2018 SGMS Meeting

Program Plenary Lectures Short Communications Posters Sponsors

The 36th meeting of the SGMS will take place at the Dorint Resort Blüemlisalp Beatenberg, 25-26 October 2018 high above Lake Thun in the Bernese Oberland, with a scenic view of the Swiss Alps!

Confirmed Speakers

• Evan Williams (University of California Berkeley) [ abstract]
• Serge Rudaz (University of Geneva) [ abstract]
• Mario Thevis (German Sport University Cologne) [ abstract]
• Ron Heeren (Maastricht University) [ abstract]
• Bernd Bodenmiller (University of Zurich) [ abstract]

Program

For program pdf [click!]

Check this page for the SGMS School Program

Plenary Lectures

Evan R. Williams: Chemistry in Aqueous Nanodrops

 Evan R. Williams

Department of Chemistry
University of California, berkeley
Berkeley, CA 94720

United States of America
 

Electrospray ionization can produce a virtually unlimited variety of ions that are confined in gaseous aqueous nanodrops of various sizes.  A new rapid mixing method that uses theta-glass capillaries to mix two solutions into various size droplets makes it possible to monitor fast reactions that occur in a range of time frames down to sub microseconds all while using 2000 times less material than conventional mixing apparatus.  Precise thermochemical measurements using a method we call ion nanocalorimetry enable accurate electrochemical red-ox potentials to be measured, and this method is used to establish an absolute electrochemical scale entirely from experimental measurements.  The use of small droplets to desalt ions in native mass spectrometry applications will be presented.  This method makes possible the use of conventional biochemistry buffers, such as Tris, phosphate, MOPS, etc., with high levels of nonvolatile salts, e.g., 150+ mM Na/KCl directly with mass spectrometry analysis.

Serge Rudaz: Harnessing the complexity of MS metabolomic data

 Serge Rudaz

University of Geneva
Department of pharmaceutical Sciences
1 Michel Servet
1211 Geneva

Switzerland

Due to the ever-increasing number of signals that can be measured within a single run by modern platforms in analytical chemistry, life sciences datasets not only become gradually larger in size, but also more intricate in their structures. Challenges related to making use of this wealth of data include extracting relevant elements within massive amounts of signals possibly spread across different tables, reducing dimensionality, summarizing dynamic information in a comprehensible way and displaying it for interpretation purposes. Metabolomics constitutes a representative example of fast moving research fields taking advantage of recent technological advances to provide extensive sample monitoring. Due to the wide chemical diversity of metabolites, several analytical setups are required to provide a broad coverage of complex samples. While early metabolomic studies relied mainly on NMR, hyphenated methods involving separation techniques and mass spectrometry (GC-MS, LC-MS, CE-MS) have now been demonstrated to be powerful and complementary analytical techniques. Classical hypothesis-driven approaches are no longer applicable to such data collection and dedicated data analysis strategies have to be used. Since several years, the integration and visualisation of multiple highly multivariate datasets constitute key issues for effective analysis leading to valuable biological or chemical knowledge. Multivariate methods based on the computation of latent variables or components, such as principal component analysis (PCA) and partial least squares (PLS) regression constitute potent tools to provide compact data representations and diagnostic tools for the detection of relevant variables. Nevertheless, most of these approaches lack the ability to fully exploit more complex data structures such as (1) multifactorial, (2) longitudinal and (3) multiblock setups. As presented in this lecture through examples, dedicated modelling algorithms, able to cope with the inherent properties of these MS metabolomic datasets are therefore mandatory for harnessing their complexity and provide relevant information. In that perspective, chemometrics has a central role to play in the choice of the appropriate methodology.

Mario Thevis: Analytical approaches towards old and new challenges in doping controls

 Mario Thevis

German Sport University Cologne
Am Sportpark
Müngersdorf 6
50933 Cologne

Germany
 

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, the combined qualities of low resolution tandem mass spectrometric analysis and high resolution/high accuracy mass spectrometry have become mainstays 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. Moreover, an excursion into advantages and limitations of alternative matrices potentially applicable to doping controls will be presented.

Ron Heeren: Translational biomolecular imaging mass spectrometry: Seeing is believing

 Ron M.A. Heeren

Maastricht University
Faculty of Health, Medicine and Life Sciences
Universiteitssingel 50
6229 ER Maastricht

The Netherlands

New Mass Spectrometry based chemical microscopes that target biomedical tissue analysis in various diseases as well as other chemically complex surfaces have now firmly established themselves in translational research. In concert they elucidate the way in which local environments can influence molecular signalling pathways on various scales, from molecule to man. The integration of this pathway information in a surgical setting is imminent, but innovations that push the boundaries of the technology and its application are still needed. In particular, researchers investigate comprehensive and isolated biomolecular molecular patterns of health and disease. This is a key element needed to pave the way for personalized medicine and tissue regeneration. One barrier to predictive, personalized medicine is the lack of a comprehensive molecular understanding at the tissue level. As we grasp the astonishing complexity of biological systems (whether single cells or whole organisms), it becomes more and more evident that within this complexity lies the information needed to provide insight in the origin, progression and treatment of various diseases. The best way to capture disease complexity is to chart and connect multilevel molecular information within a tissue using mass spectrometry and data algorithms.

A key element to accelerate the generation of novel insights into the complexity of biomolecular surfaces is the continuous improvement of resolution. Spatial resolution, Molecular resolution and maybe more importantly, structural resolution are rapidly improving. The combination of high resolution technologies (TWIMS-MS, FTICR-MS and Orbitrap MS) with smart ion chemistry, ion mobility separation, stable isotope labelling approaches, ambient ionization, new data acquisition approaches and funnel based MALDI ion sources allows us to address some of the open challenges that still exist in the field of imaging MS. A novel elevated pressure MALDI imaging ion source is described and employed to reveal local isomeric structures. More precisely, we have employed OzID in combination with MALDI-MSI to reveal the regulatory role of lipid isomeric forms in a variety of diseases.

It is evident that a single analytical technology merely yields a subset of the molecular information needed to obtain an in depth understanding of a clinical problem. Multimodal approaches enable the study of clinical samples at a variety of molecular and spatial scales. The distribution of several hundreds of molecules on the surface of complex (biological) surfaces can be determined directly in complementary imaging MS experiment with different desorption and ionization strategies. High throughput, high resolution MALDI techniques offer three-dimensional molecular data on the tissue level. The combination with tools from structural biology makes it possible to perform imaging experiments at length scales from cells to patients.

Bernd Bodenmiller: Highly multiplexed imaging of tissues in health and disease using mass cytometry.

 Evan R. Williams

University of Zurich
Institute of Molecular Life Sciences
Winterthurerstrasse 190
8057 Zurich

Switzerland

Cancer is a tissue disease. Heterogeneous cancer cells and normal stromal and immune cells form a dynamic ecosystem that evolves to support tumor expansion and ultimately tumor spread. The complexity of this dynamic system is the main obstacle in our attempts to treat and heal the disease. The study of the tumor ecosystem and its cell-to-cell communications is thus essential to enable an understanding of tumor biology, to define new biomarkers to improve patient care, and ultimately to identify new therapeutic routs and targets.

To study and understand the workings of the tumor ecosystem, highly multiplexed image information of tumor tissues is essential. Such multiplexed images will reveal which cell types are present in a tumor, their functional state, and which cell-cell interactions are present. To enable multiplexed tissue imaging, we developed imaging mass cytometry (IMC). IMC is a novel imaging modality that uses metal isotopes of defined mass as reporters and currently allows to visualize over 50 antibodies and DNA probes simultaneously on tissues with subcellular resolution. In the near future, we expect that over 100 markers can be visualized. We applied IMC for the analysis of breast cancer samples in a quantitative manner. To extract biological meaningful data and potential biomarkers from this dataset, we developed a novel computational pipeline called histoCAT geared for the interactive and automated analysis of large scale, highly multiplexed tissues image datasets. Our analysis reveals a surprising level of inter and intra-tumor heterogeneity and identify new diversity within known human breast cancer subtypes as well as a variety of stromal cell types that interact with them.

In summary, our results show that IMC provides targeted, high-dimensional analysis of cell type, cell state and cell-to-cell interactions within the TME at subcellular resolution. Spatial relationships of complex cell states of cellular assemblies can be inferred and potentially used as biomarkers. We envision that IMC will enable a systems biology approach to understand and diagnose disease and to guide treatment.

Short Communications

Oral 1 (Student Talk):
Establishment of a Novel UHPLC-HRMS Method for the Simultaneous Detection of Gestagens

Karoline Rehm¹, Anna-Katharina Hankele², Susanne E. Ulbrich², Laurent Bigler¹

1. University of Zurich, Department of Chemistry, 8057 Zurich, Switzerland
2. ETH Zurich, Animal Physiology, Institute of Agricultural Sciences, 8092 Zurich, Switzerland
3. Contact: karoline(dot)rehm(at)uzh(dot)ch, laurent(dot)bigler(at)chem(dot)uzh(dot)ch

Not only due to their complex, apolar structure, but also because of their great diversity of isomers, the measurement of steroid hormones is a highly challenging task and remains as a field full of unexplored possibilities until today [1].

Progesterone is one of the most significant hormones that plays an important role during the pregnancy and the estrous cycle of mammals. The assessment of the reproductive status through progesterone as a main gestagen is a key element in medicinal studies and an essential part to monitor the health of mother and unborn child. However, despite the function of progesterone being thoroughly researched, gaps in knowledge are present when looking at its metabolites – the progestagens – that own potential biological activity. While first investigations using GC-MS were already carried out for progestagen profiling in human and manatee plasma [2][3], few or no publications can be found employing LC-MS regarding other mammalian species such as the domestic cattle (Bos taurus) or the elephant (Elephantidae).

A new UHPLC-HRMS method based on a Q Exactive™ mass spectrometer was developed to detect and quantify simultaneously progesterone, its hormone precursor pregnenolone and 10 reduced progestagens in plasma and serum samples. Purification was achieved by solid phase extraction (SPE) and the analysis was conducted in positive electrospray ionization (ESI) mode with the application of multiplexed selected ion monitoring (msx-tSIM).

Despite the poor ionization properties of underivatized steroids, a high sensitivity in the range of pg/mL was obtained. Applicability of this method was proven by tracing the concentrations of said steroids during the estrous cycle of the domestic cattle and the pregnancy course of the elephant.

1. S. A. Wudy, G. Schuler, A. Sánchez-Guijo, M. F. Hartmann, J. Steroid Biochem. Mol. Biol. 2018, 179, 88–103.
2. M. Hill, D. Cibula, H. Havlíková, L. Kancheva, T. Fait, R. Kancheva, A. PaÅ™ízek, L. Stárka, J. Steroid Biochem. Mol. Biol. 2007, 105, 166–175.
3. K. M. Tripp, M. Dubois, P. Delahaut, J. P. Verstegen, Theriogenology 2009, 72, 365–371.

Oral 2:
From Mice to Elephants – all of them want to fly in the MS universe Rise above the noise – in unknown screening, proteomics and native MS A story of Idefix and Obelix

Eugen Damoc¹, Rosa Viner², Albert Konijnenberg³, Kyle Fort¹, Maria Reinhardt-Szyba¹, Mikhail Belov¹, Marc Günder⁴, Guido Sonsmann⁴, Alexander Makarov¹

1. Thermo Fisher Scientific, Bremen, Germany
2. Thermo Fisher Scientific, San Jose, CA
3. Thermo Fisher Scientific, Eindhoven, Netherlands
4. Thermo Fisher Scientific (Schweiz) AG, Reinach, Schweiz

In designed small molecules application also called the “needle in a haystack” challenge MS instruments are designed to measure a lot of noise. Software tools then eliminate this noise and to filter out the meaningful features and turn them into knowledge about the sample. Novel Acquisition workflows will to automatically detect the meaningful differences between samples with confidence and don’t waste instrument time on the noise.

A novel Ion Mobility tool will enhance the performance classical Proteomics workflows by adding another dimension of separation on the LC scale.

To understand the mechanisms of Protein Assembly and Interaction studies common workflows are using a bottom up approach. But what happens in the actual native protein assembly can only be interpreted indirectly. To look at the intact protein level of Protein Complexes has been so far the domain of Electron Microscopy or Protein Crystallization. As those techniques are time consuming and also demand a high degree of sample preparation, Native Mass Spectrometry offers a unique technique to assess those samples and gain already a first glimpse on quality of such protein preparations. A novel type of HRMS instrument to capture Protein Complexes in the mega Da range will be described.

Oral 3:
Fully automated workflow for mass spectrometric characterization of proteins and antibodies

Schindler Patrick, Coulot Michèle, Panigada Tania, Goerlach Ekkehard, Boeuf Rémi, Brannetti Barbara Dreessen Jörg and Stoeckli Markus.

Novartis Institute for Biomedial Research  / BAT, Basel and Cambridge

At NIBR, we have implemented a fully automated workflow for processing and mass spectrometric characterization of proteins and antibodies. The system is set-up to perform automatically from sample preparation to data interpretation, intact or reduced/deglycosylated measurements. The sample (in a barcoded tube) is registered by the NIBR customer through a web portal (shared analytics: https://pets.nibr.novartis.net/), the customer chooses the workflow of interest and submits it into the system. The selected reactions (reduction and/or deglycosylation) are performed in a customized Hamilton liquid handling robotic station (STARlet) and the resulting product is then directly injected onto a LC-MS system. The data are subsequently processed and data interpretation is performed through software developed in-house. The pdf report is generated and accessible through the shared analytics portal to the customers. The system is operational since summer 2016 and more than 1500 samples have been analyzed 2017 by the platform.

With this presentation, we want to give an insight on the different parts of the workflow as well as on their articulation; discuss the performance and the benefits of this platform for the users and the MS labs.

Oral 4:
Taking FTMS to a New Level: Novel Conductively Cooled Magnet coupled to a modified Analyzer Cell

Arnd Ingendoh, Goekhan Baykut, Matthias Witt, Roland Jertz

Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany
Arnd(dot)Ingendoh(at)Bruker(dot)com

FTMS (Fourier Transform Mass Spectrometry) in the past was often connected with bulky instruments, large and expensive magnets and the need for magnet maintenance in terms of re-filling liquid nitrogen and helium. Despite of the outstanding performance characteristics of FTMS, this may have prevented the wider use of this technology in mass spectrometry labs.

Here we introduce a novel setup of FTMS instrumentation with the combination of an improved analyzer cell and a conductively cooled magnet. Performance features which could be reached in the past only with 12T and 15T magnet systems are now available on a compact 7T magnet. The new conduction cooling technology removes the requirements to fill liquid cryogens or to provide quench ducts completely since it operates virtually without any liquids. It can be operated in a standard laboratory space, enabling a wider range of scientific disciplines to gain access to the power of FTMS.

The instrument is equipped with an FTMS cell with improved magnetron control to exploit harmonics detection schemes and thus increase the experimental speed without sacrificing resolving power. Combined with AMP detection technology, it provides broadband ion stability for a mass resolution in the range of several 10 million RP. At the same time, resolving power in the range of several 100,000s can be combined with a data acquisition rate in the range of 5-10 Hz.

Key applications are set around the Isotopic Fine Structure (IFS) capability which enables high-confidence molecular formulas assignments for known and unknown targets.

Here we present applications which combine the high mass resolution with speed of analysis. One example is a novel workflow in metabolomics which allows to analyze of over 200 samples per day with the automatic annotation of molecular formulae for hundreds to thousands of targeted and unknown metabolites in complex samples for metabolomics and phenomics studies. Other applications include drug imaging and native MS studies in pharmaceutical questions.

Oral 5:
An extension to the Dromey-Foyster algorithm for improved assignment of high resolution mass spectra

David PA Kilgour

Department of Chemistry and Forensics, Nottingham Trent University, Clifton Campus, Nottingham, NG11 8NS, UK Email: david(dot)kilgour(at)ntu(dot)ac(dot)uk

One of the key benefits of high resolution, high mass accuracy mass spectrometry is the ability to predict elemental compositions based on ion mass alone, at least for small molecules.  In 1980, Dromey and Foyster published a key paper (DOI: 10.1021/ac50053a006) showing that the calculation of elemental compositions for mass spectral peaks could be structured in order to minimize the number of steps required to generate each new candidate. This algorithm showed considerable gains in efficiency.  One of the reasons why this algorithm is so efficient is that it uses the mass defects of nitrogen and hydrogen (and carbon, that of course has no defect) to add the numbers of C, H and N present in any predicted formula, in a single step, after numbers of other heteroatoms have been accounted for.  This is because, once a particular combination of heteroatoms have been proposed, any residual mass defect must be coming from the remaining H and N.

This use of the mass defect is the key strength of the Dromey-Foyster algorithm, but it turns out to also be a potential weakness.  The Dromey-Foyster algorithm works well for high mass accuracy spectra – requiring that these spectra have been very well, probably internally, mass calibrated.  But, what if you wish to use this algorithm to assign primary peaks in complex spectra in order to be able to undertake a high quality, robust internal recalibration?  If the mass error on peaks is more than about 5ppm out then the Dromey-Foyster algorithm can become unreliable, because the mass defect of the peak mass is too far from the real mass defect, and this leads to the wrong numbers of N and H being incorporated into the proposed formulae.

In this presentation I will show the simple modifications that can be applied to the original Dromey-Foyster algorithm to allow it to be used as the basis for assignment of ions in imperfectly mass calibrated spectra.

Oral 6 (Student Talk):
Targeted on-line breath analysis supports altered collagen turnover in idiopathic pulmonary fibrosis

Martin Thomas Gaugg¹, Anna Engler², Lukas Bregy¹, Yvonne Nussbaumer-Ochsner²,  Lara Eiffert¹, Tobias Bruderer^1,3, Renato Zenobi¹, Pablo Martinez-Lozano Sinues^1,4,5, Malcolm Kohler^2,6,*

1. Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland
2. Department of Pulmonology, USZ, Zurich, Switzerland
3. Division of Respiratory Medicine, Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland
4. University Children’s Hospital Basel, Basel, Switzerland
5. Department of Biomedical Engineering, University of Basel, Basel, Switzerland
6. Centre for Integrative Human Physiology, UZH, Zurich, Switzerland

* corresponding author

Introduction: On-line breath analysis is a powerful technique to obtain insights into the metabolism of a person. Idiopathic pulmonary fibrosis (IPF) is a chronic and poorly understood lung disease. Following a recent study that reports increased levels of collagen related amino acids in lung tissue of IPF patients using GC‑MS profiling [1], we hypothesized that these altered amino acid levels might be mirrored in exhaled breath, which would allow for a non-invasive screening of IPF.

Methods: Breath analysis was performed on-line using secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS). For all previously reported target compounds where a signal was detected at the accurate mass in real-time, UHPLC-MS measurements of exhaled breath condensate (EBC) were performed, to confirm their identity. Their classification performance was estimated using 1 million leave-one-out cross-validations.

Results: On-line breath spectra were recorded for 21 IPF patients and 21 healthy controls, matched in terms of age, gender and smoking history. Within these, we could detect robust signals for proline, 4-hydroxyproline, alanine, valine, leucine/isoleucine, allysine, phenylalanine and pyroglutamic acid, most of which could also be confirmed in EBC. Six of the eight compounds showed significantly increased signal levels (p<0.05) in exhaled breath of IPF patients. Additionally, amino acid levels correlated across subjects, supporting a common underlying pathway. Using the signals of all detected amino acids, we were able to obtain a cross-validated area under the receiver operating characteristic curve of 0.86.

Conclusions: Using targeted on-line breath analysis with SESI-HRMS, we could detect increased amino acid levels in IPF patients, which allowed for a good discrimination from healthy controls. This is consistent with previous metabolomic findings from lung biopsies, strongly reducing the probability of false discoveries. However, we were able to capture this information in a non‑invasive and rapid fashion, underlining the strength of real-time breath analysis.

1.      Kang Y. P. et al., J. Prot. Res., 15 (5), 1717-1724, 2016

Oral 7 (Student Talk):
Using the microbial model of the fungi C. elegans for metabolism studies with LC-HR-MS/MS

Katharina Elisabeth Grafinger^1,2, Katja Stahl³, Andreas Wilke³, Stefan König¹, Wolfgang Weinmann¹

1. Institute of Forensic Medicine, Forensic Toxicology and Chemistry, University of Bern, Switzerland
2. Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland
3. Department of Mechanical and Process Engineering, University of Applied Sciences, Offenburg, Germany

Contact: Institute of Forensic Medicine, Forensic Toxicology and Chemistry, Bühlstrasse 20 3012 Bern, Switzerland, katharina(dot)grafinger(at)irm(dot)unibe(dot)ch

Metabolism studies are of significant importance in fields such as clinical toxicology, forensic toxicology and pharmaceutical research. In the forensic context, they are used to identify the main metabolites of a drug of abuse, which can be then used as biomarkers for the development of new methods for the detection of the consumption of said substance. These biomarkers are necessary, because their presence confirms the consumption of the parent substrates and additionally, not always the parent substrate can be detected.

Different microsomal and microbial approaches can be used to generate in vitro metabolites. The zygomycete non-pathogenic fungi Cunninghamella elegans (C. elegans) contains the cytochrome P450 CYP CYP509A1 enzyme, which makes it possible to enable reaction catalyzed by the human CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. The advantage of microbial models are low costs, easy handling, scale-up capability and the avoidance of animal and human studies in drug discovery and development processes.

In order to complement the microsomal method of pooled human liver microsomes (pHLM) we investigated C. elegans for its usefulness to generate the metabolites of phenethylamines and synthetic tryptamines. All samples were measured using Liquid Chromatography- High Resolution- tandem MS (LC-HR-MS/MS) and metabolites were identified according their isotopic pattern, the presence of the molecular ion and their fragmentation pattern.

Identification of metabolites according their tandem mass spectra resulted in about one third of the metabolites identified in pHLM for phenethylamines and about three quarters for synthetic tryptamines. Using the fragmentation patterns, the sites of biotransformations could be exactly identified and metabolites with the same molecular formula be differentiated. Obtained data was then used to propose biotransformation pathways for the all investigated substances.

ORAL 8 (Student Talk):
Deep learning – creation of an artificial neural network for the prediction of data generated by high resolution mass spectrometry with data independent acquisition

Elmiger Marco¹, Dobay Akos², Ebert Lars³, Kraemer Thomas¹

1. Department of Forensic Pharmacology and Toxicology, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland
2. Department of Forensic Genetics, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland
3. Department of Forensic Imaging/Virtopsy, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland

Email: marco(dot)elmiger(at)irm(dot)uzh(dot)ch

Background & Objectives: General unknown screening (GUS) in biological matrices becomes more and more crucial in forensic toxicology with an ever growing number of NPS entering the drug market. LC coupled to high resolution quadrupole time-of-flight mass spectrometry (LC-HR-QToF) provides a suitable analytical platform to meet this challenge. While data-dependent acquisition (DDA) approaches are still widely used, the greater possibilities provided by data independent acquisition (DIA) approaches are more promising for GUS. The disadvantage of DIA approaches is the huge amount of data produced which have to be dealt with. The promising field of deep learning offers new possibilities where neural networks can be trained to classify big amounts of data. The aim was to exemplify such an approach for HR-MS DIA files by comparing different deep learning approaches (KNIME, Keras and TensorFlow).

Method: Train and test sets were created by using blood samples from authentic cases (blank, cocaine, zolpidem, combinations of the latter two). Data collection was done via HR-MS combined with DIA on a Sciex TripleTOF 6600. The data were prepared by first converting them into the open data format .mzML and then cleaning and structure it by using an in-house R script. Data analysis was done by comparing Deep Learning models on different platforms (KMIME, Keras and TensorFlow).

Results and Discussion: Different neural networks were built in KNIME and Keras by creating a workflow or writing an R script, respectively. After the neural networks learned on the train set similarities and differences between the groups, they were able to do a prediction of the test set including samples that the neural network hadn’t seen before. Accuracy and precision of over 75 and 95 percent, respectively were reached for both approaches for the test set.

Conclusion: In this project, it was possible to prepare HR-MS DIA data files via an R script to make them available for deep learning approaches. Furthermore, it shows the possibility of sample type prediction by a deep learning approach after learning only by one training set. The accuracy and precision for the prediction via different deep learning approaches were in a good range. Thus, deep learning promises a huge potential for automatic handling of big data generated by DIA with high resolution mass spectrometry.

ORAL 9:
MS2field: Towards a transportable, fully automated HRMS platform

Michael A. Stravs¹, Heinz Singer¹, Christian Stamm¹, Christoph Ort², Reto Bolliger³, Guenter Boehm³ , Thomas Moehring⁴

1. Department of Environmental Chemistry, Eawag, Dübendorf, Switzerland
2. Department of Urban Water Management, Eawag, Dübendorf, Switzerland
3. CTC Analytics, Zwingen, Switzerland
4. Thermo Fisher Scientific, Bremen, Germany

Email: stravsmi(at)eawag(dot)ch

Liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) allow to routinely measure and quantify a large variety of micropollutants in water samples, and even to identify unknown contaminants. However, the generation of comprehensive datasets still hinges on traditional sampling procedures that have seen little progress in recent years. The acquisition of long time profiles with high temporal resolution implies extraordinary efforts for sampling and transport and potential issues with storage and degradation. Therefore, the temporal dynamics of micropollutants are often insufficiently captured , which impedes the understanding of sources and environmental processes (e.g., intermittent short peaks of herbicide runoff during rain events).

In MS2field, we are developing a transportable mass spectrometry measurement station to acquire comprehensive micropollutant profiles over prolonged periods at high temporal resolution directly in the field without human supervision, thus avoiding issues with traditional sampling. A high-resolution mass spectrometer (Q-Exactive Plus, Thermo Scientific) is installed in an air-conditioned trailer. A fully automated sampling procedure is developed using a programmable autosampler (PAL RTC, CTC) and a self-cleaning filtration device coupled to online solid-phase extraction and LC-HRMS. This provides a measurement every 20 minutes.

In a first test phase, we measured untreated wastewater with low ng/L detection limits. A runtime of over one week between services was attained, yielding over 500 data points per compound. The evaluation of target compounds showed periodical behavior for pharmaceuticals such as Candesartan, confirming that the system is able to capture temporal dynamics often missed by traditional sampling. For non-target processing, established in-house pipelines were combined with spectral analysis of time series and hierarchical clustering. This revealed 50 clusters of periodical features with different frequency contents, containing up to 230 features per cluster. This proof-of-concept dataset is, to our knowledge, unprecedented.

To monitor analytical performance, an online dashboard displays basic analytical data extracted from measurements on the fly, as well as data from ambient and instrument sensors. For failsafe operation, a stored program control system with multiple sensors is being developed to react to possible issues such as pump clogging, overheating, water leaks, instrument failures etc. Upcoming field measurements have the potential to bring new insights into temporal dynamics of micropollutants, with broad opportunities for applications across disciplines.

Poster Communications

Poster 1:
Systematic Evaluation of Washing and Extraction Protocols for Metabolomic Analysis of Hair

Eisenbeiss Lisa¹, Binz T. M.², Baumgartner M. R.², Kraemer T¹, Steuer A. E.¹

1. Department of Forensic Pharmacology and Toxicology, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland
2. Center for Forensic Hair Analytics, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland Email: lisa(dot)eisenbeiss(at)irm(dot)uzh(dot)ch

Aims: Metabolomics aims to analyze the composition and the dynamics of the metabolome. Typically, the plasma-metabolome is analyzed to detect changes of endogenous molecules. In contrast to this classical matrix, hair allows a long-term view on the metabolome as small molecules are constantly incorporated into the growing hair shaft. This accumulation allows to search for robust biomarkers, e.g. for drug intake avoiding short-term perturbations, such as circadian variations. The aim of the proposed preliminary study was to systematically investigate the influence of different pre- analytical parameters on detected metabolites and to establish a robust protocol for hair metabolomics.

Methods: Hair samples were washed with two different washing protocols (dichloromethane (DCM)/acetone/H2O/acetone and H2O/acetone/DCM/acetone). Subsequent homogenization was performed by cutting the samples into snippets or by pulverizing. Homogenized hair samples were extracted with 1 mL of different solvent mixtures (acetonitrile (ACN)/H2O, ACN/buffer pH 4 and ACN/buffer pH 8.5) in an ultrasonic bath, centrifuged and filtered. Wash water and extracts were analyzed by liquid chromatography high-resolution (HR) MS and MS/MS (Sciex 6600 TripleTOF) and GC-HRMS (Thermo Scientific Q Exactive). Tentative identification of metabolites was performed using Progenesis QI software.

Results and discussion: The detection of different metabolite classes was possible (e.g. amino acids and derivatives thereof, acylcarnitines, fatty acids, purine and pyrimidine derivatives) by using one sample preparation, untargeted metabolomics methods and database-assisted identification. Milling of hair samples prior to extraction proved favorable in terms of tentatively identified features compared to snipped samples. The order of aqueous and organic washing steps did not influence the total amount of metabolites being removed. For most metabolites, a difference in extraction yield could be found for the three different extraction mixtures. Extraction with ACN/H2O was the best compromise for metabolite extraction.

Conclusion: The establishment of a suitable sample preparation for metabolomics is a challenging task. Different parameters can have an influence on detected metabolites and hence the ability to detect metabolome changes. However, our study shows that it is possible to reliably detect a representative selection of metabolites which is a prerequisite for performing metabolome studies in hair.

Poster 2:
Studying non-covalent interactions between G protein-coupled receptors and their binding partners by MALDI mass spectroscopy

Na Wu

ETH Zurich HCI E331 Vladimir-Prelog-Weg 3, 8093 Zürich Switzerland Email: na(dot)wu(at)org(dot)chem(dot)ethz(dot)ch

G protein-coupled receptors (GPCRs) are a family of important membrane proteins, and the study of noncovalent interactions of GPCR complexes and their underlying mechanisms is crucial to understand their function. Nanodiscs have become a leading technology to solubilize membrane proteins in a biomimetic surrounding, to protect their biological functions [1]. Herein, noncovalent binding efficiencies between nanodiscs loaded with G protein-coupled receptors (ND-GPCRs) and their interaction partners, including different kinds of engineered G proteins, and a G protein mimicking nanobody Nb80[2,3] are investigated by high-mass Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). Different agonists and inverse agonists are applied that influence the conformational states of GPCRs to active and inactive states, respectively, which will affect the noncovalent interactions between GPCRs and their partners [3]. Different conformational states of ND-GPCRs and their partners are precisely and cleanly reflected by intensity changes of various m/z features in the MALDI data, helping to uncover binding to GPCRs in the active and inactive states. Our approach provides a fast, convenient, and sensitive way to explore the biological functions and formation conditions of GPCR-complexes, benefiting selecting GRCRs-related drugs.

1. J. Venkatakrishnan, et al., Molecular signatures of G-protein-coupled receptors. Nature 2013, 494, 185-94.
2. M. T. Marty, et al., Ultra-thin layer MALDI mass spectrometry of membrane proteins in nanodiscs. Anal. Bioanal. Chem. 2012, 402, 721-729.
3. S. Isogai, et al., Backbone NMR reveals allosteric signal transduction networks in the beta1-adrenergic receptor. Nature 2016, 530, 237-41.

Poster 3:
Development of a specific solid phase micro extraction tool for the determination of a small molecule-drug conjugate directed against carbonic anhydrase in cancer chemotherapy

Sahar Ghiasikhou¹, Jorg Scheuermann², Samuele Cazzamalli², Dario Neri², Renato Zenobi¹

1. Department of Chemistry and Applied Bioscience, ETH Zürich, CH-8093 Zürich
2. Switzerland Institute of Pharmaceutical Sciences, ETH Zürich, CH-8093 Zürich, Switzerland

Introduction: The targeted delivery of cytotoxic agents into tissues, especially for malignant cells is an attractive strategy to avoid dose limiting toxicity. Targeting moieties can be antibodies, aptamers and low molecular weight non-peptidic ligands. Previous studies showed the advantages of conjugating small molecules to the drug over attaching macromolecules. One of the common used target moiety for selective delivery of cytotoxic agent is acetazolamide. In this area, there is a pressing need to develop rapid techniques to quantify the amount of drug in the tissue in order to investigate the tumor targeting performance of the different ligands. So-called the “capillary gap sampler” is capable of automated and site-specific extraction of very small sample amounts, which is interesting for this application.

Methods: The capillary gap sampler is a miniaturized sampling device enabling automated low- volume sample handling, for direct interfacing to ESI mass spectrometry. It consists of a liquid bridge of several nanoliters formed between two capillaries, where one acts as the ESI-MS spray needle. Sample extraction is performed by a coated stainless-steel pin which is controlled by a robot arm. Selective drug extraction is possible through immobilizing carbonic anhydrase (CA) on the pin. This modification is performed by overnight reaction between protein with epoxy modified beads which are glued to the tip of the pin. Using fluorescence microscopy, bright field and blue channel images of beads carrying protein linked to 8-anilino,1-naphthalene sulfonate and controls confirmed the attachment of the protein.

Results and discussion: The development started with optimization of the coating procedure, desorption solution, etc. In order to find the optimum desorption solution two tests were performed: in the first one, influence of pH on the binding of acetazolamide with CA was investigated. In the second test, different phases (ACN, MeOH, EtOH, Acetic acid, Acetone 60% in water) were used for desorption. Acetic acid was found to be the optimum desorption solution. Extraction of the drug from PBS was performed by dipping the CA modified extraction tool inside the solution. After a quick washing step with water, it enters into the liquid bridge, where the analyte desorbs and is sprayed in the ESI source. Peaks corresponding to the

drug (drug+), (drug+Na++H+), (drug+2H+), (therapeutic warhead =cytotoxic agent

coupled to the targeting agent+H+) were observed in the spectrum. Finally, repeatability of the acetazolamide extraction using CA modified beads are evaluated by performing thirteen 5-minute extractions from 500nM acetazolamide in PBS solution. The relative standard deviation was below 10%, which confirms the repeatability of the method. Drug extraction studies from human plasma and homogenized tissue will also be shown.

Novel aspects: Development of a specific extraction method for evaluation of targeted drug delivery.

Poster 4: Steroid Hormone Profiling in Fingernails with LC-MS/MS Using ¹³C₃-labeled Surrogate Analytes

Clarissa D. Voegel¹, Pearl La Marca-Ghaemmaghami², Ulrike Ehlert², Markus R. Baumgartner¹, Thomas Kraemer³, Tina M. Binz¹

1. Center for Forensic Hair Analytics, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland
2. Department of Clinical Psychology and Psychotherapy, University of Zurich, Zurich, Switzerland
3. Department of Forensic Pharmacology and Toxicology, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland Email: clarissa(dot)voegel(at)irm(dot)uzh(dot)ch

The analysis of endogenous steroid hormones in hair is increasingly used in the area of stress and health-related research for the long-term determination of those biomarkers. If hair is not available, nails can be used as an alternative matrix for the retrospective evaluation of substances. The aim of the project was to develop a sensitive LC-MS/MS method for the quantification of steroid hormones in human nails. A new approach with the use of ¹³C₃-labeled surrogate analytes was used for the quantification of endogenous compounds in authentic matrix.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method was developed for the simultaneous identification and quantification of 12 steroid hormones (aldosterone, cortisone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, dehydroepiandrosterone, 17α-hydroxyprogesterone, dihydrotestosterone, progesterone) in human fingernails. Steroid hormones were extracted in a range of 0.5 mg - 10 mg nails by methanolic extraction, followed by a liquid-liquid extraction. The analysis was conducted with LC-MS/MS in electrospray ionization positive mode. The method was validated in terms of linearity, limit of detection, limit of quantification, precision, accuracy, matrix effect, recovery and robustness.

After successful validation, the applicability of the method could be proven. It was used for steroid profiling in nails of mothers and their newborn infants where cortisone, cortisol, androstenedione, 11-deoxycorticosterone, testosterone, and progesterone could be detected. Furthermore it could be shown that there is no significant difference between left and right hand for cortisol, cortisone, progesterone, and testosterone concentrations. A linear correlation between cortisol and cortisone in nails was found. In conclusion, it could be shown that nails are a suitable matrix for the retrospective monitoring of steroid hormones and that surrogate analytes can be a useful tool for quantification of low endogenous amounts of steroid hormones.

Poster 5:
Biotransformation of the reference compound 2,4-dinitrochlorobenzene in zebrafish (Danio rerio) early life stages

Tierbach, A; Schönenberger, R; Groh, K J; Schirmer, K; Suter M J-F

EAWAG, Dübendorf, Switzerland

A comprehensive risk assessment of bioactive compounds requires that biotransformation processes are functional within the test organism. Early developmental stages of zebrafish are one type of test organism frequently applied to explore the biological activity and toxicity of drugs, natural toxins and environmental pollutants. To broaden the knowledge regarding the biotransformation potential of zebrafish early life stages, we mapped the protein abundance of cytosolic glutathione S-transferases (GST) in zebrafish embryos and larvae. We demonstrated that the GST expression in early life stages of zebrafish is dynamic and reflects important developmental events e.g. hatching and liver development.

Having established knowledge about the presence of GSTs in the developing fish, we now ask if the GSTs are capable of performing biotransformation reactions with xenobiotic compounds. To address this question, we establish liquid chromatography mass spectrometry protocols for the in vivo analysis of glutathione-conjugation using 2,4-dinitrochlorobenzene (CDNB) as substrate. We analyze the difference in the biotransformation of CDNB in zebrafish embryos and free-swimming larvae under exposure to non-toxic concentrations. Additionally, we monitor the protein expression of cytosolic GSTs during the exposure with the targeted proteomics approach to allow conclusions on CDNB regulated isoforms. First results show that zebrafish embryos and free-swimming larvae are able to biotransform CDNB within the mercapturic acid pathway.

Poster 6:
Leveraging Orbitrap FTMS performance for small molecule applications

Yury Tsybin¹, Konstantin Nagornov¹, Anton Kozhinov¹, Natalia Gasilova², Laure Menin², Markus Zennegg³, Davide Bleiner³

1. Spectroswiss, EPFL Innovation Park, 1015 Lausanne, Switzerland
2. Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
3. Swiss Federal Laboratories for Materials & Technology, Dübendorf, Switzerland

High resolution performance is one of the main benefits for the small molecule analysis provided by Orbitrap FTMS. Direct infusion without and with on-line separation using liquid chromatography (LC) and gas chromatography (GC), as well as surface imaging are all widely employed sample ionization and introduction approaches for hyphenation with Orbitraps. The limitations may include a moderate throughput (higher resolution means longer ion detection), a certain care being required to provide accurate isotopic abundance ratios, restricted increase of the resolution for achieving isotopic fine structure information and separating isobaric compounds at high mass, and, perhaps most importantly, a sensitivity.

For example, trace level quantitatively-accurate measurements of organic pollutants, for example dioxins in biofluids, are essential for monitoring of the environmental hazards and timely initiating personal preventive care. However, a single measurement of a low concentration sample using GC Orbitrap FTMS may be not sensitive enough to detect the compounds of interests or to accurately quantify their levels. The fundamental nature of FTMS suggests a possible way of increasing the sensitivity of targeted and untargeted analysis for both isolated compounds and those embedded into a complex matrix, by averaging of time- domain unprocessed data (transients) across a number of technical replicates from GC-MS measurements, followed by Fourier transformation. A principal obstacle to realize this approach is the absence of an access to the transient signals from Orbitrap FTMS instruments.

We developed and implemented a transient-recording capability on the GC Orbitrap FTMS and applied it for increasing the sensitivity of the trace level persistent organic pollutant analysis. Preliminary results demonstrate that a multiplexed GC-FTMS approach is beneficial for the increased sensitivity and improved accuracy in the quantitative analysis of low abundant dioxins. Furthermore, with an available access to the transients, we were able to significantly increase the overall resolution obtainable from the GC Orbitrap FTMS by recording transients with 2-10 fold extended duration. As a result, an isotopic fine structure analysis, which can aid in targeted and untargeted molecular analysis, has been uniquely enabled on the GC Orbitrap FTMS. Comparable advantages will be demonstrated for other applications, including for multiplexed quantitative lipidomics using direct infusion FTMS, LC-MS hyphenation, and imaging of biological tissues.

Poster 7:
Phosphatidylethanol formation from endogenous phosphatidylcholines in animal tissues from pig, calf, and goat

Marc Luginbühl¹, Sytske Willem², Stefan Schürch³, Wolfgang Weinmann¹

1. Institute of Forensic Medicine, University of Bern, Switzerland
2. Laboratory of Toxicology, University of Ghent, Belgium
3. Department of Chemistry and Biochemistry, University of Bern, Switzerland

In the presence of alcohol, phosphatidylcholine (PC) is transformed to the direct alcohol biomarker phosphatidylethanol (PEth). This reaction is catalyzed by the enzyme phospholipase D (PLD) and dependent upon substrate availability. As recent studies have solely focused on the determination of PEth, information about the PC distribution in the investigated tissue has been missing.

Adressing this issue, we developed a method which allowed us to monitor PC (16:0/18:1 and 16:0/18:2) and PEth (16:0/18:1 and 16:0/18:2) simultaneously. This was performed by the use of a LC-MS/MS method based on a C8 core-shell column, coupled to a Sciex 5500 QTrap instrument. By the application of polarity switching, at first, PC was measured in ESI positive SRM mode, while PEth was determined at a later stage in ESI negative SRM mode. The PEth method was validated (accuracy, precision, matrix effects) for human blood samples to show its robustness and subsequently applied for the investigation of systematic in-vitro PEth formation in animal tissue samples (brain, kidney, liver, and blood) from a pig, a calf, and a goat: Homogenized tissue was incubated at 37°C with varying ethanol concentrations from 1-7 g/kg (determined by HS-GC-FID) for five hours, whereby a sample was taken every 30 minutes. The LLOQ for PEth was set at 7.5 ng/mL, the LOD at 3.5 ng/mL.

For all tissue samples, an increase in PEth was measurable. PEth concentrations formed in blood remained below the LLOQ, in agreement with literature. Data analysis of Michaelis-Menten kinetics and PC within the tissue provided a detailed insight about PEth formation, as the occurrence of PEth species can be linked to the observed PC composition. The results of this study show that PEth formation rates vary from tissue to tissue and among different species. To prevent the post sampling formation of PEth in organ tissue, we recommend immediate homogenization and subsequent storage in a solvent such as acetonitrile, to precipitate any PLD. Additionally, we recommend the inclusion of transitions for both fatty acid chains within the method to observe and correct isomer specific fragment ion abundances.

Poster 8:
Analytical workflow to trace production dynamics of cyanobacterial toxins

Elisabeth Janssen

Group Leader, Environmental Chemistry, Eawag Email: Elisabeth(dot)janssen(at)eawag(dot)ch

Cyanobacterial bloom events conquered freshwater resources across the globe, yet the potential risk of many cyanobacterial metabolites remains mostly unknown. The key challenge for a comprehensive risk assessment of emerging cyanopeptides is their large structural diversity and the lack of reference materials for analytical varification. Among cyanopeptides, only microcystins have been studied intensively and the wealth of evidence regarding exposure concentrations and toxicity led to their inclusion in risk management frameworks for water quality. However, cyanobacteria produce an incredible diversity of hundredth of cyanopeptides beyond the class of microcystins. The question arises, whether the other cyanopeptides are in fact of no human and ecological concern or whether these compounds merely received (too) little attention thus far.

We developed an LC-HRMS/MS workflow with empirical and in-silico identification strategies to determine cyanopeptide fingerprints. The peptide profiling includes >450 target compounds of aerucyclamides, cyanopeptolins, anabaenopeptins, microginins, aeruginosins, and microcystins. We find that microcystins never occur alone and other cyanopeptides can dominate total abundance depending on the production dynamics throughout the growth phase. These emerging cyanopeptides can be potent enzyme inhibitors and this chemical profiling needs to be complemented by effect-based screening to further prioritize tentatively abundant and toxic cyanopeptides.

Poster 9:
Feasibility of an online HPLC-DPPH antioxidant assay hyphenated with a high-resolution QTOF mass spectrometer

Richard Gössl, Manuela Baur, Andre Düsterloh, Ulrich Höller

DSM Nutritional Products Ltd., R&D Global Analytics, CH-4002 Basel, Switzerland,
E-Mail: richard(dot)goessl(at)dsm(dot)com

The reduction of the 2,2-diphenyl-1-picrylhydrazyl (DPPH^Ÿ) free radical by radical scavenging compounds is the basis of the widely used DPPH antioxidant assay which was developed in 1958 by M. Blois. In 2000 a post-column or “online” variant of the assay was introduced by interfacing the assay with an HPLC-UV. With this modification it was possible to assess not only the overall antioxidant capacity of a given sample but also the contribution of individual constituents to the total effect. We aimed in developing a method which provides in parallel a read-out of the radical scavenging capacity and high-quality spectral data (i.e. UV spectra as well as mass spectra) on a single compound level. The poster will show some examples of natural product characterization. With the presented method a link can be made between function (antioxidant capacity) and molecular structure.

Poster 10:
Advances in Orbitrap Instrumentation for Native Top-Down Analysis of Non-Covalent Protein Complexes

Eugen Damoc¹, Rosa Viner², Albert Konijnenberg³, Kyle Fort¹, Maria Reinhardt-Szyba¹, Mikhail Belov¹, Marc Günder⁴, Guido Sonsmann⁴, Alexander Makarov¹,

1. Thermo Fisher Scientific, Bremen, Germany
2. Thermo Fisher Scientific, San Jose, CA,
3. Thermo Fisher Scientific, Eindhoven, Netherlands
4. Thermo Fisher Scientific (Schweiz) AG, Reinach, Schweiz

Purpose: Evaluate the performance of the new Thermo Scientific^TM Q Exactive^TM UHMR mass spectrometer for native MS and native top-down analysis of large protein complexes

Methods: Native MS and native top-down analysis using Q Exactive UHMR mass spectrometer

Results: Demonstrated excellent performance of the new Q Exactive UHMR mass spectrometer for structural characterization of homomeric and heteromeric protein assemblies

Introduction: Native mass spectrometry has emerged as a powerful technique to study protein-ligand interactions and elucidate the structure of macromolecular assemblies, including both soluble and membrane protein complexes. Top-down studies of intact protein complexes have been reported since the early 1990’s, but their characterization using MS³ have only recently been reported ^1,2 and most work has been done on homomeric assemblies. However, poor fragmentation into subunits and stripped complexes in the front end of the MS limits the use of current MS instrumentation for native top-down analysis using a pseudo-MS³ approach. In this work we examine this limitation and explore new ways for extending native top-down performance to allow interrogation of heteromeric protein assemblies like proteasome by top-down pseudo-MS³.

2018 SGMS Meeting and SGMS School Registration & Deadlines
 

A surcharge of CHF 50 will be enforced to all payments submitted after the meeting.

Deadlines

• Early abstract registration: July 15^th (Poster/Oral acceptance will be notified by September 1^st).
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Short Oral Contributions: Early deadline for abstract submission for both talks and posters is July 1^st. The extended deadline is September 1^st. Abstracts submitted before July 1^st will have priority. Please submit your abstract including author's name and address directly to   abstract(at)sgms(dot)ch The abstract should not exceed 2500 characters.

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Please send your registration to  registration(at)sgms(dot)ch not later than October 1st, 2018. There is no need to register personally at the Dorint Hotel Blüemlisalp, Beatenberg! The SGMS committee will manage all hotel reservations and payments. We will strictly follow a first come first serve policy for the hotel room assignment.

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