Pre-SGMS School:

Ion Mobility Fundamentals & Applications

Ion Mobility coupled with Mass Spectrometry (IM-MS) is a very powerful and versatile technique. Although first described by McDaniel et al in 1962, only relatively recently (the last 10 years or so) it has gained wide popularity, primarily due to the commercialisation of the technology. The 2019 SGMS School is designed to give a broad overview of the various flavours of IM-MS and a detailed description of how major MS vendors have implemented the IM technology, accompanied by examples of practical applications.

SCHOOL PROGRAM NOW ONLINE

2019 SGMS Meeting

Program SGMS school on Ion Mobility Registration Plenary Lectures Short Communications Posters Sponsors

MAIN MEETING PROGRAM NOW ONLINE

The 37th meeting of the SGMS will take place at the Dorint Resort Blüemlisalp Beatenberg, 24-25 October 2019 high above Lake Thun in the Bernese Oberland, with a scenic view of the Swiss Alps!

The meeting will be preceeded by the 2^nd SGMS School on Ion Mobility: Fundamentals and Applications

Confirmed Speakers for 2019

• Vicki Wysocki (The Ohio State University) - [ abstract ]
• Dietrich Volmer (Humboldt University Berlin)  - [ abstract ]
• Andrea Büttner (Friedrich-Alexander-Universität Erlangen-Nürnberg) - [ abstract ]
• Valérie Gabelica (University of Bordeaux, France) - [ abstract ]

2019 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).
• Abstract registration: September 1^st (Poster/Oral acceptance will be notified by September 21st).
• Standard registration: August 31st (Includes one night at the Dorint hotel, Thursday Apéro and SGMS dinner, Friday breakfast, coffee breaks).
• Late registration: October 1st

Abstract submission

Short Oral Contributions: Early deadline for abstract submission for both talks and posters is July 15^th. 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   registration(at)sgms(dot)ch The abstract should not exceed 2500 characters.

Guidelines for the submission of abstracts:

• Save your file as LASTNAME_TITLE.xxx
• Include the name of the contact person (spell out first name) as well as the complete address and e-mail.
• Do not use any logos (company, institute, ...) on the abstracts.
• Please no formatting, flat text only..
• Figures most be supplied as separate *.jpg images.
• Do not use halftoning or colour: We publish in pure black and white.
• Include your e-mail address.

Posters: The same rules apply for the abstracts to be submitted. Poster size should not exceed 146 H x 118 W cm (size of pin wall). There will be a defined poster session.

SGMS School: For details see the SGMS website. Register via the SGMS meeting registration form

Registration

The registration form is available in  PDF and WORD

Please send your registration to  registration(at)sgms(dot)ch not later than October 1st, 2019. 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.

SGMS School Registration on the same registration form. Please tick the correct box if you are attending the School, Meeting or both.
There will be an additional fee of CHF 50.- for late registration (after October 1^st, 2019).

All PhD students attending the annual SGMS meeting pay a reduced fee of CHF 100.-, but will have to share rooms.

Program

Download the full program [ HERE (pdf) ]

Plenary Lectures  

Vicki Wysocki: Native MS: A Structural Biology Tool

Vicki Wysocki

Department of Chemistry and Biochemistry
NIH Resouce for Mass Spectrometry Guided Structural Biology
The Ohio State University

United States of America

Characterization of the overall topology and inter-subunit contacts of protein complexes, and their assembly/disassembly and unfolding pathways, is critical because protein complexes regulate key biological processes, including processes important in understanding and controlling disease. Tools to address structural biology problems continue to improve.  Native mass spectrometry and associated technologies are becoming an increasingly important component of the structural biology toolbox.  When the mass spectrometry approach is used early or mid-course in a structural characterization project, it can provide answers quickly using small sample amounts and samples that are not fully purified.   Integration of sample preparation/purification with effective dissociation methods, ion mobility, and computational approaches provide a MS workflow that can be enabling in biochemical, synthetic biology, and systems biology approaches.  Beyond what MS can provide as a stand-alone tool, MS can also guide and/or be integrated with other structural biology approaches such as NMR, X-ray crystallography, and cryoEM.  MS can determine whether the complex of interest exists in a single or in multiple oligomeric states and can provide characterization of topology/intersubunit connectivity, and other structural features.  Examples will be presented to illustrate the role MS and surface-induced dissociation can play in guiding a structural biology workflow and will include designed protein complexes and isolated or recombinant protein and nucleoprotein complexes.

Dietrich Volmer: Assessing the molecular value of lignin by high resolution mass spectrometry

Dietrich A. Volmer

Department of Chmistry
Humboldt Universität zu Berlin
12489 Berlin

Germany

Lignin's aromatic building blocks provide a chemical resource that is, in theory, ideal for substitution of aromatic petrochemicals. Moreover, degradation of lignin has the potential to generate many high-value chemicals for technical applications. To fully assess the molecular value of lignin wastes, chemical characterization methods are required that are able to resolve the highly complex mixtures of thousands of components. Due to lignin’s inherent intricate structural motifs and additional heteroatoms being introduced during chemical degradation procedures and due to the highly variable relative abundances within the material, different analytical techniques often give different estimates of the lignin product composition, even for the same sample. Among the different modern instrumental techniques, high-resolution mass spectrometry (HRMS) is currently the most suitable technique for lignin analysis. This presentation summarizes different mass spectrometry and data-processing methods developed in our laboratory for application to full-scan raw data from high-resolution mass spectrometry experiments of decomposed lignin samples. Data from different ionization techniques such as ESI, APCI, APPI and MALDI are compared as well as rapid characterizations by different ambient ionization techniques. The discussed graphical and statistical methods provide initial classification of the major lignin components and elucidation of the main structural features without the need for time-consuming tandem mass spectrometry analyses.

Andrea Büttner: A world of unforeseen sensations – odorous molecules in contemporary products

Andrea Büttner

Department of Chemistry and Pharmacy
Friedrich-Alexander-Universität Erlangen-Nürnberg
91054 Erlangen

Germany

Contemporary products are composed of a multitude of chemical constituents providing functionalities such as colour, texture, haptic sensations or flexibility, to name but a few. Commodities and products travel the world in a globalized market and add to an ever-growing complexity in our chemical world.

While certain materials are commonly (accepted to be) associated with characteristic smells, others increasingly elicit odours that would not typically be expected to appear – most recently in a range of articles of daily use, including children’s products and sports gear.

Our research group has been increasingly active in recent years in compiling data on such unforeseen smells and establishing their chemical underpinnings. Studies have targeted glues and adhesives, paints, children’s products, and recycled regrinds, to name but a few. Sniffing out the causative substances has much in common with detective work, as potent odorants are commonly not straightforward to detect analytically. Accordingly, one needs either skilled assessors that at the same time are talented chemical analysts or intelligent machines – ideally both. Facing these challenges in a modern world of chemistry, the team of the Department of Sensory Analytics at Fraunhofer IVV has joined forces with the Campus of the Senses initiative (www. campus-der-sinne.fraunhofer.de) with an aim to develop novel strategies to unveil what is often not visible to the eyelet alone sniffable by a machine.

Valérie Gabelica: A Mass Spectrometry Insight into G-Guadruplex Biophysics

Valérie Gabelica

Institut Européen de Chimie et Biologie
University of Bordeaux
33607 Pessac

France

This presentation will review the use and recent developments of mass spectrometry and associated techniques (ion mobility spectrometry ion spectroscopy) for nucleic acids biophysics. Specifically, with mass spectrometry, we characterize the stoichiometries of all complexes simultaneously present. Experiments can be carried out at equilibrium, as a function of time, or as a function of temperature. Another dimension of information is provided by ion mobility spectrometry, which measures the electric drag of ions at each m/z in an inert gas such as helium. The collision cross sections inform us on the shape of each population, and we use molecular modeling to deduce the shapes of each structure. Finally, ion spectroscopy measures spectroscopic properties (IR, UV and CD spectra) of each ion packet, to get additional insight into the secondary structures. Applications will be shown on DNA G-quadruplexes, complexes with organic ligands, i-motif structures, and silver-bound G-duplexes.

Short Communications

Oral 1 (Student Talk):
2D UV-MS Fingerprinting for Identification of Isomeric Biomolecules

Erik Saparbaev, Vladimir Kopysov, Aleksandr Pereverzev, Oleg V. Boyarkin

Laboratoire de Chimie Physique Moléculaire, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland

Cooling ionized biological molecules to cryogenic temperatures enables vibrational resolution in UV and IR gas phase spectra of these ions.1 Such detailed spectroscopic fingerprints may serve for stringent validation of 3D molecular structures, calculated by quantum chemistry.2,3  In addition, combination of UV cold-ion spectroscopy (CIS) with high-resolution mass spectroscopy (2D UV-MS) allows for a highly selective identification of isoforms of biomolecules.4

This contribution will present the fundamentals of the 2D UV-MS method and illustrate its application for identification and relative quantification of isomeric peptides, drugs and carbohydrates.

1. Boyarkin, O. V.; Mercier, S. R.; Kamariotis, A.; Rizzo, T. R. J. Am. Chem. Soc. 2006, 128, 2816-2817.
2. Rizzo, T. R.; Stearns, J. A.; Boyarkin, O. V. International Reviews in Physical Chemistry 2009, 28, 481 - 515.
3. Boyarkin, O. V. International Reviews in Physical Chemistry 2018, 37, 559-606.
4. Kopysov, V.; Makarov, A.; Boyarkin, O. V. Anal Chem 2015, 87, 4607-4611.

Oral 2 (Student Talk):
Non-target Approach to Detect Industrial Contaminant Emissions in Treated Wastewater based on High Resolution Mass Spectrometry Time Course Data

Sabine Anliker^1,2, Martin Loos³, Rahel Comte^1,2, Matthias Ruff¹, Kathrin Fenner^1,4, Heinz Singer¹

1. Department of Environmental Chemistry, Eawag, Duebendorf, Switzerland
2. Department of Environmental Systems Science, ETH, Zurich, Switzerland
3. envibee, Zurich, Switzerland
4. Department of Chemistry, University of Zurich, Zurich, Switzerland

Effluent from wastewater treatment plants (WWTPs) is well known to be an important source of organic contaminants to the aquatic environment. Municipal WWTPs often treat both domestic as well as industrial sewage. Compared to the well-studied emissions from domestic wastewater, the current knowledge about the extent of industrial organic contaminant discharges is very limited. This knowledge gap is related to the difficulty to capture emissions from industrial batch processes due to their fluctuating nature and unknown substance composition.

The aim of this project was to propose a non-target approach that enables the efficient detection of discontinuous industrial discharges based on their temporal dynamics in the effluent of municipal WWTPs and to estimate their share on the total emissions. To this end, we collected daily effluent samples during three months at two Swiss WWTPs. After centrifugation samples were analyzed by large volume direct injection reversed-phase liquid chromatography high resolution mass spectrometry (HRMS) in positive and negative electrospray ionization mode. We then generated time series of all signals detected in the MS1 full scan data. After data cleaning and cross-sample componentization (isotopologue and adduct grouping) the resulting time profile inventories consisted of several thousand time series of a priori unknown compounds. Based on temporal patterns observed in the time series of target substances known to be continuously emitted from households, we defined a threshold for intensity variation to differentiate between constant domestic and fluctuating industrial emission profiles.

The percentage of profiles finally classified as potential industrial emissions differed substantially between the two investigated WWTPs, being 3-fold higher for the plant receiving sewage from a pharmaceutical manufacturing site compared to the pure domestic WWTP. Further investigations of time profiles classified as potential industrial emissions using target quantification, suspect screening and non-target structure identification showed that our approach correctly detected true industrial emissions. For example, we found peak concentrations > 10 µg/L for several pharmaceuticals that are usually not present, or at much lower concentrations, in conventional WWTP effluent. Moreover, the identified non-target compounds could clearly be assigned to pharmaceutical production.

Overall, the results demonstrate that systematic non-target time pattern analysis of HRMS time course data is of great value to assess the relevance of discontinuous industrial discharges on the overall contaminant emissions via treated wastewater to surface waters.

Oral 3 (Student Talk):
Systematic Study on Time-dependent Postmortem Redistribution of Morphine and Methadone – Two Analytical Approaches involving LC-MS/MS and GC- HRMS

Lana Brockbals, Sandra N. Staeheli, Thomas Kraemer, Andrea E. Steuer

Department of Forensic Pharmacology and Toxicology, Zurich Institute of Forensic Medicine, University of Zurich, Winterthurerstrasse 190/52, 8057 Zurich, Switzerland

Postmortem redistribution (PMR) describes artefactual increases or decreases in drug concentrations during the postmortem (PM) period that can lead to difficulties in interpretation of PM cases in forensic toxicology. However, only few systematic studies have been carried out so far. Particularly morphine and methadone are forensically relevant compounds due to frequent misuse. Hence, these were assessed for their potential to undergo PMR by use of two analytical approaches; (1) direct targeted measurement of time- dependent PM concentration changes of the drugs and (2) untargeted metabolomics analysis aiming to find endogenous analytes as potential routine indicators for PMR.

For PM cases involving morphine (n=20) and methadone (n=12), femoral blood samples were collected at two time points; at admission of the deceased to the institute (t1) utilizing a CT- guided biopsy tool and manually approximately 24 h later (t2) during the medico-legal autopsy. First, morphine and methadone were quantified using targeted LC-MS/MS (5500 QTrap) analysis. Secondly, samples were analyzed with GC-HRMS (GC Q Exactive; untargeted data acquisition; targeted data processing for 56 endogenous metabolites such as sugars, amino-, fatty- and organic acids). Statistical analysis was carried out to find correlations between time-dependent concentration changes of endogenous compounds and morphine and methadone, respectively.

Morphine and methadone both showed significant concentration increases in femoral blood over time. Spearman’s rank-order correlation analysis found statistically significant (p < 0.05) negative correlations between time-dependent concentrations changes of morphine with creatinine, glutaric acid, hypoxanthine, fructose, pentadecanoic acid, palmitoleic acid and alanine (correlation coefficients: -0.6346, -0.5970, -0.5955, -0.5444, -0.5338, -0.5203 and - 0.4782, respectively). Concentration changes of methadone over time correlated with time- dependent concentration increases of hexadecanoic acid (correlation coefficient: 0.5545).

Both morphine and methadone seem to be prone to PMR, possibly caused by passive diffusion processes e.g. from surrounding muscle tissue. Statistically significant correlations, positive and/or negative, between time-dependent concentration changes of a drug and endogenous metabolites might indicate a parallel respectively an opposite PM behavior in the human body, which could be used as surrogate indicators for routine assessment of PMR.

Oral 4:
Quasi Non-Destructive 3D Chemical Visualization with X-ray Laser Mass Spectrometry

Davide Bleiner,  Matthias Trottmann, Adrian Wichser

Empa – Swiss Federal Laboratories for Materials & Technology, Überlandstrasse 129, CH-8600 Switzerland

Laser Ablation microanalysis has made major progress and improved the quality of the microsampling, and the spatial resolution, such to be able to map the elemental composition of a material in 3D. The ICP source is however limited in the range of elements (no H,C,N,O or S and so on) and provides no molecular information. Further, the spatial resolution is still too coarse for the analysis of thin films, or is significantly damaging the irradiated material.

Therefore, we have tried to solve such shortcomings with the use of ultrashort pulses, with some variable results. An alternative approach, still subject of better fundamental understanding, is the one that implies the use of shorter wavelengths. Since the early works with frequency quadrupled or quintupled Nd:YAG or the excimer lasers, the lack of lasers of even shorter wavelengths did not permit any further advance. Aim of this talk is to present new results related to the use of X-ray lasers and compare the results with state-of-the-art systems. In particular, we will show results related to the nano-scale mapping without elemental or molecular restrictions.

Oral 5 (Student Talk):
SICRIT - A Novel Soft Ionization Method for GC-HRMS Measurement of Alkanes

Markus Weber, Christine Merkert, Jan Christoph Wolf, Christoph Haisch

TU München

Soft Ionization by Chemical Reaction In Transfer SICRIT is a new soft ionization technology based on a dielectric barrier discharge ionization (DBDI). SICRIT merges the atmospheric pressure inlet of LC-MS instruments with a small dielectric barrier discharge based plasma ionization (DBDI) source. This allows for direct coupling of a GC, LC, SPME or Laser-ablation. [1,2,3] Usually the soft ionization leads to solely [M+H]+ peak without fragmentation.

In this study we investigate highly nonpolar compounds as alkanes. The analysis of alkanes is important for the petrochemical industry as well as for environmental protection. Although alkanes have been an analytical target for decades the measurement of complex alkane mixtures is still a challenging problem in analytical chemistry due to the huge variety of isomers. Gas chromatography (GC) hyphenated with flame ionization detection (FID) or electron impact ionization mass spectrometry (EI-MS) became a standard method, but both detectors lack the selectivity to distinguish between overlapping peaks of different alkanes. While FID has no selectivity at all EI-MS usually leads to strong fragmentation of alkanes with very similar fragmentation patterns making differentiation of alkanes with different carbon number nearly impossible. [4]

With soft ionization by SICRIT and in absence of a protonation site alkanes ionize as a specific [M3H+O]+ target-ion. Due to the low fragmentation and the high resolution of an Orbitrap mass spectrometer the separation of overlapping signals in complex mixtures like diesel fuel or crude oil is possible. This allows for fast, qualitative and quantitative analysis of alkane mixtures without additional time consuming and complex sample preparation steps.

1. J.-C.Wolf, M. Schaer, P. Siegenthaler, R. Zenobi Anal. Chem. 2015, 87, 723-729.
2. M.F. Mirabelli, J.-C.Wolf, R. Zenobi Anal. Bioanal. Chem. 2016, 408, 3425-3434.
3. M.F. Mirabelli, J.-C.Wolf, R. Zenobi Anal. Chem. 2016, 88, 7252-7258.
4. M.S. Alam, S. Zeraati-Rezaei, Z. Liang, C. Stark, H. Xu, A. R. MacKenzie, R.M.Harrison, Atmos. Meas. Tech. 2018, 11, 3047-3058.

Oral 6 (Student Talk):
Quantification of Endocannabinoids and Steroid Hormones in Human Hair – LC-MS/MS Method Development and Validation

Clarissa D. Voegel¹, Markus R. Baumgartner¹, Thomas Kraemer², Tina M. Binz¹

1. Center for Forensic Hair Analytics, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland
2. Department of Forensic Pharmacology and Toxicology, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland

Aims: Activation of the hypothalamic-pituitary-adrenal axis (HPA), including the release of glucocorticoids, is the fundamental hormonal response to stress. Endocannabinoids (ECs) play an important role in the physiology and behavioral expression of stress response. For the measurement of chronic stress hair analysis has been found to be the method of choice as it enables a long-term and retrospective determination of endogenous stress markers. The aim of this study was the development of an analytical method for the identification and quantification of endocannabinoids and steroid hormones in hair as a tool for long term stress monitoring.

Methods: Snipped hair samples were extracted in methanol followed by a fully automated supported liquid extraction (SLE) on a Biotage® Extrahera (Uppsala, Sweden). A liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method was developed for the simultaneous identification and quantification of six steroid hormones (cortisone, cortisol, androstenedione, dehydroepiandrosterone, testosterone, progesterone) and five endocannabinoids (anandamide, 2-arachidonylglycerol, oleoylethanolamide, palmitoylethanolamide, stearoylethanolamide). Validation was done with special emphasis on the quantification of endogenous compounds in hair and ways of circumventing the lack of blank hair matrix. Different approaches for determination of endogenous compounds in hair matrix for validation were compared: standard addition, background subtraction, surrogate matrix and surrogate analyte. The following validation parameters were addressed: linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, recovery, and matrix effect.

Results & Discussion: It could be shown that the established workflow using the Biotage® Extrahera was very robust and resulted in much shorter sample preparation time and higher reproducibility. All compounds were separated in a 16 min LC-MS/MS run and showed good selectivity and sensitivity. It could be demonstrated that a combination of background subtraction and surrogate analyte showed the best results and was therefore used for the validation. Overall, we successfully implemented and validated an LC-MS/MS method that enables the quantification of several endocannabinoids and steroid hormones in hair.

Oral 7:
Complementarity of the Different Ionization Techniques in Targeted and Non-Targeted Environmental Analysis by GcxGC-HR-TOFMS

Juergen Wendt

LECO European Application & Technology Center, Max-Dohrn Str.8-10, D-10859 Berlin, Germany

Forensic analysis refers to a detailed investigation for identifying and quantifying the presence of toxins, primarily in the human body. Hyphenated mass spectrometric techniques, particularly GC-MS and LC-MS are indispensable tools in forensic toxicology. In this study, the usage of gas chromatography -high-resolution time-of-flight mass spectrometry (GC-HRT) was used to detect and identify  compounds of forensic relevance. The employed  hardware configuration included additionally a direct inlet probe, allowing an alternative sample introduction without chromatographic separation.
Drug samples were analyzed using a LECO Pegasus GC-HRT system. The hardware configuration also included a direct inlet probe.This setup allowed the analysis of a broad range of forensic samples, either in combination with a previous chromatographic  separation or by means of a direct sample injection into the ion source. Both electron and chemical ionization acquisitions were conducted using a high resolution chemical ionization source. Sample analysis with chromatographic separation allowed targeted and non-targeted compounds to be determined in a single acquisition. An established EI/CI-HRT workflow facilitated the fast and confident characterization of unknowns,such as novel synthetic substances. The Target Analyte Finding feature of the software enabled  a retrospective analysis of targeted drugs. The isotopic pattern of the high resolution mass data could be used  to determine the common origin of different samples.
The direct inlet probe was mainy utilized for rapid tests of solid drugs. The integration of a temperature program for the probe tip led to a kind of pre-separation of the sample components. Also liquids, containing dissolved forensic  substances, could be analyzed with the direct inlet probe. Here the DIP tempeature program was effectively used for solvent evaporation and sample concentration. Target drugs could be identified using the extracted ion chromatogram, deconvoluted EI spectra and empirical formula, derived from the accurate mass of the CI analysis.

Oral 8:
On-line Analysis of Exhaled Breath: Identification of a Large Set of Breath Metabolites Characteristic for Children with Cystic Fibrosis with SESI-HRMS²

Bruderer Tobias^1,2, Weber R¹, Haas N¹, Baghdasaryan A^1,3, Inci D¹, Micic S¹, Perkins N⁴, Bähler P¹, Spinas R¹, Zenobi R³, Moeller A¹

1. Division of Respiratory Medicine and Childhood Research Center, University Children’s Hospital Zurich, Switzerland
2. ETH Zurich, Department of Chemistry and Applied Bioscience, Zurich, Switzerland
3. Joint Medical Center Arabkir, Division of Pulmonology, Yerevan, Armenia
4. Division of Clinical Chemistry and Biochemistry, University Children's Hospital Zurich, Switzerland.

Introduction: Early pulmonary infection and inflammation result in irreversible lung damage and are major contributors to Cystic Fibrosis (CF) related morbidity. An easy to apply and non- invasive assessment for the timely detection of disease-associated complications would be of high value. We aimed to detect disease specific exhaled VOCs in children by real-time secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS).

Methods: A total of 101 children, aged 4-18 years (CF = 52; healthy controls = 49) and comparable for sex, body mass index and lung function were included in this case-control study. On-line breath analysis was done with SESI-HRMS. Carefully pre-processed MS data was used for biomarker detection and classification. 171 m/z features were significantly different in children with CF (FDR adjusted p-value of 0.05). Compound identification was done with SESI-HRMS2.

Results: We were able to annotate 95% of the 171 CF features (m/z features) with a putative molecular formula based on a correlation matrix to assign common adducts, clusters and losses. This was in contrast to our previous CF study where only 37% of 49 features could be assigned with an unambiguous formula which was probably most likely due to the formation of non-generic fragments with the previous SESI-MS instrumentation. Our previously reported identification workflow based on exhaled breath condensates showed limited use for the present study. Therefore, we present first results from an on-line compound identification approach with a pre-separation step. We could identify molecules from several metabolic classes including highly oxidized compounds, long-chain fatty acids and their oxidation products. We are currently investigating the involved metabolic pathways in more detail.

Conclusions: We have detected a large set of exhaled molecules characteristic for CF from several metabolic classes including highly oxidized compounds, fatty acids and their oxidation products.

Oral 9:
Does Ion mobility-HRMS based Screening of Residues in Complex Matrices Permit an Unambiguous Identification of Targeted Analytes?

Anton Kaufmann

Kantonales Labor Zürich, Switzerland

The sensitivity of mass spectrometry based detection has improved tremendously over the last decade. Yet, such increased sensitivity is only beneficial if the selectivity of detection is sufficient for a given sample matrix, to prevent the reporting of false positive findings. Hence, additional confirmation criteria (e.g. ion mobility) have been proposed for the unequivocal identification of trace level compounds in very complex matrices.

The value of currently commercially available ion mobility technology for identification has been investigated by the use of a LC-IMS-Q-TOF instrument. It was shown that the HRMS full scan based detection (veterinary drugs in bovine liver extract) can produce a number of false detects, even when employing a narrow mass deviation window of 5 ppm. The number of these false detects could be reduced by a factor of 10 when employing, in addition, a 2 % drift time (collision cross section) filter. Yet, isobaric interferences can lead to false negative findings. Such effects were provoked by the injection and chromatography of a low concentration veterinary drug standard mix. In addition, a bovine liver extract was constantly post column infused to simulate the presence of potentially isobaric interferences. As a consequence, a number of analytes became undetectable (false negatives) when employing a mass deviation of 5 ppm and 2 % drift time. This is not due to inaccuracies in mass accuracy or CCS measurements, but caused by isobaric interferences. Currently available traveling wave ion mobility separations provide ion mobility peaks which are significantly wider than the proposed 2 % error margin. In other words, although the CCS difference between the targeted analyte and a co-eluting compound is significantly larger than 2 %, the travelling wave cell may be unable to physically resolve these two compounds. As a consequence, the detected peak (aggregation of targeted analyte and interfering compound) shows a shifted drift time which may be located well beyond the proposed 2 % cut-off level. Unfortunately, this problem is compounded by the fact that m/z and CCS are not orthogonal but rather highly correlated. In other words, if the mass spectrometer encounters an unresolvable, or only partially resolvable isobaric ion pair, the ion mobility drift tube may likely encounter a similar situation.    

It is a conclusion that IMS provides additional identification information. Yet, the high correlation between the m/z and the CCS in combination with the currently limited resolving power of travelling wave & drift tube IMS does not yet lead to a significant improvement of detection selectivity of trace level compounds (small molecules) in complex matrices. IMS would truly be beneficial if the currently available resolving power (e.g. 40) could be improved to a value clearly above 100.

Oral 10:
An Investigation into the Use of Cyclic Ion Mobility for the Separation of Biopharmaceutical Peptide and Protein Modifications

Jim Langridge, Henry Shion, Martin Palmer, Weibin chen, Dale A Cooper-Shepherd

Waters Corporation

Introduction: As biotherapeutics become more complex and companies strive for increased intellectual property protection, ever more sophisticated tools are being investigated to provide in-depth detailed molecular characterisation. Such studies focus on acquiring knowledge of the post-translational modifications (PTM’s) including glycosylation, oxidation and deamidation present in the protein product, with control of these being paramount. Mass spectrometry (MS) is a central technique in biopharmaceutical characterization due to its ability to report on such a wide range of attributes. However, the presence of isobaric PTM’s with differing biological properties can often be refractory to traditional LC-MS workflows even when chromatographically separated. In this work we investigate cyclic ion mobility technology as a means to distinguish isomeric PTM’s to improve biotherapeutic characterisation


Methods:  Studies were performed on a cyclic ion mobility-enabled quadrupole time-of-flight (Q-cIM-oaToF) mass spectrometer. The circular geometry of the cIM device minimizes instrument footprint whilst providing a longer separation path (1m), Ions can cycle the path length multi-times, provides significantly higher resolution over a selected mobility range.. The multifunctional T-WAVE ion entry/exit array allows mobility selectivity by ejecting species within a range of mobilities,to a pre-store array, enabling multiple stages of ion mobility selection and separation (IMSn). The native and iso-aspartic variants of the synthetic peptide WGGDGFYAMDYWGQGTLVTVSSASTK (T12-D and T12-isoD, respectively) were used in this study to mimic deamidation products from protein biotherapeutics. The peptides were infused under nano-electrospray conditions in 97:2.9:0.1 Water/Acetonitrile/Formic acid at a concentration of 1 micromolar.


Preliminary Data:The two isomeric peptides T12-D and T12-isoD formed primarily the [M+3H]3+ ion at m/z 928. After a single pass of the cIM device it was observed from its arrival time distribution (ATD) that T12-D formed two conformers, one compact (1) and one more extended (2) with approximately 90 % of the signal present as conformer 1. By comparison T12-isoD was found to populate only conformer 2. These data suggest that the presence of the isoD variant biases the conformation of the T12 peptide to the more extended form under these conditions.
The T12 peptide was selected by the resolving quadrupole and subjected to fragmentation in the trap ion guide situated prior to the cIM device. This enables separation and structural analysis of the resulting product ions by ion mobility followed by mass analysis. In order to probe the product ions in detail we subjected the peptides to both multi-pass and IMSnstudies. Peptide product ions were initially separated over a single cIM pass (resolution approx. 65). A defined mobility range containing the product ions of interest was then selected by ejection to the pre-cIM storage ion guide, with all other ions outside of this range discarded. The stored ions were then reinjected into the cIM device for multiple passes to acquire at a higher mobility resolution. Upon comparing T12-D and T12-isoD product ions, several b-type ions showed differences in their ATD, indicating different gas phase structures. When conducting the experiments with an equimolar mixture of T12-D and T12-isoD the product ion ATD indicated, and was consistent with, a mixture of the two forms. These preliminary data indicate that multifunction cIM could be used to identify and aid in sequencing peptide isomers. Further data will be collected on a range of additional modified peptides with on-line LC separation for chromatographic separation.

Posters

Poster 1:
High-throughput structure elucidation of ozonation transformation products in wastewater with high-resolution mass spectrometry and in silico fragmentation

Jennifer E. Schollée, R. Gulde, U. von Gunten, C. S. McArdell

Eawag, Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse 133, 8600 Dübendorf
School of Architecture, Civil and Environmental Engineering (ENAC), Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne

Contemporary wastewater treatment plants (WWTPs) are not optimized for the removal of trace organic compounds (TrOCs) originating from human activities, e.g., pharmaceuticals, biocides, industrial chemicals, etc. To reduce TrOC load to Swiss surface waters, a Water Protection Act was implemented in 2016 and stipulates that ca. 15% of Swiss WWTPs will be upgraded with ozonation or activated carbon treatment. Ozonation is effective in abating many TrOCs; however, rather than being mineralized, generally ozonation transformation products (OTPs) are formed1. The formation and fate of these OTPs is not well understood and many remain unknown.

Liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS2) is used for detection of TrOCs because it is sensitive, selective, enables simultaneous detection of many TrOCs, and a priori analyte selection is not required, so users can retroactively screen for unknowns, e.g., OTPs. Due to the large amount of data obtained with HRMS2, structure elucidation is often automated, e.g., with in silico fragmentation. However, automated structure elucidation of OTPs has been limited because in silico fragmenters rely on structures to be present in chemical repositories, which is not the case for many OTPs. In this study, a new software for the prediction of OTPs was used to screen for OTPs in wastewater treated with ozone. Thereafter, these predicted OTPs were included in automated structure elucidation, substantially increasing the chemical space for in silico fragmentation.

Samples were collected from a WWTP after biological treatment and ozonated in the laboratory (0.8 gO3/gDOC). One set of triplicate samples were spiked with 50 wastewater-relevant TrOCs (each 10 µg/L), while one set of triplicates was unspiked; all samples were analyzed with LC-HRMS2 and processed with a non-target screening workflow2. With a prediction system (O3-PPD)3, plausible OTPs were calculated for 85 known TrOCs, yielding over 70,000 possible structures.

Between 2000-4000 features were detected after ozonation, compared to 5000-7000 before ozonation. Features were compared to the predicted OTPs and matches were selected based on exact mass and predicted retention time, resulting in >20,000 hits. Theoretical HRMS2 generated in silico from predicted OTP structures were then compared to measured HRMS2 to assess likelihood of a correct assignment.

In total 1516 potential OTPs with measured HRMS2 were evaluated. For 71 suspects (4.7%), the proposed OTP had the highest fragmentor score, outranking all candidates retrieved from the chemical repository. These 71 candidates originated from 24 parent compounds and included known OTPs such as N-oxides, as well as OTPs not previously reported, though structural confirmation will be necessary. This workflow represents a large step forward in the automated processing of LC-HRMS2 data for the identification of OTPs.

1. von Sonntag, C. and von Gunten, U. (2012) Chemistry of Ozone in Water and Wastewater Treatment: From Basic Principles to Applications, IWA Pub.
2. Schollée, J.E., Schymanski E.L., Avak, S.E., Loos, M. and Hollender, J. (2015). Prioritizing Unknown Transformation Products from Biologically-Treated Wastewater Using High-Resolution Mass Spectrometry, Multivariate Statistics, and Metabolic Logic. Analytical Chemistry 87(24): 12121-12129.
3. Lee, M., Blum, L.C., Schmid, E., Fenner, K. and von Gunten, U. (2017) A computer-based prediction platform for the reaction of ozone with organic compounds in aqueous solution: Kinetics and mechanisms. Environmental Science: Processes & Impacts 19(3), 465-476.

Poster 2:
The Benefit of CCS Value Prediction for DB Searches in Metabolomics, Lipidomics and Proteomics

Heiko Neuweger, Sven Meyer, Aiko Barsch, Nikolas Kessler, Heiner Koch, Favio Salinas*, Jürgen Cox*, Arnd Ingendoh

Bruker Daltonik GmbH, Fahrenheitstr.4, D-28355 Bremen
*MPI of Biochemistry, Martinsried, Germany

Trapped ion mobility (TIMS) MS offer new options for higher confidence in identifying compounds in complex samples. First, LC co-eluting compounds can be separated by the additional IMS which results in cleaner MS/MS spectra and improved DB search results. Second, TIMS enables the highly reproducible determination of collisional cross sections (CCS) of ions with ≤ 0.2% accuracy. These experimental CCS values provide increased confidence in compound identification if compared to predicted values generated by machine learning (ML) algorithms.
We developed a CCS value prediction algorithm which was evaluated against literature values as well as rich dataset comprising several hundreds of compounds. The usefulness of these high numbers of predicted CCS values for DB searches was checked for complex metabolomes, proteomes and lipidomes.

Lipid standards and from a milk extract, metabolic hop extracts and tryptic HeLa digests were investigated by the “4D LC-TIMS-QTOF” workflow, where aside of the LC retention time, the accurate mass, isotope pattern, MS/MS fragments and the CCS values were used for confident compound identification from DB search.
An Elute UHPLC or nanoElute was coupled to a timsTOFpro (all Bruker).  Post-processing was done by MaxQuant (v1.6.4.0) with a data set of >80.000 unmodified peptides, MetaboScape 4 (Bruker) using the T-ReX algorithm and by LipidBlast. For CCS prediction several deep learning models were applied, like LSTM (MPI) and a branched CNN (Bruker).

CCS values clearly increased the confidence in lipid annotation as orthogonal information. MetaboScape provided a fully integrated solution for confident lipid annotation enabled by 4D Lipidomics.
Possible applications of CCS value prediction of peptides are multifold. E.g., the dynamic range of Host Cell Protein (HCP) analysis in biopharma development could be extended beyond the sensitivity for MS/MS based peptide identification. In many cases, identification was feasible by high mass accuracy and CCS value determination alone without applying MS/MS. This approach could also be applied to the identification of low abundant, non-tryptic or singly charged peptides.

Poster 3:
Thermal Denaturation of Multi-domain DNA Complexes Using Temperature-Controlled nanoESI-MS

Adam Pruška, Adrien Marchand, Renato Zenobi

Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093 Zürich, Switzerland

In addition to the classical double helix structure, oligonucleotide sequences can form many secondary and tertiary structures such as G-quadruplexes, multi-way junctions, and more. Whereas these individual structures are often structurally and thermodynamically characterized using standard biophysical methods, more relevant structures made of multiple domains remain substantially challenging to study. In particular, understanding the mutual effect of adjacent domains is complicated, due to the presence of coexisting intermediates consisting of partially folded complexes. For example, G-quadruplexes as non-canonical guanine-rich DNA sequences are important as gene promoters and telomeres, where they are adjacent to duplexes or other G-quadruplexes. Also, branching of nucleic acids occurring during DNA replication, can result in the presence of three or more overlapping strands that are partially complementary to each other, constituting multiple interlocked domains.

Here, we used native mass spectrometry to detect coexisting species in samples of model DNA complexes made of two or three domains. Specifically, a formed duplex (fD) is easily distinguished from its constituting single strands and, the G-quadruplexes (fG) are revealed by the presence of two potassium cations thanks to their mass difference. The abundances of the coexisting species were recorded as a function of temperature using a laboratory-built temperature-controlled nanoelectrospray ionization (TCnESI) source.

The MS melting experiments of fD-fG complexes were investigated to characterize stabilities of several duplexes adjacent to fG on both the 5’ and 3’ end of the sequence. Second, we studied a complex made of two fGs within the same strand. The result indicates the presence of a fifth bound K+, suggesting that the fGs are stacked on top of each other. As another example, the fG-fD-fG complex exhibited an interesting phenomena: in the absence of K+, no folded duplex was observed. The presence of two fGs induced the formation of the central duplex.

The MS thermal denaturation method allows the importance of characterizing multiple domain structures and provides novel information of DNA domains unfolding and their thermodynamics. This gives access to comprehensive information about their thermodynamics.

Poster 4:
Improved Middle-Down Characterization of Antibodies Using Multiple Ion Activation Techniques and PTCR

Guido Sonsmann

Thermo Fisher

See more with less - PTCR a new tool for intact protein characterization

PTCR (Proton Transfer Charge Reduction) is a Fragmentation mechanism, which allows the manipulation of multiply charged ion species. Reagent ions reduce the charge status exploring and enhancing the characterization of intact proteins. Various application examples will be presented for top-down sequencing in native MS.

Poster 5:
Determination of the stereoisomeric distribution of d- and l-methamphetamine in the context of "not inconsiderable quantities"

Tim J. Gelmi, Marlies Verrijken, Wolfgang Weinmann

Institute of Forensic Medicine, Forensic Toxicology and Chemistry, Bühlstrasse 20, CH-3012 Bern

Amphetamine-type stimulants (ATS) comprise a class of synthetic drugs, which are structurally and functionally close to endogenous amines. The leading compounds of these drugs are amphetamine and methamphetamine, which both occur in two stereoisomeric forms, the more potent d-stereoisomer and the l-stereoisomer.

In Germany, there recently has been an amendment to the Annexes to the Narcotics Law regarding the "not inconsiderable quantities". Based on pharmacological-toxicological findings, the following limit values were set: 10 g for the racemic mixture of d/l-methamphetamine, 5 g for the pure d-stereoisomer and 50 g for the pure l-stereoisomer. In Switzerland, on the other hand, this distinction is not practiced and there is only one limit value of 12 g. To examine whether this single value is still contemporary, we have analyzed methamphetamine pills seized in the canton of Berne and surrounding cantons from the years 2000, 2001, 2007 and 2017.

The stereoisomeric distribution of the pills was analyzed by HPLC-MS/MS and GC-MS. Both methods demonstrated comparable results. They indicated that the pills mainly consisted of d-methamphetamine, some seizures even being pure and only containing the more potent stereoisomer. Others showed racemic mixtures of d/l-methamphetamine, but rarely in 1:1 ratios. The concentration of the more potent d-methamphetamine most often exceeded that of the l-stereoisomer. There even was a case, where mainly l-methamphetamine was detected, but only very little of the d-stereoisomer.

In conclusion, the analyses revealed that the single limit value of 12 g for a "not inconsiderable quantity" of methamphetamine is probably not up to date anymore and should be revised. Most of the seized pills showed a much higher concentration of the more potent d-methamphetamine and were either present pure or in a non-equimolar ratio. The risk of taking such a pill is much higher and therefore the limit value should also be lowered and brought into line with European/German standards.

Poster 6:
Combining LC-MS and visual proteomics for single cell analysis

Christian Berchtold¹, Luca Rima², Stefan Arnold², Götz Schlotterbeck¹, Thomas Braun²

1. FHNW, Institute for Chemistry and Bioanalytics, Hofackerstrasse 30, CH-4132 Muttenz, Switzerland
2. C-CINA, Biozentrum, University of Basel, Mattenstrasse 26 4058 Basel, Switzerland

Usually, several analysis techniques are combined to study cellular processes. Metabolomics and proteomics have a tremendous potential revealing the mechanism of biological processes, especially if they are correlatively combined on a single-cell level. However, due to the low amount of molecules within one cell (proteins and metabolites), this appears to be very challenging due to sensitivity of the applied analysis methods.

We present a proof of concept for the combination of the metabolomic and proteomic analysis of individual cells. We combined a light microscope with a single-cell picker [1-3] for the lysis of adherent eukaryotic cells. Subsequently, the cell lysate was placed on microscope slides and extracted into an LC-System using a modified CAMAG TLC-MS interface for metabolite analysis. Proteins remained on the microscope slide and were detected by antibody staining using a fluorescent microscope after LC-MS analysis. Glutamic acid, glutamine, and dopamine were detected qualitatively from single cells. Proteins such as actin were identified in the remaining lysate of the same cell using the reverse phase protein array approach.

We demonstrate a strategy to merge proteomics and targeted LC-MS metabolomics to detect metabolites and proteins from the same sample, or single-cell, respectively. However, the concept needs further optimization regarding sensitivity, robustness, and speed for quantitative high-throughput measurements.

1. S. Kemmerling, S. A. Arnold, B. A. Bircher, N. Sauter, C. Escobedo, G. Dernick, A. Hierlemann, H. Stahlberg, and T. Braun. J. Struct. Biol. 183 (2013), 467–73
2. C. Schmidli, L. Rima, A. Arnold, T. Stohler, A. Syntychaki, A. Bieri, S. Albiez, K.N. Goldie, M. Chami, H. Stahlberg, T. Braun JoVE 137 (2018), e57310
3. A. Syntychaki, L. Rima, C. Schmidli, T. Stohler, A. Bieri, R. Sütterlin, H. Stahlberg, and T. Braun. J. Proteome Res. (2019) (in press)

Poster 7:
Development of a Novel LC-MS/MS Method to Determine 7α-hydroxy-4-cholesten-3-one (C4), a Bile Acid Synthesis Biomarker in Human Serum

Anita O. Ducati, Matthias Sury, Petra Struwe

Celerion Switzerland AG, Allmendstrasse 32, 8320 Fehraltorf, Switzerland

Bile acid biosynthesis is a catabolic pathway of cholesterol taking place in the liver and there it affects various physiological processes, such as lipid and glucose metabolism and nutrient absorption through the intestines. Due to its occurrence in the liver and role in lipid homeostasis, the dysregulation of this pathway may lead to non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). The imbalance of bile acid synthesis was also connected to other diseases, such as irritable bowel syndrome (IBS), Crohn’s disease or insulin resistance (IR) and type 2 diabetes. 7α-hydroxy-4-cholesten- 3-one (C4) is an intermediate metabolite of the classic bile acid biosynthesis pathway and its level characterizes the activity of the cytochrome P450 7A1 enzyme (CYP7A1), the key enzyme in the rate- limiting step of bile acid biosynthesis. Therefore, C4 is an important biomarker of conditions with pathological bile acid synthesis.

We developed an LC-MS/MS method that can measure C4 levels in human serum in the range of 0.200- 200 ng/mL. An acid-assisted protein precipitation with a solid phase extraction clean-up step was applied. C4-d7 was used as an internal standard. The extracted samples were analyzed by using a Waters Acquity i-Class UPLC system coupled to a 5500 QTRAP (SCIEX) operated in ESI positive mode. As C4 is an endogenous metabolite, a surrogate matrix was used to prepare calibrators (STDs) and quality control samples (QCs) at the lower end of the dynamic range. We tested three potential surrogate matrices: acetonitrile:water 50:50 (v/v), 1 % bovine serum albumin (BSA) in phosphate- buffered saline (PBS) solution and a protein-free synthetic serum (SeraSub, CST Technologies). Interestingly, the slope of standard curves in the three surrogate matrices were varying compared to the standard curve prepared in a human serum pool by standard addition.

The developed method can quantitatively measure C4 in the range of 0.200-200 ng/mL, which is sufficient to measure C4 levels in diseases with impaired bile acid synthesis. During method development, we showed the importance to examine and compare the standard curves of surrogate and actual matrices. A not adequate choice of surrogate matrix can lead to the over- or underestimation of sample concentrations in a biomarker assay.

Poster 8:
Detection of oxidative hair treatment using an untargeted metabolomics approach

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

Aims Hair analysis has become an important tool in forensic toxicology to retrospectively monitor consumption behavior and assess drug or alcohol abstinence. However, cosmetic hair treatments (e.g. bleaching with hydrogen peroxide (H2O2)) are known to significantly decrease drug concentrations, possibly leading to false negative results. A marker for oxidative hair treatments, at best detectable within routine analyses, would therefore be desirable. For this, an untargeted metabolomics approach should be applied to identify endogenous markers to objectively differentiate cosmetically untreated from treated hair samples.

Methods Cosmetically untreated hair samples (n = 21) were either left untreated or bleached in vitro for 30 min with a mixture of bleaching oxidase (containing 9 % H2O2) and bleaching oxidation powder. Subsequently, hair samples were analyzed by liquid chromatography coupled to high-resolution (HR) time of flight (TOF) mass spectrometry (MS) (Sciex 6600 TripleTOF). (Tentative) identification of metabolites was performed using Progenesis QI software (Waters) and PeakView (Sciex). To study the applicability of the identified markers, in vitro experiments with different H2O2 concentrations (1.9 % up to 12 %) and during varying incubation times (15 – 60 min) were performed. To confirm the in vitro results, authentic hair samples with obvious hair treatment were additionally studied for the identified markers. Results and discussion Applying our untargeted metabolomics workflow, three metabolites showed significantly higher signals after bleaching and were identified as the eumelanin degradation products 1H-pyrrole-2,3,5-tricarboxyllic acid (PTCA), 1H-pyrrole-2,3,4- tricarboxylic acid (isoPTCA) and 1H-pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA). PTeCA was found to be formed exclusively through the oxidation process already using low H2O2 concentrations (1.9 %) and short incubation times (15 min), thus omitting the implementation of complex cut-off values for routine applications.

Conclusion The results of this study show that the application of an untargeted metabolomics workflow was successful to identify markers for oxidative hair treatments and serves as a proof-of-concept for the developed untargeted metabolomics workflow. As PTeCA was only detectable in treated hair samples, its detection in routine samples can be classified as highly indicative for hair manipulation through oxidative hair treatments.

Poster 9:
Application of metabolomics and molecular networking in investigating the defense-related compounds of Citrus sinensis in response to Xanthomonas citri infection

Luiz Leonardo Saldanha^1,2, Pierre-Marie Allard², Santiago Codesido Sanchez², Emerson Ferreira Queiroz², Jean-Luc Wolfender², Henrique Ferreira¹

1. São Paulo State University (UNESP), Rio Claro, São Paulo, Brazil
2. University of Geneva, Geneva, Switzerland

Xanthomonas is a genus of phytopathogenic bacteria affecting more than 200 plant families. Citrus canker is caused by Xanthomonas citri subsp. citri (strain 306) and is one of the most aggressive diseases that affect citrus crops economically important worldwide. It may cause premature fruit drop and defoliation when plants are severally affected, resulting in significant losses in yield and fruit quality.

To better understand Citrus sinensis L. Osb. ("sweet- orange") responses to X. citri infection, extracts of infected and non-infected plants harvested at 0, 6, 12, 24 and 48 hours post infection (h.p.i.) from local and distal leaves, were analyzed by UHPLC-HRMS2. After appropriate treatment, (+)-LC-MS data were submitted to multivariate data and molecular networking (MN) analyses. Metabolome differences between infected and non-infected plants were reflected in principal component analysis (PCA) plots and the discriminants loadings were filtered in orthogonal projections to latent structures discriminant analysis (OPLS-DA). When the sample clusters were in the two-dimensional space, principal component 1 (PC1 52%) and PC2 (13%), the samples were classified into two groups, where 24 and 48 h.p.i. local infected leaves were clustered together in one group, and all other samples formed another cluster. MSMS data were organized as a single MN and were searched against GNPS libraries and in-silico fragmentation database, which allowed the dereplication of 2291 substances (57,8% of total features). The MN-based dereplication lead to the identification of alkaloids, flavonoids, sesquiterpenoids among other compounds. Altogether, the multivariate and MN analyses revealed a series of tryptamines derivatives which were shown to be produced by C. sinensis in response to X. citri inoculation at 24 and 48 h.p.i. in local leaves. These preliminary results indicate that C. sinensis trigger defense substances biosynthesis after 1 day of X. citri inoculation. However, systemic response may be latter triggered.

Poster 10:
FTMS Data Simulator: Apples-to-Apples Comparison of Experimental and Calculated Orbitrap Mass Spectra

Konstantin O. Nagornov¹, Natalia Gasilova², Anton N. Kozhinov¹, Laure Menin², Yury O. Tsybin¹

1. Spectroswiss Sàrl, EPFL Innovation Park, 1015 Lausanne, Switzerland
2. Ecole Polytechnique Federale de Lausanne, 1015 Lausanne, Switzerland

Recent progress in calculating profiles of isotopic distributions for mass spectra analysis has been mainly in the efficiency of the computational algorithms. Modern (Swiss-made) isotopic envelope simulators, such as www.cheminfo.org and www.envipat.eawag.ch, are rapid tools with full on-line functionality. However, when applied to FTMS data analysis, they do not consider the FT processing-related specificity. Thus, they may not account for the FT-related artifacts, reducing the accuracy of matching calculated and experimental results.

Here, we will describe implementation of the FTMS Isotopic Data Simulator and benchmark it for FTMS applications. This tool simulates isotopic envelopes and mass spectra by applying similar FT processing parameters to both theoretical and experimental FTMS time-domain data (transients).

We will demonstrate the benefits of accurate simulation of FTMS mass spectra for the following: (i) rationalizing the experimental results; (ii) searching the experimental data using similarity score for ranking the results, for example for LC-MS analysis with Orbitrap FTMS; (iii) designing FTMS experiments by selecting appropriate resolution settings and FT processing parameters; (iv) teaching the FTMS subject by demonstrating both transient and corresponding mass spectra, containing typical FT artifacts; and (v) understanding interference of peak shapes in FTMS mass spectra.

Poster 11:
Thermal Denaturation of RNAse A Bound to a Library of DNA/RNA Ligands: Attainment of Binding Coefficients and Thermodynamic Analysis of Multiple Stable Intermediates via Mass Spectrometry Coupled to a Temperature Controlled Source

Irina Oganesyan, Adrien Marchand, and Renato Zenobi

Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland

Introduction: Obtaining the proper 3D structure for a protein is crucial since it determines its biological function. With an increasing interest in diseases induced by improper protein folding like Alzheimer’s and Parkinson’s, understanding the mechanisms of a protein’s folding-unfolding pathway, as well as monitoring the differences in these pathways during protein-ligand interactions can help to elucidate thermodynamic characteristics of binding enthalpies (∆H°) and entropies(∆S°) for multiple structure stoichiometries in a mixture. In this research, RNAse A bound to a library of DNA and RNA nucleotides was thermally unfolded using a temperature-controlled source native ESI (TC nESI). The resulting stoichiometries of intermediate species during unfolding and unbinding process was monitored and analyzed by mass spectrometry (MS). Upon this experimental setup, the dissociating constants (Kd) of various ligands were obtained enabling the ranking of ligands by their bimolecular interaction strengths. In addition, calculated Gibbs free energy (∆G ) of folding and binding events at 37 °C indicated the most stable conformations of bound/unbound RNAse A.

Methods: The temperature-controlled source was built in-house at the ETH workshop. It consists of a copper block with the emitter embedded inside where the analyte is held at the derived temperature. Cooling or heating of the block is controlled by a Peltier element (Adaptive ET-127-10-13-RS, 15.7 V, 37.9 W), and directed by LabView software (National Instruments). The information of the current temperature comes from a thermistor sensor (5000 Ω, ON-950-44005 Omega). The temperature for all experiments was ramped from 25 °C to 85 °C, 2 °C per minute. MS measurements were made on a Synapt G2-S (Waters, MA) using soft conditions: ES+, 1.3 kV (capillary voltage), trap and collision energy set to 2.0.

Conclusions: Thermal denaturation of RNAse A with five DNA/RNA nucleotides by TC nESI allowed for a fairly high throughput method of ranking the more prominent binding partners. According to preliminary data, 5’GMP and 5’UMP are more protuberant binders with higher melting temperatures of these complexes which contradicts the literature reports for pyrimidine bases being the more favorable ligands to RNAse A.

Poster 12:
Identification of isomeric carbohydrates by interplay of hydrogen bonds with aromatic compounds.

Erik Saparbayev¹, Vladimir Kopysov¹, Viktoriia Aladinskaia¹, Ruslan Yamaletdinov^2,3, Aleksandr Pereverzev¹, and Oleg V. Boyarkin¹

1. Laboratoire de Chimie Physique Moléculaire, Ecole Polytechnique Fédérale de Lausanne, Station 6, CH-1015 Lausanne, Switzerland.
2. Nikolaev Institute of Inorganic Chemistry, Novosibirsk, 630090, Russian Federation.
3. Novosibirsk State University, 630090, Russian Federation.

Carbohydrates play an important role in biological processes, because, as a result of posttranslational modifications, about half of all proteins in living organisms contain oligosaccharide chains. Regarding an isomeric diversity of carbohydrates there is currently no single comprehensive analytical technique for their qualitative and quantitative analysis.

In this work we propose and explore a use of non-covalent carbohydrate-aromatic complexes for identification of isomeric glycans by developed in our group unique method of 2D UV-MS cold ion spectroscopy. These complexes were formed in solution and then isolated and cooled in the gas phase. The experimental and theoretical investigations show that an interplay of different types of inter- and intramolecular hydrogen bonds (CH-π, OH-π, cation-π etc.) in such complexes makes UV absorption of the aromatic ion highly sensitive to the small structural differences between isomeric carbohydrates, including enantiomers. These findings enable the identification and relative quantification of any isoforms of carbohydrates in their mixtures.

Poster 13:
Determination of Time since Deposition (TsD) of biological crime scene traces by mass-spectrometry based proteomics

Tom Dario Schneider¹, Jonas Grossmann², Bernd Roschitzki², Thomas Kraemer¹, and Andrea Eva Steuer¹

1. Zurich Institute of Forensic Medicine (ZIFM), University of Zurich, Switzerland
2. Functional Genomics Centre Zurich (FGCZ), University of Zurich/ETH Zurich, Switzerland

Introduction / Background: By comparing the individual-specific DNA profiles, a piece of evidence can be assigned to a person without any doubt. Evidence should not only be associated with a person, but also with the crime itself, e.g. by determining the type of evidence and the age of the evidence. To date, there is no reliable method for determining the age of a trace. The aim of this study was to investigate time-dependent changes in bloodstains by analyzing the proteome of a sample and to then draw conclusions about the age of biological traces

Methods: Blood samples were applied to protein saver cards and by drying them at ambient conditions. The dried blood spots were then left to age under controlled and environmental conditions. Proteins were extracted from fresh and aged blood spots with ammonium bicarbonate. The samples were vortexed, sonicated and subsequently centrifuged. For each sample, 50 µg of protein were reduced, alkylated and proteolytically digested with trypsin. After sample clean-up using C18 micro-columns, the peptides were dried and stored at -20° C. Prior to analysis, the dried peptides were resolubilized and then analyzed with nano-High-Performance Liquid Chromatography and Fourier-Transformed Tandem Mass-Spectrometry (FT-MS/MS, Thermo Scientific Orbitrap Fusion Lumos) in data-independent acquisition (DIA) mode.
Progenesis QI for Proteomics and Spectronaut were used to get quantitative profiles for individual proteins and peptides for each sample. The quantitative changes over time were evaluated with a regression model on both peptide and protein level. The resulting log-ratios were plotted for each time-point and used to create a linear regression model for TsD-estimation.

Results: Contamination with other organisms could be detected by the presence of proteins and peptides of microbial origin. Different proteins showed varying degrees of time- and storage-dependent changes. At the peptide level, typical time-dependent changes were found in the altered abundances of peptides, semitryptic peptides and post-translational modifications (PTMs). Regression-modelling could be applied to time-dependent changes of peptides/PTMs and to estimate the TsD.

Discussion: The presented (initial) results are promising in terms of feasibility for the determination of the Time since Deposition of biological crime scene traces. The proteome of a biological sample shows complex dynamics over time, especially when exposed to environmental conditions. Aged traces provide numerous possibilities (and challenges) to tackle the question of TsD-determination.

Conclusion: These initial investigations show that the analysis of the proteome at both peptide and PTM-level seems to be a suitable technique to understand and elucidate time-dependent changes of a body-fluid derived trace, such as blood. Further investigations will be necessary to show robustness of the analytical technique as well as to improve the quality of the model used for the determination of Time since Deposition. Dynamic environmental conditions must be considered when research is conducted to understand the Time since Deposition, since they will interfere with regression-modelling and may require to change the estimation approach – or at least the group of analytes considered for modelling. Other forensically relevant biological matrices (saliva, semen, vaginal secretion and menstrual blood) need to be investigated, too.

Poster 14:
Asthma biomarker discovery in exhaled breath by secondary electrospray ionization mass spectrometry

Streckenbach B.^1,2, Weber R.³, Micic S.³, Bruderer T.^1,2,3, Inchi D.³, Kohler M.^2,4, Zenobi R.^1,2, Moeller A.^2,3

1. Department of Chemistry and Applied Biosciences, ETH Zurich
2. Zurich Exhalomics Flagship Project, Zurich
3. Division of Respiratory Medicine, University Children’s Hospital Zurich
4. Department of Pulmonology, University Hospital Zurich

Despite the fact of asthma being one of the most prevalent chronic airway diseases in children, correct diagnosis and thus early treatment are hindered on several levels. First, it is challenging to distinguish respiratory symptoms that are associated with asthma from those of other respiratory tract infections. Furthermore, the globally agreed approach for asthma diagnosis is mainly based on spirometry, which requires a forced exhalation procedure. Unfortunately, this cannot be performed with children under the age of about five years, resulting in an age limit for asthma diagnosis.

We propose that detecting exhaled metabolites by secondary electrospray ionization mass spectrometry (SESI-MS) could serve as an alternative diagnostic tool. This method allows non-invasive, rapid and highly sensitive analysis of small molecules in exhaled breath without the need of forced exhalations, which could therefore even be performed by preschool-aged children.

We are aiming at screening for a discriminative asthma-specific breath pattern by identifying a potential set of (semi-)volatile biomarkers using SESI-MS. For this purpose, an observational case control study is performed on a study population consisting of 30 children with asthma and 30 healthy subjects. Study related data of participants is collected by SESI-MS measurements together with current standard tests for diagnostic discrimination (spirometry, quantification of fractional exhaled nitric oxide (FeNO), throat swab and allergy testing).

With the help of such an asthma-specific breath pattern, the diagnosis of asthma could not only be strongly improved but also be conducted at preschool age, thus further improving disease management.

Poster 15:
A ‘Smart’ and Compact TOF Mass Spectrometer - Designed for Manufacturability with State-of-the-Art Technology

D. Lasi, L. Hofer, A. Péteut, M. Gruber, S. Gasc, and J. Jost

Spacetek Technology AG, Brüggliweg 18, 3073 Gümligen, Switzerland

We present a new compact time-of-flight (TOF) mass spectrometer designed for manufacturability and mass production, using state-of-the-art electronics and software technologies, all making it a competitive alternative to a quadrupole instrument where speed and sensitivity matter.

This new device is based on orthogonal-extraction reflectron TOF architecture (sensor: ~300mm long) and operates at up to 20 kHz, for a complete mass spectrum up to every 0.1 s. Early tests showed 900 M/ΔM (at 44u, CO₂) mass resolution and 105 dynamic range. Among others, this instrument includes an adapter that allows to combine a commercial quadrupole ion source with the ion source of the TOF mass analyzer, thus allowing for a seamless replacement of an existing slow quadrupole filter with a faster and more sensitive TOF sensor.

The instrument combines selected ideas from space exploration with state-of-the-art technologies. For example, it combines particle swarm optimization algorithms originally developed for the instrument onboard the ESA Rosetta cometary mission (Bieler et al., 2011) with a System-on-Chip (SoC) to autonomously optimize the ion optics without any calibration gas. Moreover, the same capabilities of remote control, data retrieval, and diagnostic required in space are implemented with an HTTP API, making it the first TOF mass spectrometer 100% ready for the Internet of Things (IoT).

Finally, the integration with standard technologies for the storage and visualization of time series, such as InfluxDB and Grafana, and the planned integration with tools such as R, SAS, and Python, open new possibilities for inline measurements, data analytics, and preventive maintenance using machine learning algorithms.

Poster 16:
MS2field: Development and deployment of a transportable, automated LC-HRMS platform for water quality screening

Michael A. Stravs¹, Heinz Singer¹, Christian Stamm¹, Christoph Ort², Reto Bolliger³, Guenter Boehm³, Nicole Zehethofer⁴, Siegrun Mohring⁴, Thomas Moehring⁴

1. Department of Environmental Chemistry, Eawag, Überlandstr. 133, Dübendorf, Switzerland
2. Department of Urban Water Management, Eawag, Überlandstr. 133, Dübendorf, Switzerland
3. CTC Analytics, Industriestrasse 20, Zwingen, Switzerland; 4 Thermo Fisher Scientific, Hanna-Kunath-Straße 11, Bremen, Germany

Liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) is the state-of-the-art method for micropollutant analysis in water samples, allowing for quantification of hundreds of target substances and identification of unknown contaminants. However, the time and effort required for traditional sampling and sample preparation presents a bottleneck to the acquisition of comprehensive datasets, in particular with high time resolution.

To alleviate these issues, we equipped a transportable trailer with a fully automated LC-HRMS platform for deployment in the field, allowing for autonomous acquisition of time series with high temporal resolution (20 min). A Q Exactive HF mass spectrometer (Thermo Fisher, Bremen) was coupled to a PAL RTC autosampler (CTC Analytics, Zwingen) and an online solid-phase extraction (SPE) – LC system. Every 20 minutes, a sample is taken from a bypass stream, filtered through a self-cleaning filtration device, and enriched on an online SPE cartridge. LC-HRMS acquisition is performed with a fast gradient, and alternatingly data-dependent or data-independent MS2. The equipment is monitored with multiple sensors, and an online dashboard provides access to system status and automated evaluations.

The platform was deployed in two field campaigns for a total of ~3 months, collecting over 6000 datapoints. At the wastewater treatment plant Fehraltorf, raw wastewater influent (2x 2 weeks, total ~2000 samples) and treated wastewater effluent (3 weeks, ~1500 samples) were measured. Targeted analysis of pharmaceuticals and selected pesticides in influent data revealed daily usage patterns, e.g. for antihypertensives (Valsartan, Candesartan), as well as episodic events (e.g. for Mecoprop), but also unexpected patterns. In addition, non-target analysis revealed approximately 17’000 reliable time series from originally >105 profiles, using autocorrelation and periodograms. Hierarchical clustering (~300 clusters) revealed numerous patterns possibly attributable to industrial sources, and in-silico metabolite identification (SIRIUS 4.0) was used to tentatively identify an exemplar homologous series. The small creek Eschelisbach (TG), was monitored during ~5 weeks (>2500 samples), revealing time dynamics of pesticides in base flow and during rain events.

The results demonstrate the applicability and robustness of the developed platform, and highlight opportunities for future developments in environmental chemistry.

Poster 17:
For on-line processing of High Resolution Mass Spectra

Laure Menin, Daniel Ortiz, Natalia Gasilova, Francisco Sepulveda, Luc Patiny

Ecole Polytechnique Federale de Lausanne, 1015 Lausanne, Switzerland

Cloud based services are becoming of main importance for many applications. Since the last 10 years, we are developing core functionalities to allow the processing of High Resolution Mass Spectra (HRMS) directly in the browser. This is particularly useful for MS platforms since most of the users do not have access to commercial software. Recently, we compiled various in-house tools to solve usual tasks but more importantly to treat special requests involving complex projects as commonly encountered in research laboratories. In particular, the tool was recently extended to the analysis of oligonucleotides, a domain for which there are no free and open-source programs. Those tools, available on the website http://ms.cheminfo.org, are open-source (MIT license), don't require any installation. In order to use this toolbox, the only mandatory application is a web browser installed on a computer. Those free and open source applications are always up-to-date and in constant development, quickly evolving with the needs of EPFL researchers. Several tools available were recently updated or implemented, among others:

• Generation of isotopic pattern distribution from a molecular formula
• Generation of molecular formulas from a monoisotopic mass and matching theoretical to experimental data. The tool returns possible PubChem compounds.
• Tracking of classical contaminants: the experimental mass spectrum is screened against the ms.cheminfo.org database of contaminants. The user can easily create its own database of compounds to be screened, in the form of a Google spreadsheet.
• Protein analysis: calculation of theoretical fragment ions (including internal ones) with any user defined modification.  The tool matches the theoretical isotopic pattern to the experimental mass spectra and returns a list of matches with similarity scores.
• Oligonucleotide analysis: calculation of theoretical DNA/RNA fragment ions including losses of bases / H2O and internal fragments, and matching with experimental data.

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