Mass Spectrometry and the Elucidation of Steroid Metabolome Cedric Shackleton UCSF Benioff Children’s Hospital Oakland Research Institute, Oakland, California, and Institute of Metabolism and Systems Research (IMSR), College of Medical and Dental Sciences, University of Birmingham, UK
Dr. Cedric Shackleton has been a fundamental contributor to steroid metabolome analysis by mass spectrometry and has pioneered the technology and its application to the diagnosis of inborn disorders of sterol and steroidogenesis, with major translational impact. His work over 5 decades has defined the steroid metabolome of many congenital steroid disorders from synthetic to metabolic and receptor defects, pioneering the use of GC-MS in steroid profiling over 30 years ago. He has authored more than 350 PubMed-listed research publications and remains highly active, working at the Institute of Metabolism and Systems Research (IMSR) of the University of Birmingham, UK, helping to set up their specialist steroid methods. Furthermore, Dr. Shackleton has had great influence on those he has mentored over the years.
Cedric Shackleton obtained his PhD at the University of Edinburgh followed by a post-doc at the Karolinska institute learning GC/MS under Professor Jan Sjövall who introduced him to the concept of metabolic profiling using the technique.
From 1969 he ran a steroid GC/MS laboratory at the MRC in London followed in 1978 by 5 years with the University of California, where he introduced novel techniques, such as Fast Atom Bombardment (FAB), for the study of steroid metabolism. From 1983 he has been at Children’s Hospital Oakland studying steroid disorders, primarily those causing hypertension or disorders of sexual differentiation, such as Congenital Adrenal Hyperplasia (CAH), including the most recent identified version P450 Oxido-Reductase deficiency
Primarily, his studies have involved steroid metabolism, but in 1991 he was a pioneer in the use of electrospray for protein analysis and ran a clinical service dedicated to the characterization of unusual and novel (variant) haemoglobins by ESI/MS. Since the 1970s he has had an interest in testing for the misuse of anabolic steroid drugs in sport and with support from U.S. and International Olympic Committees contributed to methodologies for analyzing misused natural steroids using MS carbon isotope ratio analysis.
A major topic in recent years has been the study of Smith-Lemli-Opitz syndrome, a cholesterol deficiency condition (7-dehydrosterol reductase deficiency) associated with physical malformation and mental retardation. He developed a GC/MS technique which allowed non-invasive detection at mid-term pregnancy and has been involved in gene-therapy research as possible therapy for the condition.
Sterols and steroids were among the first biomolecule groups to be studied by mass-spectrometry. The early success of GC separation in 1960 by Sweeley and Horning and the combination of GC and MS by Ragner Ryhage in Stockholm with invention of a technique for separating the bulk of carrier gas from analytes prior to ionization were fundamental. This led in the mid-60s to the first commercial dedicated GC/MS instrument, the LKB 9000. Professor Jan Sjövall (a colleague of Dr Ryhage) in particular spearheaded studies of sterols and steroids leading to multiple publications of mass spectra, and steroid quantification of serum, urinary and biliary steroids. Developments in derivatization, capillary columns and computerization were all important constituents of these developments. This presenter (a student of Sjövall) was early involved in the clinical diagnostic use of GC/MS in the 1970s and has been involved in studying urinary steroids and defining new disorders through metabolic profiles ever since.
Particle beam MS (eg. FAB) was introduced in the 80s and allowed for the first time analysis of underivatised steroids and steroid conjugates. Steroid LC/MS (Thermospray) arrived in 1987 but it was a decade later before ESI and LC/MS/MS made the use of MS in the routine steroid analytical lab a practicality. Now the technique has essentially replaced RIA in routine endocrinological applications.
GC/MS is labour-intensive and was never really suited to individual serum hormone analysis but has always been powerful for profiling urinary steroid metabolites, which now forms a branch of the recently-termed metabolome discipline. Recently there has been a re-birth in urinary steroid analysis in diagnosis and several groups are working on transferring GC/MS methodologies to LC/MS/MS to avoid need of derivatization and shorten chromatography times. This is a challenge because many of the “metabolite-type” steroids have weak ESI ionization and poor CID sensitivity. Two steroid groups are particularly suited to LC/MS/MS and those are the steroid sulfates and glucuronides since they have an existing ionic center. Analysis of intact conjugates avoids the labour-intensive enzyme hydrolysis, and we a pursuing their analysis. A drawback here is the paucity of authentic reference materials.
A huge challenge in steroid metabolomics is the presentation of diagnostic data in a form friendly to researchers and clinicians. Many recent publications have addressed this issue, including use of heat-maps and machine learning for metabolome diagnostic interpretation.
>> Thursday 16:00 in Mozart 4-5
Mass Spectrometric Analysis of Glycomic Signatures of Cancer and Autoimmune Diseases: Towards Clinical Application Manfred Wuhrer LUMC
Manfred Wuhrer is professor of Proteomics and Glycomics and head of the Center for Proteomics and Metabolomics of the Leiden University Medical Center (LUMC)). He is also chairman of the Dutch Society for Mass Spectrometry (NVMS).
Manfred Wuhrer studied Biochemistry at Regensburg University, Germany. He obtained a PhD from Giessen University in 1999 on the Characterization of Stage-Specifically expressed Glycolipids of the Human Parasite Schistosoma mansoni. In 2003 he came to the LUMC establishing a research line on mass spectrometric glycosylation analysis.
In 2013 Manfred Wuhrer became Professor Analytics of Biomolecular Interactions at VU University Amsterdam. In 2015 he was appointed Professor in Proteomics and Glycomics at the LUMC heading the Center of Proteomics and Metabolomics.
Many major human diseases including various types of cancer and autoimmune diseases are associated with protein glycosylation changes. These changes are often instrumental in disease development and progression. In cancer, glycosylation changes of both tumor tissues and tumor-derived circulating antigens have been described. However, diagnostic test often merely rely on the measurement of antigen concentrations and fail to register glycosylation changes, largely due to the lack of suitable technology and workflows. In this presentation, examples will be given of recently developed mass spectrometric workflows to determine disease-associated glycosylation changes from tumor tissues and in the circulation, but also from other body fluids including urine and saliva. Promising markers will be presented including immunoglobulin G glycosylation, cancer glycoprotein antigens, as well as tissue glycosylation signatures revealed by mass spectrometry imaging. Steps towards the clinical translation of these markers will be discussed.
>> Tuesday 14:00 in Europa
Metabolomics Messages on Human Health and Disease Jurek Adamski Helmholtz Zentrum München
Dr. Adamski studied biochemistry in Poznan, Poland and accomplished his postdoctoral training in endocrinology at the Max-Planck-Institute for Experimental Endocrinology in Hannover, Germany and Karolinska Institutet, Hudinge, Sweden. At present he is director of Genome Analysis Center at the Helmholtz Zentrum Munich and is affiliated as a professor at the Institute of Experimental Genetics at the Technical University of Munich, Germany. Part of his research is performed at the German Center for Diabetes Research (DZD), München- Neuherberg, Germany. Prof. Adamski acts as Editor-in-Chief for Journal of Steroid Biochemistry and Molecular Biology (Elsevier). Prof. Adamski is committed to the training of pre- and post-doctoral fellows of Technical University of Munich. The students originate from the divisions of biology, biotechnology and biochemistry. Prof Adamski is interested in identification the factors responsible for the pathogenesis of complex metabolic diseases such as diabetes, obesity, cardiovascular disorders and cancer. Prof Adamski also studies the genetic components and environmental challenges such as medication, life style, nutrition, and gender–specific disease.
The human metabolome represents functional read out of processes in health and disease. The metabolome is both stable and highly dynamic. Stable components are determined by genetics, genomic imprinting or physiological homeostasis. The dynamics originates from circadian rhythm, hormonal status, nutrition, environmental exposure, ageing, medication or disease. Despite its interlaced origin metabolome specifically reflects distinct processes. Metabolomics studies request therefore a special study design. Unique metabolomics signatures have been identified pre-disease (like type 2 diabetes), in disease progression (chronic kidney disease) or in companion diagnostics (drug action monitoring). Metabolomics informed diagnostics has great potential for selectivity, specificity and multiplexing of indications to be screened for.
>> Wednesday 9:00 in Track 3 (Papageno) : Session 1
Clinical Proteomics: The Path Towards Implementation Harald Mischak mosaiques therapeutics GmbH & University of Glasgow
Harald Mischak, born 1961 in St. Pölten, Austria, received his PhD in technical science from the Technical University of Vienna, Austria, in 1986.
Between 1988 and 1993, after postdoctoral work on the Rhinovirus receptor at the University of Vienna (Institute for Biochemistry), he was on leave as an invited scientist on signalling by protein kinase C and Raf at the Laboratory of Viral Carcinogenesis (funded by the Fulbright Foundation) and as a Schroedinger and Fogarty Fellow at the Laboratory of Genetics at the NIH National Cancer Institute in Bethesda, Maryland, USA.
He continued his research on kinases as Group Leader at the GSF, Munich, Germany from 1993-1998. He wrote his habilitation in clinical microbiology at the Technische Universität München on Protein Kinase C in Signal Transduction. After one year as a scientific group leader at Franz-Volhard Klinikum (MDC) at Berlin-Buch, he worked on the structure of kinases and related molecules at the NIDDK, Bethesda, Maryland, USA. In 1999 he took up a position at the Department of Nephrology at Medical School of Hannover. Here he founded Mosaiques diagnostics and therapeutics AG in 2002, which was started with the aim to identify disease-specific polypeptides. Currently, he holds a position as Professor for Proteomics and Systems Medicine at the University of Glasgow, and he is the chief scientific officer of Mosaiques AG as well as executive director of Mosaiques diagnostics GmbH and Mosaiques DiaPat GmbH. With more than 300 scientific publications on signaling and proteomics that have been cited over 20000 times, he is one of the leading experts worldwide in the field of proteome research and applied systems biology. Prof. Mischak is named as inventor on more than 100 patent applications, the majority on proteomic biomarkers.
Among his major achievements is the identification of distinct biological roles of Protein Kinase C. He demonstrated that Protein Kinase C isoforms display highly divergent biological properties in differentiation and oncogenic transformation (Mischak et al. 1993, J Biol Chem 268, 1749-1756 and 20110-20115) and associated these with distinct intracellular localization (Goodnight, Mischak, et al. 1995, J Biol Chem 270:9991-10001). Together with Walter Kolch (for Raf signaling, e.g. Kolch et al. 1993, Nature 364:249-252) and Hans Hacker (for CpG signaling, e.g. Hacker, Mischak et al. 1998, EMBO J 17:6230-6240) he pinpointed the role of kinases in several major signaling mechanisms. Based on his experience on proteomics in basic research, he initiated the use of urinary proteomics and capillary electrophoresis coupled mass spectrometry for clinical application, and is the leading authority in clinical proteomics and biomarker identification. Among his achievements in this field are the development of guidelines for clinical proteome analysis, where he led a large international and multidisciplinary group to develop clinically relevant proteomic biomarkers (Mischak et al. 2007, Proteomics Clin Appl 1:148-156; 2010, Sci Transl Med 2:46ps42; 2012, Eur. J Clin Invest 42, 1027-1036), and the demonstration of successful application in the diagnosis and prognosis of several diseases (e.g. Decramer et al. 2006, Nat. Med. 12:398-400; Theodorescu et al. 2006, Lancet Oncol. 7:230-240; Rossing et al. 2008, JASN 19:1283-1290; Good et al. 2010, Molecular & Cellular Proteomics 9:2424-2437; Kuznetsova et al. 2012, Eur. Heart J. 33, 2342-2350).
The two main focuses of Prof Mischak’s work are:
1) identification, validation, and especially implementation of proteomic biomarkers, aiming especially at biomarkers associated with chronic kidney disease, coronary artery disease, heart failure, and certain types of cancer
2) uncovering the molecular changes on a proteomic level that are relevant in, or even cause of, the major diseases mentioned above. This approach is based on the biomarkers identified, but also on addition proteomic, metabolomic and genomic data. Using appropriate bioinformatic approaches, the high-dimensional data are combined to identify the underlying molecular structures and ultimately develop a molecular model of the respective disease, which in turn will allow identifying the most appropriate therapeutic targets for intervention.
Clinical proteomics, the application of proteome analysis to clinical purpose, represents a major field in the area of proteome research. The aim of clinical proteomics is the improvement of clinical care based on (1) the identification and application of biomarkers, and (2) the suggestion of relevant therapeutic targets. These two areas have different requirements regarding specimens to be employed, technology, and data evaluation.
While substantial efforts (especially in biomarker discovery, but also in the identification of therapeutic targets) are evident based on the large number of associated publications, only a few approaches have actually resulted in clinical application. In this presentation, key issues and major challenges in clinical proteomics will be discussed. Among these are: a) the definition of a clinical need and a context-of-use, b) selection of appropriate samples, sample preparation and analytical platform, c) application of appropriate statistics, d) demonstration of benefit in a well-powered clinical study and e) obtaining regulatory approval and reimbursement, to enable actual implementation.
For several conditions and diseases, clinical proteomics has delivered solutions that are already being applied. Most successful developments are based on multi-marker panels that have demonstrated value in large studies (i.e., including at least several hundred patients). The results of these studies are expected to initiate a change in disease assessment: from diagnosis based on existing damage and therapy aiming to prevent deterioration, towards diagnosis based on molecular mechanisms, prior to observations of clinical symptoms, and therapy via correction of molecular anomalies, thereby preventing disease onset. This will also open a path towards personalized precision medicine, where intervention will be guided by molecular mechanisms, not morphological changes.
It is becoming clear that the tools required to meaningfully apply clinical proteomics (i.e., potential biomarkers, relevant technology and bio-banked samples) are available. The move from discovery towards validation and application is not only urgently necessary, but within reach. Now, a change in objective, away from additional discovery studies and towards properly testing the plethora of potential biomarkers that have been described, is needed to demonstrate the practical value of clinical proteomics.
>> Wednesday 11:00 in Track 3 (Papageno) : Session 2
Mass Spectrometry Harmonisation: Paediatric Steroid Tales Ronda Greaves Murdoch Children’s Research Institute
Ronda Greaves is the Associate Professor of Clinical Biochemistry at RMIT University and Research Fellow at the Murdoch Children’s Research Institute in Melbourne Australia. A/Prof Greaves is passionate about the: advancement of paediatric biochemical genetics; translation and harmonisation of related emerging technologies; and overall quality of laboratory testing to support appropriate clinical decisions.
Ronda has published extensively in the area, defining quality across the total testing process including the development of position papers, reference methods, reference intervals and external quality assurance programs for paediatric mass spectrometry methods. Ronda currently serves on the Clinical Mass Spectrometry Journal’s Editorial Board and is Secretary to the Emerging Technologies Division Executive Committee of the IFCC.
Our goal in laboratory medicine is to improve patient health by using laboratory tests as our tool to support medical decisions. The change from immunoassay to mass spectrometry analysis of steroids has significantly improved the specificity, accuracy and sensitivity of many steroids frequently analysed in children. However, mass spectrometry assays are not infallible and recognised differences exist across the total testing process. There are demonstrated variations pre-analytically, analytically and post-analytically in methods and understanding what differences are critical and how results compare between laboratories is essential to close the gap between result differences between laboratories. In this presentation, we will explore the challenges and possible solutions for the harmonisation of paediatric steroid analysis by mass spectrometry to support medical decisions.
Tackling Analytical Challenges in Sports Drug Testing by Mass Spectrometric Approaches Mario Thevis German Sport University, Cologne, Germany European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany
Dr Thevis graduated in organic chemistry and sports sciences in 1998. He earned his PhD in Biochemistry in 2001 and did post-doctoral research at the Department of Chemistry and Biochemistry of the University of California Los Angeles (UCLA) in 2002. After being a senior researcher from 2003 to 2005 he was appointed as Professor for Preventive Doping Research at the German Sport University Cologne in 2006. Mario Thevis further qualified as Forensic Chemist, acts as director of the European Monitoring Center for Emerging Doping Agents (EuMoCEDA), and is Editor-in-Chief of DRUG TESTING & ANALYSIS. Since 2014, Professor Thevis is also vice president for research at the German Sport University Cologne.
Sports drug testing laboratories are facing multifaceted challenges including the misuse of naturally/endogenously occurring substances, non-approved/discontinued drug candidates, urine manipulation, etc. In order to provide best-possible analytical performance, mass spectrometry-based approaches are predominantly utilized to detect prohibited substances and methods of doping. With the constantly increasing analytical requirements concerning the number of target compounds, the complexity and range of physico-chemical properties of analytes (e.g., inorganic ionic transition metals, gases, lipids, alkaloids, peptides, proteins, DNA/RNA-based drugs, etc.) as well as the desire to accelerate analyses and obtain information allowing also for retrospective data mining, high resolution/high accuracy mass spectrometry has become a mainstay in doping controls. In that context, various assays have been reported, enabling either multi-component analyses of low- or high molecular mass measurands or the specific and dedicated (confirmatory) detection of prohibited substances.
Selected applications will be presented reporting on examples of recent findings in routine sports drug testing, demonstrating both the inventiveness of cheating individuals that undermine current anti-doping efforts as well as the relevance of in-depth investigations into unusual findings, where the athletes’ innocence was to be shown albeit prohibited substances were unequivocally identified in their doping control urine samples.
>> Wednesday 14:30 in Track 4 (Paracelsus) : Session 3
Combining Mass Spectrometry Imaging & Microproteomics to Investigate Intratumor Heterogeneity Liam McDonnell Fondazione Pisana per la Scienza ONLUS, Pisa, Italy
Liam McDonnell obtained an MCHEM in Chemistry from the University of Oxford, UK, and a PhD in Chemistry from the University of Warwick, UK. Following a post-doctoral researcher position at the FOM institute for Atomic and Molecular Physics in Amsterdam, he started his own research group at Leiden University Medical Center, where he is Associate Professor. He is director of Proteomics at the Fondazione Pisana per la Scienza, and Vice-President of the Mass Spectrometry Imaging Society.
Mass spectrometry imaging (MSI) is able to simultaneously record the distributions of hundreds of molecules directly from tissue. This spatially-resolved molecular information can be combined with multivariate/clustering analysis to reveal regions of tissue with distinct molecular signatures, a process that has been termed MSI-based molecular histology and has been used to reveal tumor subpopulations, metabolically distinct cell layers, and tumor interface zones. Rapid direct tissue analysis is essential for MSI in order to maintain spatial localization and acceptable measurement times. The absence of an explicit analyte separation/purification step means MSI lacks the depth of coverage of LC-MS/MS. Here, we demonstrate how MSI can be combined with high sensitivity microproteomics, even of the same tissue section, to further investigate the molecular changes associated with tumor subpopulations.
Tandem MS-Proteotyping: Proteomics- and Genomics-Based Characterization and Typing of Infectious Bacteria Edward Moore University of Gothenburg, Sweden
LC-MS/MS proteomics- and genomics-based characterisation and typing of microorganisms, i.e., ‘proteotyping’, using peptides from expressed proteins, matched to genomic sequence data, can be applied for sensitive and accurate detection and typing of pathogenic bacteria. Proteotyping is capable of resolving closely-related bacterial taxa with simultaneous detections of virulence and antibiotic resistance features, providing for comprehensive characterisations of infectious bacteria. The methodology may be applied directly to analyses of clinical samples without prior cultivation and isolation, thus providing for rapid, reliable, infectious disease diagnostics.
>> Thursday 11:00 in Track 4 (Paracelsus) : Session 5
High-throughput Glycomics in Patient Stratification - What Did We Learn from the First 60,000 Analyses Gordon Lauc University of Zagreb & Genos Glycoscience Research Laboratory, Zagreb, Croatia
The majority of proteins are glycosylated and their glycan parts have numerous structural and functional roles. This essential posttranslational modification is generated by a complex biosynthetic pathway comprising hundreds of glycosyltransferases, glycosidases, transcriptional factors, ion channels and other proteins. Since glycans are created without the genetic template, alternative glycosylation creates an additional layer of protein complexity by combining genetic variability with past and present environmental factors. Individual variability in glycome composition is very large, but glycosylation of an individual protein seems to be under strong genetic influence, with heritability being up to 80% for some glycans. Structural details of the attached glycans are of great physiological significance and many pathological conditions are associated with various types of glycan changes. For example, glycans attached to the Fc part of immunoglobulin G are important modulators of IgG effector functions. Slight modifications in the composition of the IgG glycome significantly tune IgG towards binding to different Fc receptors and can convert IgG from a pro-inflammatory effector into an anti-inflammatory agent.
Since the onset of genome wide association studies, thousands of genetic loci have been associated with different diseases and traits. However, 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 epiproteomic modification, has an immense potential in this respect, which is only beginning to be investigated.
How Tandem Mass Spectrometry Revolutionized Newborn Screening David Millington Duke University - Retired
David S. Millington, PhD, is Emeritus Professor of Pediatrics, recently retired from Duke School of Medicine. He and his colleagues pioneered the application of tandem mass spectrometry for the targeted analysis of acylcarnitines that became a frontline diagnostic test for defects of fat oxidation and branched-chain amino acid catabolism. Subsequently, the method was modified to include several essential amino acids and applied to dried blood spots, paving the way for the expansion of newborn screening for from a handful to over 30 metabolic conditions. As the expanded newborn screening method has spread, Dr. Millington has developed educational material and taught many laboratorians and follow-up coordinators worldwide how to apply the technology and interpret results. Expanded newborn screening by MS/MS is now applied to tens of millions of neonates annually and has help save thousands of lives. More recently, he has collaborated with a North Carolina biotechnology company to bring digital microfluidics into biochemical diagnostics and newborn screening. He has also pioneered novel biomarker assays for numerous lysosomal storage conditions to facilitate patient diagnosis and monitoring. Dr Millington has published over 200 research articles and book chapters and has served on the North Carolina Newborn Screening Program Advisory Board since 1990. He was honored jointly with Dr Mohamed Rashed by the ISNS with the Robert Guthrie award for outstanding contributions to newborn screening in 1996. He was also honored by the MSACL in 2015 with their first Distinguished Contribution Award in recognition of achievements made in the field of clinical mass spectrometry.
Inspired by a clinician’s account of a child rescued from near death by a revolutionary therapeutic intervention, the author applied chemistry and mass spectrometry to solve an analytical challenge that led to the first front-line diagnostic test performed by tandem mass spectrometry (MSMS) – the analysis of acylcarnitines to recognize and diagnose inherited disorders of fatty acid and branched-chain amino acid catabolism. By applying this method to dried blood spots and adding an additional analytical component to include several essential amino acids, a novel multiplex assay was developed to screen newborns for over 30 inherited metabolic conditions with a single test. The introduction of this method into public health systems and hospitals across the world during the past 20 years has literally revolutionized neonatal screening, and new technology is being introduced to add even more value to this critical “first test”. This concept subsequently became the basis of targeted metabolomics platforms that have been used, for example, to help identify new animal models of metabolic disease by screening the offspring of genetically modified adults. MSMS with UPLC has been widely applied to develop new assays for useful biomarkers of metabolic disease for both diagnosis and therapeutic monitoring. Examples from the author’s laboratory will be used to illustrate the value and scope of these methods.
>> Thursday 14:30 in Track 4 (Paracelsus) : Session 6
Advances in Computational Methods for Tissue Imaging by Mass Spectrometry Raf Van De Plas Delft TU
Imaging Mass Spectrometry (IMS) has made rapid progress as an imaging modality that can map the spatial distribution of molecules in tissue. In recent years, novel computational developments have become an increasingly important part of major advancements in this field. This talk presents several computational techniques developed in our group, specifically relevant to molecular imaging in medicine and the clinical practice. We show recent work in low-level signal processing, where in silico integration of isolation windows enables High-Dynamic-Range mass spectrometry, substantially increasing MS sensitivity. We also address advancements in data-driven image fusion, a multi-modal data mining methodology that drives the automated discovery of biomolecular relationships between stained microscopy and IMS, thus directly linking exploratory tissue analysis to established clinical targets.