Accelerating the Implementation of Mass Spectrometry in the Clinical Lab



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Plenary & Keynote Lecture Series

Distinguished Contribution Award Lecture

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.

Plenary Lectures

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.
Mass Spectrometric Analysis of Glycomic Signatures of Cancer and Autoimmune Diseases: Towards Clinical Application
Manfred Wuhrer

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.

Keynote Lectures

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.
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Ronda Greaves
RMIT University

Title Coming Soon
David Millington
Duke University - Retired

In 1941, George Beadle and Edward Tatum confirmed a hypothesis proposed in 1902 by the physician Archibald Garrod that hereditary diseases are due to "inborn errors of metabolism". In the 1960s, microbiologist Robert Guthrie developed the first blood test for phenylketonuria (PKU), which is caused by an inborn error of metabolism that results in decreased metabolism of the amino acid phenylalanine. Untreated, PKU can lead to intellectual disability, seizures, behavioral problems, and mental disorders. This diagnostic test established the field of newborn screening. In 1990, David Millington and colleagues applied the then novel technique of tandem mass spectrometry to evaluate acylcarnitine profiles from newborn blood samples, demonstrating the ability to screen for numerous inherited fatty acid oxidation and organic acid disorders via a single procedure. Their methodology was rapidly developed and extended by the community to the profiling of amino acids (including phenylalanine, for diagnosis of PKU) and other metabolic markers. David Millington’s foundational work has revolutionized the field of newborn screening and saved thousands of lives.

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.

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.
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.