes after kidney transplantation. In summary, dd-cfDNA monitoring will allow more personalized treatment that shifts emphasis from reaction to prevention.

Keynote Lectures


>> Wednesday 9:00 in Track 1 (Mozart 1-3) : Session 1
From Spectrometric Data to Metabolic Networks: An Integrated Quantitative View of Cell Metabolism
Oscar Yanes
Rovira i Virgili University & IISPV

INTRODUCTION: Metabolite profiling – or metabolomics – presents a powerful global approach to measure shifts in metabolites as functional readouts of cellular state. Metabolites can complement upstream biochemical information obtained from genes, transcripts, and proteins and advance our understanding of how cells are altered in health and disease. Unfortunately, the great success in the characterization of genes, transcripts and proteins has currently no parallel in metabolites. Metabolomic studies are revealing large numbers of naturally occurring metabolites that cannot be characterized because their chemical structures and spectrometric data are not available. This is preventing metabolomics from evolving as fast as other omic sciences, and thus it is restricting the integration of multiple layers of omic data to gain more insights into the emergence of observed phenotypes.
OBJECTIVES: To fill this gap, new experimental approaches based on mass spectrometry (MS), and innovations in bioinformatics to enable a comprehensive analysis of cellular metabolites are needed.
RESULTS: Here I will present novel computational tools for: 1) identifying and quantifying metabolites from reconstructed GC-MS, LC-MS and MALDI-MS spectral profiles; 2) the structural characterisation of unknown metabolites; and 3) the use of isotopically labeled metabolites to study the flow of chemical moieties through the complex set of metabolic reactions that happen in the cell. Finally, I will show that the integrated analysis of proteomics and metabolomics data through metabolic networks provides a new conceptual structure for an alternative quantitative and predictive description of cell metabolism.
>> Wednesday 11:00 in Track 6 (Doppler Hall) : Session 2
Large-Scale Inference of Protein Tissue Origin in Sepsis Plasma Using Quantitative Targeted Proteomics
Johan Malmström
Lund University
The blood plasma proteome is maintained by influx and efflux of proteins from surrounding cells and organs. The liver secretes the majority of the highly abundant plasma proteins, the so-called classical plasma proteins, involved in the principal functions of plasma such as serving as transport medium, provide colloid osmotic pressure and maintaining hemostasis through the complement and coagulation systems. Blood plasma also contains numerous tissue proteins that most likely do not contribute to the principal functions of blood plasma. This group of proteins is more numerous than the classical plasma proteins and their role, if any, in the plasma is unclear. A subset of these proteins may be waste products resulting from the turnover of proteins and cells under physiological and pathological conditions.
To investigate the complex processes that control the composition of the plasma proteome, we have developed a mass spectrometry-based proteomics strategy to infer the most likely tissue origin of the proteins detected in blood plasma. The strategy relies on the construction of a large-scale protein tissue distribution atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was subsequently transformed to a spectral library to enable sensitive and reproducible quantification of tissue-enriched proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. Here, we show that analysis of septic blood plasma reveals a drastic reorganization of the blood plasma proteome related to disease severity, where part of the plasma proteome followed a disease-dependent reorganization. On the contrary, certain tissue-enriched proteins were increased predominately in the most severely ill subjects. The increase of tissue proteins in critically ill subjects may indicate early signs of organ failure, a hallmark of sepsis pathology.
>> Wednesday 14:30 in Track 2 (Mozart 4-5) : Session 3
The Development of Targeted Proteomic Assays, Attempting to Take Biomarkers from the Research Lab to the Clinic
Kevin Mills
University of London

The development of targeted proteomic assays, attempting to take biomarkers from the research lab to the clinic.

Almost all large hospitals have LC-triple quadrupole based mass spectrometry platforms. These robust and versatile instruments are used for a multitude of small molecule analyses including amino acids, glycosphingolipids, glucosaminoglycans, vitamins as well as for monitoring immunosuppressant levels, drug trials and neonatal screening for inborn errors of metabolism. However, rarely are these instruments used in a clinical setting for the analyses of proteins and are usually analysed using immunochemistry based assays (ELISA, radioimmunoassays etc).

LC-MS based analyses, particularly with stable isotope labelled internal standards, are unparalleled for their accuracy and assay reproducibility with intra- and inter-assay coefficients of variations of often less than 2% and 5-8%, respectively. This is substantially lower than that of many immunochemistry based protein assays which often have intra- and inter-assay based coefficients in the range of 10-25%. Therefore, it is logical to try to combine the accuracy and reproducibility of LC-MS based platforms to analyse proteins in a clinical setting. This type of analysis has other added advantages over immuno-based technology including;

• Cost implications (no need to purchase kit based systems from a third party)
• Multiplexing up to 50-80 proteins in a single analysis
• Validation of biomarkers from discovery experiments
• the ability to translate biomarkers into routine tests in a clinical setting much quicker than having to wait for the development to market of a kite marked kit.

The work of my research group attempts to bridge the gap between finding a biomarker in a research lab to its validation and potential translation of that test into a clinical setting. I will give several examples of this where we have found biomarkers in plasma, urine and CSF and translated them into multiplexed tests. The diseases I will cover will include inborn errors of metabolism (Fabry Disease), hypertrophic cardiomyopathy and the neurodegenerative conditions of Alzheimer’s and Parkinson’s disease.
>> Wednesday 14:30 in Track 3 (Papageno Hall) : Session 3
Selected Ion Flow Drift Tube Mass Spectrometry SIFDT-MS for Real-Time Measurement of Trace Concentrations of Volatile Compounds in Breath and Culture Headspace
Patrik Španěl
J. Heyrovský Institute of Physical Chemistry
INTRODUCTION
The need for rapid and accurate measurement of trace concentrations of compounds present in air and human breath has led to construction of specialised mass spectrometers based on the Selected Ion Flow Tube Mass Spectrometry, SIFT-MS, its drift tube variant, SIFDT-MS.

OBJECTIVES
The primary objective was to quantify in real time volatile compounds including ammonia, hydrogen sulphide or hydrogen cyanide present in concentrations as low as a part per billion by volume (ppbv) in human breath and in the headspace of bacterial cultures. The specific objective was to reduce the size of the instrumentation whilst improving sensitivity.

METHODS
The selected reagent ions (either H3O+, NO+ or O2+) for selective chemical ionization of volatile compounds present in the air matrix are created in an external ion source and injected into the flow-drift tube reactor with well-defined reaction time and reagent ion-analyte molecule interaction energy, Er. The absolute concentrations of the analytes can thus be accurately quantified using the chemical kinetics principles.

RESULTS
Several advantages of SIFDT-MS over the established SIFT-MS are becoming apparent (including better sensitivity, improved accuracy and feasibility of design of portable instrument). However it is very important to assess the potential complications due to increased Er, including fragmentation and changes of k. Using the electrostatic reagent ion switching it is possible to analyse the samples by several reagent ions simultaneously without a need for the injection quadrupole mass filter.

CONCLUSION
SIFDT-MS has a potential for accurate analyses in matrices such as exhaled breath and bacterial culture headspace.
>> Thursday 08:30 in Track 4 (Paracelsus Hall) : Session 4
Rethinking Sex Steroids: Understanding the Clinical Relevance of 11-Oxygenated Androgens
Karl Storbeck
Stellenbosch University

The C19 steroid 11β-hydroxyandrostenedione (11OHA4) is a major product of adrenal steroidogenesis, but was ignored for decades due to an apparent lack of activity. However, recent studies have demonstrated that 11OHA4 is the precursor to the potent 11-oxygenated androgens, 11-ketotestosterone and 11-ketodihydrotestosterone, that bind and activate the human androgen receptor with affinities and potencies similar to that of testosterone and 5α-dihydrotestosterone (DHT), respectively. The significance of these findings becomes apparent when considering androgen dependent diseases such as castration resistant prostate cancer and endocrine conditions associated with androgen excess such as polycystic ovary syndrome and congenital adrenal hyperplasia. Recent findings pertaining to the importance of the overlooked 11-oxygenated androgens will be presented, highlighting the role of 11-oxygenated androgens in disease states and challenging the paradigm that testosterone and DHT are the only clinically relevant androgens.
>> Thursday 10:30 in Track 5 (Trakl Hall) : Session 5
The Human Glycome Project - Exploring the New Frontier in Personalised Medicine
Gordan Lauc
University of Zagreb; Genos
The majority of proteins that evolved after appearance of multicellular life are glycosylated and glycans significantly affect structure and function of these proteins. However, due to structural complexity of glycans and the absence of a direct genetic template, the analysis of protein glycosylation is much more complicated than the analysis of DNA or proteins. Consequently, the knowledge about the importance of individual variation in glycans for both normal physiological processes and diseases is still limited. In the last few years it is becoming increasingly clear that variations in a DNA sequence are only a beginning of the understanding of complex human diseases. Genetic polymorphisms have to be put in the context of complex biology of life and a more elaborate approach that combines different ‘omics phenotypes is needed to understand disease mechanisms and perform patient stratification that transcends genomics. Glycomics, as by far the most complex posttranslational modification, has an immense potential in this respect, which is only beginning to be investigated. By generating glycomic data for over 80,000 individuals from some of the best characterised clinical and epidemiological cohorts we enabled glycomics to meet other ‘omics. The analysis of this rich gold mind is painting a picture of a very complex genetic and epigenetic regulation of glycosylation that fine tunes protein activity in multiple biological systems and, if altered, contributes to development of different complex diseases.
>> Thursday 13:15 in Track 2 (Mozart 4-5) : Session 6
Antibody Sequencing and Quantitation
Theo Luider
Erasmus Medical Center
The metabotropic glutamate receptor-1 (mGluR1) has recently been identified as an oncogene that is abundantly expressed in >60% of human melanoma, breast, renal cell and prostate cancer tissues. Many of these cancers cannot be successfully treated and new treatment modalities are clearly needed. In 2003, glutamate signalling through mGluR1 was reported to be a promising new molecular target for the treatment of cancer. We identified that affinity purified mGluR1 autoantibodies from plasma from a unique ataxia patient effectively block the mGluR1 receptor both in vitro and in vivo. These fully matured human antibodies are selective and have high target affinity. The precise sequence of these antibodies would offer an attractive basis for the development of a new antibody-based therapy for mGluR1 dependent cancers.

We have developed a novel technology pipeline for mass spectrometry (MS) based protein sequencing of full length human antibodies from plasma and reveal the full length sequence of the anti-mGLuR1 antibodies. The recovery of fully matured human derived autoantibody sequences with this pipeline has great potential for therapeutics. Existing antibody-based therapies rely on antibodies raised in other species, which can evoke anti-drug responses and are less efficient than human autoantibodies. In order to successfully develop the MS based protein sequencing pipeline, we have optimized antibody enrichment from plasma samples, advance novel protocols for MS sample preparation, and develop state-of-the-art MS analysis tools to derive protein sequences from the complex data output. In this way, we will produce an anti-mGluR1 antibody with therapeutic potential, and a pipeline that allows for the recovery of additional fully matured human derived antibody sequences for other indications like multiple myeloma.


>> Thursday 14:30 in Track 4 (Paracelsus Hall) : Session 7
Impact of the New European IVD Regulation on Medical Laboratories - Opportunities and Challenges
Folker Spitzenberger
Technische Hochschule Lübeck
The current regulatory rules governing in vitro diagnostic medical devices (IVDMD) in the EU are mainly represented by the European IVD directive dating back from 1998. This directive does not cover devices with characteristics related to newer techniques and applications in current in vitro diagnostic testing, lacks conformity with current international consensus with regard to the risk-based classification of IVDMD, lacks transparency with regard to the identification and labelling of IVDMD and lacks requirements for laboratory-developed tests (LDT).

To solve these and other shortcomings, the the current IVD Directive will be replaced by the new Regulation (EU) 2017/746 ("IVDR") which entered into force in May 2017 with a transition period of five years.

The scope of the regulation includes newer technologies such as, for example, POCT devices and companion diagnostics.

The classification of IVDMD according to the IVDR will follow new classification rules on the basis of a risk-based classification system including 4 risk classes: A (lowest risk class), B, C and D (highest risk class).

As consequence, the involvement of Notified Bodies in the conformity assessment of IVDMD will increase significantly in the future.

Manufacturers will be obliged to register themselves and the devices in a central European database (Eudamed). Of note, manufacturers of higher risk devices (Classes C, D) will be obliged to make publicly available a so called summary of safety and performance including key elements of the supporting clinical data of a device. This might increase the extent of information of device performance data that are currently often lacking in the perspective of medical laboratories.

The new IVDR requirements for LDT represent challenges for medical laboratories and interpretation of these requirements is necessary to guarantee adequate care for patient groups with specific needs in medical laboratory diagnostic testing.
>> Thursday 15:45 in Track 2 (Mozart 4-5) : Session 8
Imaging the Unimaginable with Imaging Mass Cytometry
Frits Koning
LUMC

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4+ T cell compartment in the human fetal intestine. We identified 22 CD4+ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4+ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-? and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4+ T cells. Imaging mass cytometry indicated that memory-like CD4+ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4+ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.
>> Thursday 15:45 in Track 4 (Paracelsus Hall) : Session 8
Skin Lipidomics in the Diagnosis and Treatment of Cutaneous Inflammation
Anna Nicolaou
Manchester University

Skin depends on a unique profile of lipids that are necessary for the correct structure and function of the epidermal barrier, management of cellular communications and regulation of cutaneous homeostasis. Alterations in the cutaneous lipid profile can have severe consequences for skin health and such changes have been implicated in many inflammatory skin conditions. Using a targeted lipidomics platform we have investigated the prevalence of bioactive lipids in human skin, and reported an array of eicosanoids, octadecanoids, docosanoids, endocannabinoids, acyl ethanolamines and ceramides. We have conducted clinical studies and used human skin organ culture models and isolated cells in order to explore the differential contributions of lipid families to skin conditions. We have also explored lipid responses to various stimuli, and have examined temporal changes in lipid profiles aiming to understand their contribution to acute cutaneous inflammation and its resolution. Systemic supplementation with omega-3 polyunsaturated fatty acids (PUFA) has also revealed the differing cutaneous activities of these protective fatty acids, and demonstrated how they mediate their activities through perturbation of the profiles of existing species as well as formation of new lipids. Overall, we have shown that targeted lipidomics can elucidate the network of cutaneous bioactive lipids, support the development of biomarkers and diagnostics, and identify therapeutic targets for inflammatory skin disease.