The 33rd meeting of the SGMS will be held at the   Dorint Resort Blüemlisalp Beatenberg, October 29-30, 2015 high above Lake Thun in the Bernese Oberland, with a scenic view of the Swiss Alps!

Plenary Lectures

The great paradox of the 21st century:  We are unable to identify 70-90% of small molecules in biological samples and rivers on Earth, while humans have endeavored to analyze the soil on Mars, the atmosphere on Jupiter and the sea on Titan.
	Robert Mistrik HighChem Cajakova 18 Bratislava 81105 Slovakia
Mass spectrometers coupled with high- or ultra-performance chromatographic techniques allow the detection of thousands of small molecules in a complex sample; however their efficient and reliable identification is still a major bottleneck that is hindering progress in various scientific fields. The majority of the unidentified compounds are not entirely unknown to the chemical world, however their true identity remains elusive since their reference spectra are not available. There is also a growing concern that even those compounds reported as positively identified are in fact incorrect annotations confused either with structural isomers displaying similar fragmentation patterns or even with structurally unrelated isobaric compounds sharing common elemental composition. Some emerging “de novo” identification computer programs are likely to contribute to the inaccuracies, since they often apply proteomic-like fragmentation principles or use purely combinatorial bond-breaking logic, although small molecules definitively do not fragment in a uniform manner and often undergo non-trivial electron displacements or complex rearrangements. Even though many reported “automated” structure annotation methods did not hold the promise they might have hoped for, there are functional ways that can assists in identification of vast number of unknowns, which will be presented. Those methods are based on heuristic and quantum chemical methods rather than relying on combinatorial methods or molecular formula calculations. In addition, a whole array of methodological challenges the mass spectrometrists are facing when aiming to overcome the identification bottleneck will be discussed.
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Rosina, one year at comet Churymov -Gerasimenko

Kathrin Altwegg

University of Bern
Centeer for Space and Habitability
Sidlerstrasse 5
3012 Bern

and the ROSINA team

In situ mass spectrometry in space has its own challenges. Not only has the mass spectrometer to be lightweight and energy efficient, but it also has to withstand a broad temperature range, high vibration levels during launch and a wide variety of pressures. Autonomous operation over long times, immunity to cosmic rays and high compression of data are other prerequisites for successful instruments on board spacecraft. The probably most advanced instrument currently flying aboard a spacecraft is the ROSINA (Rosetta Sensor for Ion and Neutral Analysis) instrument on board the ESA Rosetta spacecraft encountering comet 67P/Churyumov-Gerasimenko. Rosetta is following the comet from almost 4 AU through its perihelion at 1.3 AU and out again for more than 1 ½ years. The closest distances of just a few kilometers to the comet has been reached during the delivery of the lander Philae in November. The cometary atmosphere consists mostly of water and CO/CO2. However, it is known that comets have quite a diversified organic part in their coma, both as volatiles and as dust. Furthermore, isotopic ratios in water and other molecules can give very strong indications on the formation process of cometary, and therefore solar system material.

In this talk I will give a short overview on the ROSINA instruments and some of the discoveries made so far in the cometary coma, which give us valuable information on the origin and evolution of our solar system.

Myxobacterial secondary metabolomics: Towards a comprehensive survey of a promising natural products resource

Rolf Mueller

Department of Microbial Natural Products
Helmholtz-Institute for Pharm. Res. Saarland (HIPS)
Helmholtz Centre for Infection Res. (HZI)
Pharmaceutical Biotechnology
Saarland University Campus C 2.3
66123 Saarbrücken
Germany

Natural products with their unique structural diversity are of exceptional importance for drug discovery. Among natural product-based drugs approved by the FDA, those originating from microbial sources make up 30% [1]. Myxobacteria represent an important source of novel natural products exhibiting a wide range of biological activities, and a number of secondary metabolites from myxobacteria are currently investigated as potential leads for novel drugs [2]. Together with activity-guided screening and genome mining, metabolomics-based approaches using modern mass spectrometry techniques can help to bridge the gap between genome-encoded potential and the usually contradictory low numbers of secondary metabolites known from a specific producer [3]. While it would be desirable to obtain a fully unbiased picture of the secondary metabolome using a single instrumental setup, this is usually prohibited by the high structural complexity of secondary metabolites. In this contribution, an effort is made to create a comprehensive overview of myxobacterial secondary metabolomes using mass spectrometric data from ~2500 complex myxobacterial extracts, representing a diversity-oriented subset of our strain repository. The crucial prerequisites enabling our study are: i) access to an extensive myxobacteria collection sampled at locations worldwide, including isolates from rare and under-exploited habitats, ii) a sensitive analytical platform using high-resolution TOF-MS (and recently also FT-ICR MS)  for both targeted and untargeted secondary metabolome analysis, and iii) a database-assisted bioinformatics platform allowing us to conduct data evaluation across large numbers of secondary metabolomes and at the same time to dig deep into individual secondary metabolite profiles. I will present results of a statistical survey covering our entire reference extracts collection and taking into account most known myxobacterial secondary metabolites. Furthermore, we exemplify how metabolomics-and statistics-based approaches can contribute to reveal novel metabolite candidates and demonstrate how these methods underpin our efforts to isolate seizable quantities of newly discovered natural products, through the identification of alternative sources featuring improved production of target compounds.

1. E. Patridge, P. Gareiss, M. S. Kinch, and D. Hoyer, An analysis of FDA-approved drugs: natural products and their derivatives, Drug Discov. Today, pp. 8–11, 2015.
2. R. Müller, J. Wink, Future potential for anti-infectives from bacteria – How to exploit biodiversity and genomic potential Int. J. Med. Microbiol., pp. 3-13, 2014.
3. D. Krug and R. Müller, Secondary metabolomics: the impact of mass spectrometry-based approaches on the discovery and characterization of microbial natural products, Nat. Prod. Rep., pp. 768–83,  2014.

Challenge and success of mass spectrometry based quantitative proteomics on clinical body fluid cohorts

Dorothea Rutishauser 

Department of Medical Biochemistry & Biophysics
and Science for Life Laboratory
Karolinska Institute
Scheelesväg 2
SE 17177 Stockholm
Sweden

In the last couple of years we have applied mass spectrometry based proteomics to clinically oriented research projects with access to patient cohorts trying to get insight into how well the body responds to a treatment for a disease or to monitor a medical condition progress. The different clinical divisions have carefully collected patient samples over years with the goal to find biological molecules that are a sign of normal or abnormal processes in a disease or suggesting new ways of diagnosis. We have analyzed these body fluid samples of patients and identified unique signatures of proteins responsible for the diagnosis, prognosis and therapeutic prediction of a disease such as neurodegenerative disorders, atopic eczema and rheumatoid arthritis.

Proteomics analysis of patient cohort samples requires high technical reproducibility, stable and accurate quantitation in a high throughput manner. We have successfully applied single shot label free analysis on latest generation high resolution mass spectrometry instrumentation in order to extract quantitative information. Simultaneously to the detection of protein abundance changes we were also identifying and quantifying “silent” posttranslational modifications, such as isoAsp as well as IgG glycosylation by employing multiple dissociation techniques.

The largest mass spectrometric data set we have acquired on plasma samples from different Alzheimer’s cohorts and we have found regulated proteins viable for diagnosis as well as protein based classification models for disease prediction and progression.

High Resolution Mass Spectrometry for in Depth Understanding of Complex OMICS Challenges and Comprehensive Characterization of Large Molecular Weight Assemblies

Detlev Suckau

Bruker Daltonik GmbH
Biopharma and Omics Solutions R&D
Bremen, HB 28359
Germany

Quadrupole Time-Of-Flight mass spectrometry (QTOF) has evolved into a versatile high performance technology during recent years. Today, they can achieve mass accuracies of < 1 ppm and resolving powers up to 80,000; opening a wealth of analytical options. QTOF technology accommodates large ion populations per scan thus providing a unique statistical base for the measurement of isotope patterns and an unusually high intra-spectra dynamic range of up to > 5 orders of magnitude.

These instrument characteristics allow reliable molecular formula determination based on accurate mass and isotopic pattern matching for small molecules. Thus enabling the identification of statistically relevant molecular features in complex mixtures.

QTOFs are further applied in large scale OMICS applications, particularly in metabolomics and proteomics as they can provide scan speeds of up to ~100 spectra/sec. High mass accuracy and resolution independent of scan speed and peak abundance with accurate isotope representation translates into sensitive and accurate quantification capabilities.

For large molecules such as antibody subunits in the 25 kDa range, correct isotope pattern representations allow validation of subunit composition and even sensitive detection and quantification of subtle modifications such as protein deamidation, which can only be detected by quantifying a slight distortion of the isotopic pattern. Such assessments may permit the determination of critical quality attributes of biopharmaceuticals.

A wide variation of technology extensions such as ETD fragmentation, ion mobility separation, scan modes such as SWATH, MSE or HR-XICs further broaden the application range of QTOFs, e.g., to further structure elucidation, targeted screening and quantification applications.

Short Communications

Non-target screening of water with high resolution mass spectrometry: Perspectives from a collaborative trial

Emma L. Schymanski¹, Heinz P. Singer¹, Jaroslav Slobodnik², Ildiko M. Ipolyi², Peter Oswald², Martin Krauss³, Tobias Schulze³, Peter Haglund⁴, Thomas Letzel⁵, Sylvia Grosse⁵, Nikolaos S. Thomaidis⁶, Christian Zwiener⁷, Juliane Hollender¹.

¹Eawag: Swiss Federal Institute for Aquatic Science and Technology, Überlandstrasse 133, 8600 Dübendorf, Switzerland. ²Environmental Institute, Slovak Republic. ³Helmholtz Centre for Environmental Research - UFZ, Germany. ⁴Umeå University, Sweden. ⁵Technische Universität München, Germany. ⁶University of Athens, Greece. ⁷Eberhard Karls University of Tübingen, Germany. Email: emma(dot)schymanski(at)eawag(dot)ch

A collaborative non-target screening trial was organised in 2013/2014 by the NORMAN Association to begin efforts to harmonize and provide guidance on (environmental) non-target screening in Europe. An extract of water from the Danube was analysed by 18 European institutes using liquid and/or gas chromatography (LC, GC) coupled with mass spectrometric (MS) detection. Target and suspect screening methods yielded similar number of identifications for LC-MS approaches (649 unique compounds reported: 347 as targets), with very few non-target substances reported. Approximately 80 substances were reported without a definite structure (isomer mix). Carbamazepine and atrazine were reported most frequently. While suspect screening was popular for LC-MS/MS, searching the NIST database dominated the unknown identification approaches for GC-MS. Of the 858 unique compounds reported overall, 46 were reported in both LC and GC, approximately 5 %. Major challenges remain isobaric, co-eluting substances and dependence on target and suspect lists (e.g. terbutylazine, sebutylazine, simazine and transformation products), formula assignment, including retention information and the confidence of identification. Some of the tentative non-target identifications were revised using evidence from other participants. While the non-target analytical techniques were already considerably harmonized between the participants, the data processing remains time-consuming. Major recommendations to improve non-target screening include better integration and connection of desired features into software packages, the exchange of target and suspect lists and the contribution of more spectra from standard substances into (openly accessible) databases.

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High resolution ion mobility-mass spectrometry for separation and identification of isomeric lipids

Michael Grössl, Stephan Graf, Richard Knochenmuss

Tofwerk, Uttigenstr. 22, 3600 Thun, Switzerland

Contact: groessl(at)tofwerk(dot)com

Current separation and identification techniques for isomeric lipids are too often insufficiently powerful, slow or ambiguous. High resolution, low field ion mobility coupled to mass spectrometry is shown here to be a powerful, fast new method for the analysis of isomeric lipids across all major lipid classes (fatty acids, triacylglycerols, phospholipids, ceramides, steroids and sterols).

The separated isomers differ in functional group position (e.g. hydroxyl for ceramides or phosphate for PIPs), acyl chain position (regiosisomers), double bond position and geometry (cis/trans). The same level of performance is maintained in complex biological mixtures and the additional separation dimension provided by ion mobility leads to an almost 2-fold increase lipid-associated peaks. Additionally, accurate and precise collision cross sections are obtained which can be used for as molecular identifiers. This physical quantity can be the basis of lipidomics workflows, as the appropriate libraries are developed.

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MALDI-TOF MS for microorganism identification: from pattern recognition towards marker based approaches

Joël F. Pothier¹*, Valentin Pflüger², Frédéric Foucault², Guido Vogel²

¹ Zurich University of Applied Sciences ZHAW, School of Life Sciences and Facility Management, Institute of Natural Resource Sciences, Environmental Genomics and Systems Biology Research Group, Einsiedlerstrasse 29, CH-8820 Wädenswil, Switzerland;

² MABRITEC AG, Lörracherstrasse 50, P.O. Box 325, CH-4125 Riehen, Switzerland

joel(dot)pothier(at)zhaw(dot)ch

In the last decade Matrix Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS) has become a revolutionary and powerful tool for the identification of bacteria, yeast and fungi identification in routine diagnostics. There are currently several commercial systems available (e.g. bioMérieux, France; Bruker Daltonics, Germany; Andromas, France) which are all based on databases containing protein mass patterns deduced from standardized spectra of bacterial reference strains. Different identification algorithms for spectral matching are applied, all based on weighted pattern recognition. The current pattern recognition based systems require standardized sample preparation and to a certain extent comparable culturing conditions. Further, these reference databases mainly focus on clinical relevant species and cover between 700-2’000 species. As publicly available bacterial whole genome data is tremendously increasing, ZHAW and Mabritec AG developed in the framework of a CTI-project (project nr. 11225.1 PFLS-LS) a new database approach completely based on genome derived in silico data. This new centralized database is called PAPMIDTM (Putatively Assigned Protein Masses for Identification) and will be made accessible through a web based commercial application. Ribosomal proteins were chosen as universal marker candidates. They have the advantage of being constitutively expressed and therefore abundant in detectable amounts independently of sample preparation and culturing conditions. Ribosomal proteins are highly conserved and therefore allow a highly accurate species identification. To some extend even subtyping within a species is possible. This new approach enables an automated spectral quality control and allows a phylogenetic interpretation of acquired bacterial spectral data. The PAPMIDTM database is independent of the MALDI-TOF MS instrument type and currently contains the relevant reference information of more than 9’200 genomes covering an excess of 3’200 bacterial species. It is updated on a monthly basis. First validations with clinical and environmental isolates showed a promising high sensitivity and specificity.

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A Novel Lipid Screening Platform Allowing a Complete Solution for Lipidomics Research

Nick Morrice¹, Kerstin Pohl², Quentin Enjalbert³, Alex Connor⁴, Baljit K. Ubhi⁵, Eva Duchoslav⁶, Fadi Abdi⁷, Leo Wang⁴, Paul Baker⁷ and Steve Watkins⁴

¹SCIEX, Warrington, UK; 2SCIEX, Darmstadt, Germany; ³SCIEX France; ⁴Lipomics, CA, USA, ⁵SCIEX, CA, USA, ⁵SCIEX, Ontario, Canada and 7SCIEX, MA, USA

Introduction: A major challenge in lipid analysis is the many isobaric and near isobaric interferences present in highly complex samples that confounds identification and accurate quantitation. This problem, coupled with complicated sample preparation techniques and data analysis, highlights the need for a complete solution that addresses these difficulties and provides a simplified method for analysis. A novel lipidomics platform was developed that includes simplified sample preparation, automated methods, and streamlined data processing techniques that enables facile, quantitative lipid analysis. Herein, serum samples were analyzed quantitatively using a unique internal standard labeling protocol, a novel selectivity tool (differential mobility spectrometry; DMS) and novel lipid data analysis software.

Methods: Applying the kit for simplified sample extraction and preparation, a serum matrix was used following the protocols provided. An LC-DMS-QTRAP® System (SCIEX) was used for targeted profiling of hundreds of lipid species from 10 different lipid classes allowing for comprehensive coverage. This system allows for: 1. quantitative results for each lipid class as a sum of individual species; 2. mole percent composition was obtained computationally from lipid molecular species data; and 3. accurate lipid species compositions. The data was compared with historical data generated by alternative methods. Samples were quantitated using software accompanying the full solution which incorporates the novel labeled internal standards available as a kit, developed for this platform (Avanti Lipids).

Preliminary Data: Quantitative lipid species measurements were obtained from the following complex lipid classes: diacylglycerols (DAGs), triacylglycerols (TAGs), phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), sphingomyelins (SMs), lysophosphatidylcholines (LPCs) and lysophosphatidylethanolamines (LPEs), free fatty acids (FFAs), cholesteryl esters (CEs) and ceramides (CERs). Covering these classes required a two-injection approach but data can be collected in short, fast gradients up 15 minutes run time per injection. The two injections classes covered TAGs and SMs, CEs, CERs and DAGs and FFAs, PE/PCs and their lyso components (LPC/LPEs).

Novel Aspect: Anovel, quantitative lipid screening platform allowing a complete solution for lipidomics research

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Gas-phase fragmentation and conformation of the sugar-modified antisense oligonucleotide tcDNA

Yvonne Hari and Stefan Schürch

Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern

stefan(dot)schuerch(at)dcb(dot)unibe(dot)ch

Tricyclo-DNA (tcDNA) is a sugar- and backbone-modified analogue of DNA that is currently tested as antisense oligonucleotide for the treatment of Duchenne muscular dystrophy. The name tricyclo-DNA is derived from the modified sugar-moiety: the deoxyribose is extended to a three-membered ring system. This modification is designed to limit the flexibility of the structure, thus giving rise to entropically stabilized hybrid duplexes formed between tcDNA and complementary DNA or RNA oligonucleotides.

While the structural modifications increase the biostability of the therapeutic agent, they also render the oligonucleotide inaccessible to enzyme-based sequencing methods. Tandem mass spectrometry constitutes an alternative sequencing technique for partially and fully modified oligonucleotides. For reliable sequencing, the fragmentation mechanism of the structure in question must be understood. Therefore, the presented work evaluates the effect of the modified sugar-moiety on the gas-phase dissociation of single stranded tcDNA. Moreover, our experiments reflect the exceptional gas-phase stability of hybrid duplexes that is most noticeable in the formation of truncated duplex ions upon collision-induced dissociation. The stability of the duplex arises from the modified sugar-moiety, as the rigid structure of the tcDNA single strand minimizes the change of the entropy for the annealing. Moreover, the tc-modification gives rise to extended conformations of the nucleic acids in the gas-phase, which was studied by ion mobility spectrometry-mass spectrometry.

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Chemoselective digestion: an alternative realm for middle-down mass spectrometry

Kristina Srzentić,¹ Konstantin O. Zhurov,¹ Martin Kussmann^1,2, and Yury O. Tsybin³

¹ Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland ² Nestlé Institute of Health Sciences, 1015 Lausanne, Switzerland ³ Spectroswiss Sàrl, 1015 Lausanne, Switzerland

E-mail: kristina(dot)srzentic(at)epfl(dot)ch

Recent advances in MS instrumentation provide the necessary platform for the application of middle-down (MD) proteomics approach and investigation of increasingly larger (>3 kDa) peptides in a high-throughput manner. However, MD meets its practical limitations in the selection of cleaving agent that would provide the desired peptides of 3-15 kDa. Bioinformatics study performed in-house revealed that targeting rare residues could be the answer for MD digestion. Herein, we evaluated chemical agents targeting primarily methionine, tryptophan and cysteine (CNBr, BNPS-Skatole and NTCB, respectively) for their optimal performance in terms of selectivity, reproducibility, peptide length, charge and obtainable sequence coverage

Preliminary results indicated chemoselective cysteine mediated digestion with NTCB to be a viable alternative to enzymes for MD approach, particularly readily applicable to the structural analysis and paratope mapping of immunoglobulins (IgGs). Currently employed bottom-up (BU) avenues for IgG analysis do not meet all analytical requirements.Due to the frequent digestion, resulting peptides are short and connectivity between complementarity determining regions (CDRs) is obscured. Oftentimes even a single CDR is split into multiple peptides. Real advantage of digestion using NTCB is due to the position of cysteines within particular IgG structure; generated peptides can contain adjacent CDRs, revealing the connectivity information and therefore potentially facilitating paratope discrimination in a mixture of IgGs. At the same time, the number of required peptide IDs is reduced, and in turn, the obtainable sequence coverage per LC run is increased. High reproducibility of chemoselective digestion reactions may find a particular utility in quantitative proteomics.

Base-promoted chemical digestion parameters (choice of base, pH, temperature, duration, cleaving agent-to-protein ratio) as well as liquid chromatography (LC)-MS method parameters (column, gradient, activation method, number of ions) were evaluated to find the optimal experimental configuration for analysis of yielded peptides on model 7 protein mixture and IgG mixtures. All LC-MS experiments were performed on a Dionex 3000 Ultimate coupled to a LTQ Orbitrap Elite FTMS mass spectrometer (Thermo Scientific). NTCB-based middle-down proteomics pipeline successfully generated and IDed peptides containing consecutive CDRs of the light and heavy chain, as well as the ones carrying CDR3. 

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Challenges in food fraud and food safety: protein quantification by LC-MS/MS

Christophe Fuerer and Véronique Parisod

Nestec SA, Nestlé Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26 

christophe(dot)fuerer(at)rdls(dot)nestle(dot)com

Food fraud is defined as the intentional modification of food, food ingredient, and packaging for economic gain, where product adulteration can be achieved through several mechanisms such as water inclusion, species substitution, addition of an unrelated substance, and mislabeling. The motivation for food fraud is economic, but it can have severe health consequences as illustrated by the Chinese melamine crisis in 2008. Food safety describes the unintentional contamination of food with microorganisms, toxins, chemicals, or allergenic food components. Adulteration and contamination can both arise during food processing, but are more likely to be already present in the raw materials. Ensuring the integrity of food ingredients and food products is a growing challenge, and multiple reaction monitoring (MRM) methods can be used to simultaneously identify and quantify components in a variety of food matrices. Examples of MRM for multiplex allergen detection and milk protein characterization will be presented to highlight the promises and challenges of those methods for food integrity assessment.

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Target Plate Material Influence on Fullerene-C60 Laser Desorption/Ionization Efficiency: Implications for MALDI

Guido P. Zeegers, Barbara F. Günthardt, Renato Zenobi

Department of Chemistry and Applied Biosciences ETH Zürich, Switzerland

guido(dot)zeegers(at)org(dot)chem(dot)ethz(dot)ch

Systematic laser desorption/ionization (LDI) experiments in the absence of protons, based on electrospray deposition of fullerene-C60 on a wide range of target plate materials, were chosen to establish the validity of models thus far proposed primary ionization in matrix-assisted LDI (MALDI) mass spectrometry.

The ion yield of all fragment and cluster an- and cations over a m/z 100 – 2500 range was monitored on a commercial MALDI-Time-of-Flight-MS instrument (Bruker Autoflex, nitrogen laser, l: 337 nm) varying both laser fluence (0 – 3.53 Jcm-2) and ion extraction delay time (0 – 950 ns). The resulting species-specific ion yields are an indication for the kind of ionization mechanisms contributing to LDI and the time frames in which they operate, providing insight in the (MA)LDI primary ionization.

Target plate material ion yield differences are attributed to their conductive properties. An increased electrical resistivity increases the single fullerene-C60 anion yield. Inconel® 625 and Ti90/Al6/V4, both highly electrically resistive, provide the highest anion yields, even exceeding the cation yield by a factor ~1.4 for the latter.

We present a mechanism based on transient electrical field strength to explain this trend. Cluster anion formation is negligible, however, cluster cations are readily formed and provide an additional pathway to form single fullerene-C60 cations through decomposition at high laser fluence. Our mechanistic overview underlines the importance between uni- and bimolecular ionization processes.

Ion yield depends greatly on the choice of substrate material and careful substrate selection could therefore allow for more sensitive (MA)LDI measurements.

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Posters

(Size: 146 cm high x 118 cm wide)

P01: Automated in-gel digestion on a commercial autosampler directly coupled to nanoLC-MS/MS 

Achermann François, Buchs Natasha, Doiron Nicholas, Lagache Braga Sophie, Heller Manfred, University of Bern, Department of Clinical Research, Proteomics & Mass Spectrometry Core Facility, Bern, Switzerland

Bolliger Reto, Böhm Günter, CTC Analytics, Zwingen, Switzerland

Contact: Böhm Günter gboehm(at)ctc(dot)ch

Introduction: SDS-PAGE separates protein samples from LC-MS incompatible contaminations, and due to its separation efficiency, ease of use and low cost, it is frequently used to fractionate proteins of an entire proteome for bottom-up proteomics. One disadvantage is that each gel lane, containing a proteome, has to be cut into many slices, followed by in-gel digestion of proteins and extraction of peptides. The number of gel slices of a comparative proteome study goes into the hundreds, rendering this process very repetitive and prone to mistakes and errors during sample handling. It is therefore beneficial to automate the in-gel digestion process, in order to reduce such risks and improve reproducibility.

Methods:  We have adapted a manual in-gel digestion protocol, including reduction, alkylation and acid-labile detergent assisted trypsin digestion of proteins, to be performed on a PAL RTC liquid handling – autosampler system (CTC Analytics AG, Switzerland). The system is equipped with park stations for a syringe tool and samples/reagents, a cooled tray stack, an evaporation tool, a heatable incubator with orbital shaking capability, a vortex mixer, a syringe wash station, a centrifuge, and a LC injection valve. The current PAL RTC setup treats batches of 6 gel samples within 7 hours. Peptide extracts in HPLC vials are transferred to a nanoLC-orbitrap XL mass spectrometer (Thermo Scientific, Germany) or can directly be injected by the PAL system. Proteins were identified and quantified with MaxQuant software.

Data preview: A 12-protein molecular weight standard (Bio-Rad) was separated over the entire width of a 12.5% SDS-PAGE at two concentrations corresponding to 1ng or 5ng of each protein standard present in vertical slices of 1.5mm width cut from the gel. Four batches, including three slices of each protein concentration, were processed by an experienced human operator or on the PAL RTC system during four consecutive days. Protein yields were determined by identification and quantification with MaxQuant software. The low molecular weight proteins (Mr <20 kDa) were not consistently identified from the 1ng samples with both procedures. This is a known problem with the gel-LC-MS/MS approach. Keratin contaminations were very similar with both procedures and protein concentrations, indicating that keratin proteins were introduced into the samples before the in-gel digestion process. One-way Anova test was used to compare MaxQuant derived non-normalized protein intensities of batches and procedures with Tukey's honestly significant difference criterion to evaluate statistical significance (alpha = 0.05). No statistically significant differences in protein yield between batches and between procedures were found. The average coefficients of variation of the 12 standard proteins were 19.4% and 11.8% at 1ng protein, and 5.8% and 6.5% at 5ng for the manual and the automated procedure, respectively. These results indicate that the PAL RTC automated in-gel digestion protocol performs as well as an experienced human operator, with potentially a better reproducibility achieved on the PAL system when dealing with low protein concentrations. The next step is the direct coupling of the 

P02: Rapid method for monitoring monoclonal antibody (mAb) production in Biotechnological processes using quantitative MALDI-TOF-MS: working towards cell characterization

Robert F. Steinhoff, Daniel Karst, Jasmin Krismer, Martin Pabst, Miroslav Soos, Massimo Morbidelli, Renato Zenobi*

ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zurich

*Corresponding author: zenobi(at)org(dot)chem(dot)ethz(dot)ch

MAb process monitoring using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides new insights on process stability, efficacy and viability when looking at intra and extracellular protein levels. MAbs and their production processes have undergone an impressive development from immunology concepts formulated over a century ago by Paul Ehrlich, to their nowadays-widespread clinical applications. Today, mAbs are used in many areas, including therapy and diagnostics, resulting in an enormous demand. (3 out of the top 10 drugs by worldwide sales are monoclonal antibodies). Current methods for monitoring mAb production are based on separation techniques like size-exclusion chromatography (SEC), and liquid chromatography (LC), and are often time consuming and expensive.

Here we demonstrate a fast relative quantification MALDI-MS monitoring method for intact mAbs with a mass accuracy of < 0.5 % at 150.000 Da based on accurate internal calibration steps. MAb samples are aliqouted without prior sample preparation together with a UV-absorbing matrix and an internal standard protein onto a microarray for mass spectrometry (MAMS) chip. Subsequent analysis (number of replicates n = 10) is performed on a commercial MALDI TOF instrument equipped with a high mass conversion dynode detector, in less than 1 minute. The intact mAb and corresponding building blocks are identified and quantified in a relative fashion, which for the first time allows monitoring minor changes and inconsistencies in the overall production process (fig.1). The linearity of the method is shown in a dilution experiment with a purified universal mAb. Fed batch and perfusion reactor cell cultures are investigated to prove the applicability of the method regarding, robustness and high throughput capabilities. Therefore, the results of the monitoring experiment clearly demonstrate the expected trends and have been confirmed and cross-validated by HPLC-UV measurements.

Figure 1 Monoclonal antibody (mAb) monitoring. The signal at 150000 m/z corresponds to the singly charged monoclonal antibody, and the signal at 23500 m/z represents the light chain. The internal standard is at 129900 m/z and 70000 m/z.

P03: Systematic Comparison of Integrated Microfluidics/Nanoscale LC Platforms and Tandem/High Resolution MS for Quantitative MRM Analysis of Candidate Peptide Biomarkers

Billy Joe Molloy; Chris Hughes; Johannes PC Vissers; James I Langridge

Waters Corporation, Manchester, UK

Biomarker discovery and validation are the first steps in understanding disease and drug development. Validation is technologically challenged since it requires high-throughput analysis of large cohorts, with high sensitivity, high resolution, large dynamic range and excellent selectivity. MRM and high resolution IM-MS are enabling technologies. Miniaturized LC offers improved mass-sensitivity but lack throughput, robustness and reproducibility. Integrated microfluidics was compared with nanoscale LC for tandem and high resolution peptide MRM quantification, considering speed, sensitivity and selectivity.

MS Qual/Quant was spiked into E. coli matrix and cardiovascular disease biomarker SIL peptides in serum. Both samples were analysed using RP chromatography with tandem quadrupole or high resolution quadrupole oaTof MS. Peptides were resolved from background over the entire dynamic range of interest for cardiovascular disease peptides and at levels present in the MS Qual/Quant mixture. Retention time reproducibility equalled 0.02 min standard deviation under microfluidic and nanoscale LC conditions. MS Qual/Quant ratio measurements were accurate with an average 11.5% variation from expected values. Sub 100 amol on-column levels were detected without matrix interference using the integrated microfluidic device with tandem MS under high-throughput conditions. Ultimate sensitivity levels equalled sub 10 amol on-column using nanoscale LC with either tandem or high resolution MS.

P04: Single Section Multi-Model Tissue Imaging by DESI and MALDI TOF Mass Spectrometry

Mark Towers¹; Emrys Jones¹; Anna Mroz²; Zoltan Takats²; Emmanuelle Claude¹; James I Langridge²

¹Waters Corporation, Wilmslow, UK; ²Imperial College London, London, UK

DESI is an established technique in the field of mass spectrometry and used for tissue imaging. For lipids and small molecules, DESI results are of very high-quality compared to the more established MALDI imaging technique. Moreover, DESI can be performed without further sample preparation such as matrix deposition. Using optimised gas and solvent flow rates, DESI does not damage the tissue surface. The potential to analyse a single tissue section by DESI imaging with subsequent analysis by MALDI imaging is presented.

Frozen tissues of colorectal tumour were sectioned and mounted onto glass slides. In the DESI imaging experiment, a slide containing two consecutive sections was placed onto the stage. A pattern was defined over one tissue and imaging performed. Next, matrix solution was sprayed onto both sections. MALDI imaging experiments were carried out at the same pixel size on the pristine and the DESI altered section. Analysis of the DESI and both MALDI imaging data sets showed intense lipid detected peaks localised to defined regions. Overall, the MALDI experiments were similar, indicating that DESI did not modify the tissue or delocalise endogenous molecules. Complementary information was obtained when contrasting MALDI and DESI imaging data, providing a more comprehensive molecular description.

P05: Glutathione S-Transferase: the First Step Towards Mapping of Biotransformation Enzymes in Zebrafish Embryos 

Alena Tierbach¹, Ksenia J Groh^1,2, Kristin Schirmer^1,3,4, Marc J-F Suter^1,3 

alena(dot)tierbach(at)eawag(dot)ch 

¹Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland 

²ETH Zürich, Swiss Federal Institute of Technology, Department of Chemistry and Applied Biosciences, 8093 Zürich, Switzerland 

³ETH Zürich, Swiss Federal Institute of Technology, Institute of Biogeochemistry and Pollutant Dynamics, 8092 Zürich, Switzerland 

⁴EPF Lausanne, School of Architecture, Civil and Environmental Engineering, 1015 Lausanne, Switzerland 

The zebrafish embryo toxicity test (zFET) is increasingly employed as an alternative test model to replace conventional fish toxicity tests. One important requirement for the establishment of an alternative test model is that the toxicological outcomes can be directly extrapolated to the test system it aims to replace. To achieve this it is essential to have knowledge about the potential and the dynamics of the xenobiotic metabolism throughout the development of relevant embryonic stages. Enzymes involved in the xenobiotic metabolism can modify the chemical structure of foreign substances by introducing functional groups (phase I) and/or through conjugation reactions with endogenous molecules (phase II). These reactions often decrease the reactivity and thus the toxicity of the foreign substance and facilitate its excretion. However, in some cases the xenobiotic metabolism can also lead to the activation of non-reactive compounds and thus increased toxicity. Yet, little is known about the developmental expression of xenobiotic metabolizing enzymes in zebrafish and thus about the chemical groups which might display different patterns of toxicity within different life stages. 

In this study, we use a targeted proteomic approach to characterize the expression of phase I and phase II enzymes during the zebrafish development. Enzymes which are involved in the biotransformation of xenobiotics, are monitored in zebrafish embryos by mass spectrometry with the use of proteotypic peptides and peptides characteristic for the enzyme families of interest. The expression of selected phase I and phase II enzymes is monitored in unexposed embryos up to 7 days post fertilization (dpf) and in liver samples of adult zebrafish. 

We have developed a targeted proteomic approach to characterize the expression of glutathione-S-transferases, a group of phase II enzymes. The presence and expression level of selected members of this enzyme family could be determined at different life stages of zebrafish. The detection of other enzymes including cytochrome P450 monooxygenases, monoamine oxidase, alcohol dehydrogenase (phase I) and sulfotransferases (phase II) was associated with methodological difficulties. The problems resulting from the low copy number of biotransformation enzymes in non-exposed embryos and the difficulties associated with isolation of subcellular fractions from fish tissue are discussed. 

P06:Tandem Mass Spectrometric Analysis of Metallocene Adducts with Short Oligonucleotides 

Rahel P. Eberle and Stefan Schürch

Department of Chemistry and Biochemistry, University of Bern

Corresponding Author: stefan(dot)schuerch(at)dcb(dot)unibe(dot)ch

Metallocene dichlorides constitute a remarkable class of antineoplastic agents that are highly effective against several cancer cell lines. They were shown to accumulate in the DNA-rich region, which suggests DNA as the primary target. These compounds exhibit two cyclopentadienyl ligands and two labile halide ligands, resulting in a bent sandwich structure. The cis-dihalide motif is structurally related to the cis-chloro configuration of cisplatin and similar modes of action can thus be assumed. Cisplatin binds to two neighboring guanine nucleobases in DNA and consequently, distorts the double-helix, thereby inhibiting DNA replication and transcription. Platinum is classified as a soft Lewis acid and binds preferentially to the nitrogen atoms within the nucleobases. The metallocene dichlorides investigated in this study comprise the metal centers Ti, V, Nb, Mo, Hf, and W, which are classified as hard or intermediate Lewis acids, and thus, favor binding to the phosphate oxygen.

Although several studies reported adduct formation of metallocene dichlorides with nucleic acids, substantial information about the adduct composition, the binding pattern, and the nucleobase selectivity has not been provided yet.

ESI-MS analyses gave evidence for the formation of metallocene adducts (M = Ti, V, Mo, and W) with single-stranded DNA homologues at pH 7. No adducts were formed with Nb and Hf at neutral pH, albeit adducts with Nb were observed at a low pH. MS2 data revealed considerable differences of the adduct compositions. The product ion spectra of DNA adducts with hard Lewis acids (Ti, V) gave evidence for the loss of metallocene ligands and only moderate backbone fragmentation was observed. By contrast, adducts with intermediate Lewis acids (Mo, W) retained the hydroxy ligands. Preliminary results are in good agreement with the Pearson concept and DFT calculations. Since the metallodrugs were not lost upon CID, the nucleobase selectivity, stoichiometry, and binding patterns can be elucidated by means of tandem mass spectrometry.

P07: Reengineering Alkaloid Biosyntheses via Metabolic Shifting and Molecular Refining

Nadine Bohni, Ellen Piel, Karl Gademann

Department of Chemistry, University of Zurich, Zurich, Switzerland

Contact: nadine(dot)bohni(at)chem(dot)uzh(dot)ch

Nature is unmet in its ability to generate new chemical structures. While biosynthesis is able to  produce a well defined array of reactions, the introduction of novel reactivity has been shown to be difficult. In contrast, organic synthesis is unrivaled in its power to unravel new reactions and reagents. Cyanobacteria – prokaryotic photoautotrophs – are promising producers of structurally versatile chemical scaffolds,[1] including chlorinated natural products.[2] In this study, we demonstrate that (1) new alkaloid derivatives can be generated in this phototroph via metabolic shifting and (2) these can be further transformed via a distinct chemical transformation involving in vivo catalysis, i.e. the Suzuki reaction.

The directed biosynthesis of 6-bromonostocarboline was achieved by supplementation of the culture medium with equimolar amounts of biosynthetic precursor 5-bromotryptophan[3] as well as by metabolic shifting of the culture from chlorine to bromine. The cross coupling proceedes in cyanobacterial culture, with a water-soluble catalyst at room temperature and in ambient air. The product formation is observed by LC-MS and via comparison with a synthetic sample.

Here, we describe the Suzuki reaction on the cyanobacterial metabolite nostocarboline directly inside the cyanobacterial culture, without prior isolation of the intermediate. This represent, to the best of our knowledge, the first example of Suzuki cross coupling directly on intact cyanobacterial culture, in cellular and unlysed medium.

1. R. K. Singh, S. P. Tiwari, A. K. Rai, T. M. Mohapatra (2011) Cyanobacteria: An Emerging Source for Drug Discovery. J. Antibiot. 64(6): 401-412.
2. P. Becher, J. Beuchat, K. Gademann, F. Jüttner (2005) Nostocarboline: Isolation and Synthesis of a New Cholinesterase Inhibitor from Nostoc 78-12A. J. Nat. Prod. 68: 1793-1795.
3. C. Portmann, C. Prestinari, T. Myers, J. Scharte, K. Gademann (2009) Directed Biosynthesis of Phytotoxic Alkaloids in the Cyanobacterium Nostoc 78–12A. ChemBioChem 10: 889-895.

P08: Analysis of the multiple electrochemically generated oxysterols by NP-TLC and RP-HPLC-MS

Daniel Vetter, ERC Gmbh, Riemerling, Germany

Oxysterol species are formed in vivo by enzymatic and non-enzymatic oxidation of cholesterol. Besides acting as intermediates in the biosynthesis of bile acids and steroid hormones, oxysterols per se possess versatile bioactivities, such as controlling gene expression, affecting calcium-signaling and immune or inflammatory responses. Many functions of oxysterols are not fully understood and others may not have been discovered yet, especially those of non-enzymatic origin. The limited number of commercially available standards challenges both analyses and functional studies.

Using an amperometric flow-through cell (ROXY EC system, Antec, NL) for oxidation of cholesterol we were able to obtain numerous oxidation products within short reaction times. The products were analyzed by NP-TLC and RP-HPLC-MS using 19 standard compounds. Besides the six oxysterols identified by both techniques, more than ten additional electrochemically generated compounds were detected. The identified compounds denote that oxidation mostly occurred near the double-bound at the B-ring, which is in agreement with susceptibility to free radical driven oxidation. Interestingly, some of the new electrochemically generated oxysterols were also present in lipid extracts obtained from cell culture models of nitrosative stress.

P09: Automatization in newborn screening - Direct mass spectrometric analysis of amino acids and acylcarnitines from dried blood spots

Christian Berchtold¹, Irene Wegner¹, Stefan Gaugler², Timm Hettich¹, Markus Wyss², Ralf Fingerhut³, Götz Schlotterbeck¹

Contact: Christian Berchtold: christian(dot)berchtold(at)fhnw(dot)ch

¹Fachhochschule Nordwestschweiz, Hochschule für lifesciences, Institut für Chemie und Bioanalytik, Gründenstrasse 40, 4132  Muttenz ²CAMAG Chemie-Erzeugnisse & Adsorptionstechnik AG, Sonnenmattstrasse 11, 4132 Muttenz ³University Children`s Hospital, Children’s Research Center, Swiss Newborn Screening Laboratory Steinwiesstrasse 75, CH-8032 Zürich

Introduction:  In Switzerland, 80'000 newborns are screened for inborn errors of metabolism every year. This screening is well established and based on dried blood spots. It provides an early diagnosis for several severe diseases such as phenylketonuria for example. This procedure is of great benefit for the survival and sustainable treatment of newborns. However, the current workflow includes laborious steps such as manual punching and extraction of the blood spots. The direct and automated extraction and mass spectrometric analysis of dried blood spot is of high importance in this field to optimize analyses performance, safety and to reduce costs [1, 2]. Therefore an automated method for the analysis of dried blood spots for direct mass spectrometric screening of amino acids and acylcarnitines has been developed. The method was tested and validated in high throughput conditions.

Materials and Methods: A fully automated dried blood spot sampler, the DBS-MS 500,(CAMAG, Muttenz BL, CH) and a 6410 triple quadrupole mass spectrometer (+ESI) equipped with an 1100 series quaternary HPLC pump (Agilent, Santa Clara CA, USA) were used. Solvents and additives were provided by Sigma-Aldrich (Buchs SG, CH). Standards for amino acids and acylcarnitines were provided by Chromsystems (München Ba, D). The Method was validated according to ICH guidelines [3].

Results: Protocols for the fully automated scanning, extraction and mass spectrometry analysis of amino acids and acylcarnitines in less than 3 minutes have been established. The sustainability of this method was tested in a series of about 300 samples and verified over a 10 days period. Intraday and interday variations were found to be below 20 %. Linearity was above R2 = 0.97 for all compounds. Detection limits have been tested and proved to be within acceptable values for all analytes.

Acknowledgment: Thanks to CTI (Grant Nr. 16898.1 PFLS-LC) for funding this project

1. Neugeborenscreening Kinderspital Zürich http://www.neoscreening.ch
2. Fingerhut et. al Rapid Commun. Mass Spectrom. 2014, 28, 965–973
3. ICH Validation Guidelines http://www.ich.org

P10: Rapid Phospholipid Characterization Using a Novel Intelligent Workflow on a Tribrid Orbitrap Mass Spectrometer

Reiko Kiyonami¹, Yasuto Yokoi², Guido Sonsmann¹ and David A Peake¹

¹Thermo Fisher Scientific, San Jose, CA, USA ²Mitsui Knowledge Industry, Tokyo, JAPAN

Background: Positive and negative HCD MS/MS data are often required for characterization of PCs. Here we report that individual PC species can be fully characterized within one positive LC/MS/MS run using an intelligent workflow.

Materials and Methods: Egg PC and human serum were used as test samples. All experiments were performed using a Thermo Scientific Dionex 3000 RSLC pump connected with the Thermo Scientific Tribrid OT Fusion mass spectrometer. An intelligent data dependent workflow was used, in which a full MS scan (120K) was followed by top15 HCD MS/MS (30K) and an additional CID MS/MS (30K) was triggered if the polar head group of PC fragment ion (184.0733) was detected from the HCD spectrum. Another two approaches were used to compare with the workflow, including 1) Top 15 MS/MS data dependent experiments on both positive mode and negative mode, respectively and 2) Top 10 MS/MS data dependent experiment using positive/negative online switching. LipidSearch software (Thermo Scientific) was used for lipid identification.

Results: More than one hundred PC lipids were identified and characterized using the combined HCD and CID MS/MS data, or alignment of positive/negative MS/MS data. More low abundance PC species were fully characterized using the new workflow. The intelligent workflow developed here was then applied to the analysis of total lipid extracts obtained from human serum samples for large scale lipid profiling with on-going experiments.

Conclusion: Poductivity and sensitivity for phosphatidylcholine characterization was improved using a novel intelligent hybrid dissociation workflow on a Tribrid Orbitrap MS.

P11: Revolutionizing Data Independent Acquisition on Q-q-OT-IT Tribrid Mass Spectrometers

Michael Blank¹, Romain Huguet¹, Ryan Bomgarden², Rosa Viner¹, Andreas Huhmer¹, Neelu Puri³,  Marc Guender¹, Vlad Zabrouskov¹

¹Thermo Fisher Scientific, San Jose, CA, ²Thermo Fisher Scientific, Rockford, IL, ³University of Illinois at Chicago, Rockford, IL

Data Independent Acquisition (DIA) represents a powerful screening technique for the comprehensive and accurate quantitation of biological samples, particularly their protein component. As the method sequentially collects MS/MS fragmentation spectra on all ions within a given m/z range, it affords the opportunity for retrospective analysis of unknowns and new targets of interest. Obtaining peak performance from a DIA analysis demands an optimum balance of instrument speed, sensitivity, and selectivity to overcome the complexity that results from using larger isolation windows while still maintaining reproducibility of quantitation for low abundance analytes.

Two non-small cell lung cancer lines, one showing marked resistance to the tyrosine kinase inhibitor Erlotinib, were established and used to assess the quantitative performance of the Thermo Fisher Scientific Orbitrap Fusion Lumos. Following a series of data dependent acquisition (DDA) discovery experiments to build a spectral library, several targeted  experiments were undertaken including wide-isolation selected ion monitoring (WiSIM), multiplexed DIA, and parallel reaction monitoring (PRM). An Easy nLC 1000 uHPLC system  and 75 uM x 50 cm PepMap column were employed with gradients varying from 60 min to 150 minutes.

The spectral library generated from the use of data dependendent acquisition on the Orbitrap Fusion included more than 6000 protein groups, with as many as 5000 protein groups and 27000 unique peptides being identified in a single LCMS run. Use of WiSIM DIA enabled quantitation over at least 4 orders of magnitude for this sample thanks in part to the high resolution of the MS1 scan and sensitivity of the ion trap for MS/MS detection. The results obtained showed a general reduction in expression of proteins belonging to the canonical MAPK pathway and an increase in abundance of downstream proteins of the mTOR pathway for the drug resistant cell line, consistent with previous results obtained on the Thermo Fisher Scientific Q Exactive HF MS. Additionally, the use of multiplexed windows on the Orbitrap Fusion allowed for the employment of much narrower isolation windows and the detection of MS/MS spectra with high resolution accurate mass. Subsequent analysis by parallel reaction monitoring (PRM) shows greater sensitivity, selectivity, and dynamic range than for either of the two DIA methods and proved to be an ideally suited follow-up/alternative experiment for the quantification of those targets which were not suitably quantified by either of the two DIA methods.

P12: A new ICP-TOFMS for the analysis of micro- and nanosamples

Olga Borovinskaya, Martin Tanner

 Tofwerk AG, Uttigenstrasse 22, Thun, Switzerland

A growing interest in the elemental analysis of very small samples such as microdroplets, nanoparticles1 or single cells2 has created a high demand for faster and more sensitive inductively coupled plasma mass spectrometers (ICP-MS). Simultaneous detection over the entire mass range and high temporal resolution are key parameters for the accurate and precise measurement of very short transient signals (200 µs- 10 ms) produced from small sample volumes.

We present the icpTOF, a new ICP-MS featuring a time-of-flight mass analyser. It records the entire mass spectrum with µs time resolution. The analytical performance of the new instrument will be evaluated on several selected applications, covering the following topics.

1. Discrete single microdroplets as sample introduction system for the elemental analysis of pl-nl volumes of liquids.
2. Determination of elemental composition, size and number-concentrations of engineered nanoparticles present in aqueous media at very low amounts (103-105 particles/ml).
3. Direct multi-element imaging of geological and biological samples in combination with laser ablation systems.

The icpTOF opens up new possibilities and ways for the analysis of micro- and nanosamples. Strengths and features of fast simultaneous detection will be discussed and demonstrated in several applied studies.

1. Laborda, F.; Bolea, E.; Jiménez-Lamana, J. Anal. Chem. 2013, 86, 2270-2278.
2. Bendall, S. C.; Simonds, E. F.; Qiu, P.; Amir, E.-a. D.; Krutzik, P. O.; Finck, R.; Bruggner, R. V.; Melamed, R.; Trejo, A.; Ornatsky, O. I.; Balderas, R. S.; Plevritis, S. K.; Sachs, K.; Pe’er, D.; Tanner, S. D.; Nolan, G. P. Science 2011, 332, 687-696.
3. Borovinskaya, O.; Hattendorf, B.; Tanner, M.; Gschwind, S.; Gunther, D. J. Anal. At. Spectrom. 2013, 28, 226-233.

P13: Electrochemical Reduction of Disulfide Bonds for Superior Analysis of Protein Therapeutics by MS and HDX-MS

Jean-Pierre Chervet1), Agnieszka Kraj1), Laurent Rieux1), Daniel Vetter2),
Esben Trabjerg3), and Kasper Rand3)

1) Antec, Zoeterwoude, The Netherlands 2) ERC GmbH, Riemerling, Germany
3) University of Copenhagen, Copenhagen, Denmark

A novel method for the efficient and fast reduction of disulfide bonds in peptides and proteins combined online with mass spectrometric detection is presented. The method utilizes an alternating potential pulse, applied to an electrochemical cell and does not require any reducing chemicals.

The cell was used in direct infusion electrospray MS and integrated in a Hydrogen-Deuterium Exchange (HDX)-MS workflow, resulting in a fast and fully-automated platform for the characterization of protein therapeutics [1].

A Monoclonal Antibody (MAb) and Nerve Growth Factor-β (NGF) were used to exemplify the applicability of this approach. The MAb disulfide bonds were efficiently and selectively reduced into light and heavy chains, providing high sequence coverage of 96% and 87% for the heavy and light chain of this IgG1-antibody, respectively. The 3 disulfide bonds of the tight cystine knot of NGF were fully reduced by this on-line electrochemistry-HDX-MS approach, resulting in a 99% sequence coverage, meanwhile the reduction by traditional tris(2-carboxyethyl) phosphine (TCEP) resulted in only 46% sequence coverage.

[1] E. Trabjerg et al., Anal. Chemistry 87 (2015) 8880 - 8888

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P14: Targeted proteomics to find biomarkers of glucocorticoid exposure in zebrafish (Danio rerio) embryos

Anita O Hidasi1,2, Ksenia J Groh1,3, Kristin Schirmer1,2,4, Marc J-F Suter1,4

1Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, Dübendorf, 8600, Switzerland 2 EPFL, School of Architecture, Civil and Environmental Engineering, Lausanne, 1015, Switzerland 3 ETHZ, Department of Chemistry and Applied Biosciences, Zürich, 8093, Switzerland 4 ETHZ, Institute of Biogeochemistry and Pollutant Dynamics, Zürich, 8092, Switzerland
E-mail contact: anita(dot)hidasi(at)eawag(dot)ch

Synthetic glucocorticoids (GCs) are frequently used in medicine. They have been detected in the aquatic environment in the ng/L range. GCs mimic cortisol, the natural stress hormone, by acting through the glucocorticoid receptor (GR) and altering target gene expression. Endogenously, cortisol is produced in fish when the hypothalamus-pituitary-interrenal (HPI) axis is activated. In our work, zebrafish embryos are used to investigate whether exposure to environmentally relevant concentrations of a model GC, clobetasol propionate (CP), results in GC-related effects, such as immunosuppression.

Targeted proteomics analyses are conducted on tryptic peptide digests of embryos exposed to ≤ 100 nM CP. The proteotypic peptide targets are monitored using LC-MS and selected reaction monitoring (SRM). GR target genes already examined in zebrafish at the mRNA level were selected to monitor their protein products. The 36 targeted proteins have different physiological roles, e.g. myogenesis, vitamin D metabo­lism, immune function, and cardiogenesis. The SRM assays were developed using the selected proteo­typic peptides synthesized in order to find the most intense transitions (i.e. m/z of the peptide and m/z of one of its fragments) to monitor in the embryo extracts. Nine out of the selected 36 proteins were detected in control zebrafish embryo digests (Figure 1). The nine detected proteins were monitored after exposing zebrafish embryos to 10 and 100 nM CP from 4 to 5 days post fertilization (dpf) (Figure 2). Only Anxa1b, an immune function related protein, was strongly down-regulated after exposure to 100 nM CP. Embryos exposed to ≤ 100 nM CP from 1 hours post fertilization to 4 dpf showed significant immunosuppression after challenging them with bacterial lipopolysaccharide (LPS). Thus, experiments are ongoing to investigate the effects of this exposure frame on the nine proteins with the developed SRM assay.

The described work characterizes the CP effects on the molecular level. This study is focusing on finding potential GC-specific and sensitive biomarkers that can be used in developing a bioassay to monitor GCs in water samples. The bioassay will be used in effect-directed analysis in order to identify unknown compounds able to interfere with the HPI axis.

Figure 1:  The selected 36 protein targets grouped by their physiological functions. The highlighted proteins were detected in control zebrafish embryo digests

Figure 2:  Relative protein abundances compared to control zebrafish embryos. Embryos were exposed to CP from 4-5 dpf. N = 3 indepent experiments with n = 35-40 embryos/exposure group. Error bars represent standard deviation.

P15: Using High-Resolution Mass Spectrometry and Tandem Mass Spectrometry to Prioritize Nontarget Compounds in Wastewater for Further Identification 

 Jennifer Schollée^1,2, Emma Schymanski¹, Sven Avak^1,3, Martin Loos^1,2, and Juliane Hollender^1,2 

¹Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland ²Institute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, 8092 Zürich, Switzerland ³Universität Zürich, 8092 Zürich, Switzerland 
Contact: jennifer(dot)schollee(at)eawag(dot)ch Überlandstrasse 133, Dübendorf 8600, Switzerland 

Transformation products (TPs) are formed during the wastewater treatment process (WWTP) and may affect downstream aquatic communities or contaminate drinking water resources. Lab studies and targeted screening can only provide a limited picture of the compounds present. This project focuses on using nontarget screening with liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) and tandem MS (MS/MS) to prioritize peaks for structure elucidation. A combination of LC-HRMS, MS/MS, metabolic logic, and multivariate analysis was used to find interesting compounds. 

Influent and effluent samples from an activated sludge treatment train were collected from a Swiss WWTP. Samples were enriched with solid-phase extraction and analyzed with LC-HRMS using electrospray ionization in positive mode. Pre-processing was carried out with the software R, including peak picking, isotope/adduct clustering, and feature building. Principal component analysis (PCA) was used to classify peaks and links between the two groups were explored based on known biotransformation reactions and similarity of the MS/MS spectra. 

From the PCA, 9758 features were classified as potential parents and 3011 features as potential TPs. Using 7 biotransformation reactions, 4249 potential links were found and 156 were selected for targeted MS/MS based on the workflow prioritization. MS/MS fragmentation from the proposed parent and TP were compared, and 19 links with an MS/MS similarity score of higher than 0.5 were chosen for further structure elucidation. In one example, the MS/MS of the parent and TP both contained fragments at masses 103.0390 and 89.0597. Other linkages were also found with these fragments, with a mass difference of 44.0264 mDa [C2H4O] between the parent compounds. This homologue series was expanded and the EICs visualized, indicating that while some of the lower masses were removed after treatment, the higher masses appeared unchanged or increased. Preliminary results indicate that by applying multivariate analysis and a nontargeted workflow, relevant compounds can be found, including parent and TP pairs but also potentially new homologue series.