2017 SGMS Meeting

Oral abstracts Registration Abstract Submission Poster Presentations Sponsors

The 35th meeting of the SGMS will be held at the   Dorint Resort Blüemlisalp Beatenberg, October 25-26-27, 2017 high above Lake Thun in the Bernese Oberland, with a scenic view of the Swiss Alps!

Confirmed Invited Speakers

• Frantisek Turecek, University of Washington, USA
• Julia Chamot-Rooke, Pasteur Institute, France
• Sönke Szidat, University of Bern, Switzerland
• Kevin Pagel, University of Berlin, Germany
• David Goodlett, University of Maryland, USA
• Hans Wessels, Radboud University Medical Center, the Netherlands

Program

Wednesday, 25 October 2017

Thursday, 26 October 2017

Friday, 27 October 2017

Registration is now closed

The registration form is available in WORD or PDF and should be sent to [ ] not later than October 1st, 2017. There is absolutely 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.

In honour of our 35th anniversary, we have a social event away from the conference hotel. Please indicate on the registration form if you are a vegetarian.

ABSTRACT SUBMISSION (see below) should be sent to [  ]

All PhD students attending the annual SGMS meeting pay a reduced fee of CHF 150.-, but will have to share rooms.

Submission of Abstracts

Next, to the plenary lectures, there will be time for several oral presentations from various participants as well as poster presentations. The time allotted will be 20 minutes.

We highly encourage students to submit an oral presentation this year !!

Early deadline for abstract submission for both talks and posters is July 1^st. The extended deadline is September 1^st. Abstracts submitted before July 1^st will have priority. Please submit your abstract including author's name and address directly to  abstract(at)sgms(dot)ch. The abstract should not exceed 2500 characters.

ABSTRACT DEADLINE EXTENSION: 20 Sept 2017

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.
• We can read most of the common word processing formats.
• Figures most be supplied as seperate *.jpg images.
• Do not use halftoning or colour: We publish in pure black and white.
• Include your e-mail address.

O1: František Tureček: Biological Radicals in the Gas Phase

František Tureček

Department of Chemistry
University of Washington
Klaus and Mary Ann Saegebarth Professor

Bagley Hall, Box 351700
Seattle, WA 98195
USA

Structures of biomolecular ions are the topic of perennial importance and interest. With the advent of electron-based methods of ion dissociation it became possible to generate transient intermediates of radical reactions of biomolecules such as peptides, proteins, and DNA of relevance to radical enzyme reactions and radiation damage, and study them by tandem mass spectrometry. The lecture will address new techniques to generate biological radical ions in the gas phase and investigate their structure and ground- and excited electronic-state properties using UV-VIS photodissociation action spectroscopy and electronic structure calculations.

O2: Laser-Ionization Mass Spectrometry (LIMS) with XUV Radiation for High Resolution Stoichiometric Microanalysis

Yunieski Arbelo¹, Rafael Muller^1,2, Ilya Kutzentzov³, Carmen Menoni³, Greta R. Patzke², Jorge J. Rocca³, Davide Bleiner^1,2 davide(dot)bleiner(at)empa(dot)ch

¹Empa – Swiss Federal Laboratories for Materials & Technology, Überlandstrasse 129, CH-8600 Switzerland; ²University of Zurich – Dept. Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; ³Colorado State University, Dept. Electrical & Computer Eng., Fort Collins, Colorado 80523, USA

Laser microanalysis profited from the sensitivity of ICP-MS, achieving detection limits in the low mg/kg across the mid and high mass range. The flexibility and rapidity of the method have been discussed in hundreds of papers. Remaining challenges are the non-stoichiometric sampling (“fractionation”) and the redundant sampled mass, and heterogeneity in particle size, with respect to the fraction that is eventually atomized/ionized in the plasma and measured at the detector. Further, the laser diffraction prevents the achievements of spot sizes smaller than ca. 5µm.

XUV using laser-produced or discharge-produced hot/dense plasmas can close such gaps. XUV laser-action has been also accomplished on table-top systems [1-3], which in the time of large beamlines with limited access, helps bridging the gap between the user and the tools.

High-res microanalysis was performed using XUV radiation at =10—50nm. The high photon energy generates ions, from H onwards, directly from the sample, without an ICP intermediate. The online detection was performed with self-developed TOFMS with a reflectron or a novel “transmistron”. XUV-LIMS allowed quasi-non-destructive analysis, and results on single-shot chemical imaging will be presented

1. Arbelo, Y, F. Barbato, D. Bleiner (2017) He-doped pseudospark as a home-lab XUV source beyond the beamtime bottleneck, Plasma Sources Sci. Technol., in press
2. Bleiner, D., Arbelo-Pena, Y., Masoudnia, L., & Ruiz-Lopez, M. (2014). Table-top X-ray lasers using a plasma gain-medium: Limits and potentials. Physica Scripta, 2014(T162), 014050.
3. Staub, F., Imesch, C., Bleiner, D., & Balmer, J. E. (2012). Soft-X-ray lasing in nickel-like barium at 9.2 nm using the grazing-incidence scheme. Optics Communications, 285(8), 2118-2121

O3: Hans Wessels: Innovative Top-Down and Glycoproteomics in Translational Healthcare

 Hans Wessels

Radboud University Medical Center
Center for Proteomics, Glycomics & Metabolomics
Geert Grooteplein Zuid 10
NL-6500 HB Nijmegen

The Netherlands
 

Proteins are highly versatile macromolecules that enact a wide range of biological functions essential to life. At least several million unique proteins exist in the human body which are, surprisingly, encoded by only fifteen to twenty thousand genes. The divergent number of proteins compared to genes is due to protein variants, or “proteoforms”, that originate from a single gene as unique combinations of amino acid sequence variations and/or post-translational modifications. It is important to note that out of all possible proteoforms only some may correlate with disease biology which stresses the need for accurate structural information in clinical science. Recent advances in emerging technologies such as Top-Down proteomics and glycoproteomics provide exciting opportunities for understanding disease processes, identifying therapeutic targets, and designing individualized diagnostics. Top-down proteomics preserves key biological information by analyzing whole proteins directly by tandem mass spectrometry whereas glycoproteomics can be exploited to obtain site-specific glycan occupancy information via glycopeptide profiling. Both technologies have specific advantages that complement one another to enable comprehensive characterization of glycoproteins.

O4: Spatiotemporal damage of enzymes resolved by proteomic techniques

Elisabeth Janssen

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

Various biological processes are governed by enzymes and their overall impact depends critically on how longs they remain active. Thus, methods to follow both changes in structure and functionality are crucial to comprehend inactivation mechanisms and to predict enzymatic life times.  Here we employ proteomics techniques and enzymology to investigate the stability of enzymes using high-resolution mass spectrometry. Other than in traditional proteomics, we follow the decay kinetics of every peptide fragment and aim for full sequence coverage of the enzyme.

This technique enables us to monitor a three-dimensional protein during a decay process by (1) taking it apart into its peptide fragments, (2) determine their stability individually, and (3) reconstruct the protein with its site-specific fingerprint of degradation rates. We create heat maps for different oxidation reactions that visualize the reactive centers within the protein.

Here, we demonstrate this spatiotemporal proteomic technique for the photoinactivation of three ubiquitous extracellular enzymes: phosphatases, aminopeptidases and glucosidases. Various bacteria and algae excrete extracellular enzymes and they play central roles in the biogeochemical cycling of nutrients and carbon in the aqueous environment. One major pathway of inactivation is represented by photochemical reaction in the sunlit surface water. Here, we can visualize on the one hand that tryptophan residues are the initial targets of direct photochemical reactions and antioxidants can reduce oxidized tryptophan intermediates hence decelerate the enzyme`s decay. We further demonstrate how subsequent reactions occur within the protein structure leading to heterogeneous damage along with inactivation.

O5: Julia Chamot-Rooke: Top-Down Proteomics: The Next Step in Clinical Microbiology

Julia Chamot-Rooke

Mass Spectrometry for Biology Unit
Pasteur Institute

25 rue du Dr. Roux
75015 Paris

France

In the last decade, the introduction of MALDI-TOF Mass Spectrometry (MS) for rapid microbial identification has revolutionized the field of clinical microbiology. The approach has been widely embraced by hospitals as it is faster, more accurate, and less expensive than conventional phenotypic or genotypic methods. However, it suffers from important limitations. The discriminatory power of the technique is insufficient to differentiate closely related bacteria or sub-species and more importantly resistance and virulence cannot be addressed. There is therefore a crucial need for innovative analytical approaches allowing an efficient and more accurate bacterial identification based on protein analysis.

Top-down proteomics is an emerging technology based on the analysis of intact proteins using very high-resolution mass spectrometry [1]. It provides the highest molecular precision for analysing primary structures by examining proteins in their intact state, leading to more straightforward and reliable results than the classical bottom-up approach based on protein enzymatic digestion.

Top-down proteomics is particularly suited to the analysis of bacterial proteins, which are of small size (< 30 kDa) and produced in large amount by bacterial pathogens [2,3]

In order to use top-down proteomics for clinical microbiology applications [4], we set up an integrated platform in which all steps have been carefully optimized: bacterial lysis, protein extraction, LC-MS/MS analysis of intact proteins and data processing. For this last point, a new software tool, which branches from an existing one [5], but tailored towards top-down proteomics data, has been developed. This new software, based on machine learning, can rapidly cluster the thousands of MS/MS spectra obtained in top-down LC-MS/MS experiments, compare datasets obtained from various bacterial pathogens and identify discriminative spectra.

Using this integrated top-down platform, we show that it is now possible to differentiate closely-related pathogens that are impossible to distinguish with MALDI-TOF MS, in only a few hours after bacterial culture. We also highlight the great potential of top-down approaches to delineate complete protein sequences (including C-terminal and N-terminal extremities) and detect single References nucleotide polymorphisms.

References

1. Proteoform: a single term describing protein complexity. L.M. Smith, N. L. Kelleher and the Consortium for Top-Down Proteomics, Nature Methods 10 (3), 186-187 (2013).
2. Posttranslational Modification of Pili upon Cell Contact Triggers N. meningitidis Dissemination. J. Chamot-Rooke et al., Science, 331, 778-782 (2011).
3. Complete posttranslational modification mapping of pathogenic Neisseria meningitidis pilins requires top-down mass spectrometry. J. Gault et al., Proteomics (2014) DOI: 10.1002/pmic.201300394.
4. Top-down proteomics in the study of microbial pathogenicity. J. Gault et al. in MALDI-TOF and Tandem MS for Clinical Microbiology, Wiley (2017)
5. DiagnoProt: a tool for discovery of new molecules by mass spectrometry. A.R. Silva et al. Bioinformatics (2017) DOI: 10.1093/bioinformatics/btx093

O6: Glutathione S-transferase protein expression in different life stages of the vertebrate model zebrafish (Danio rerio)

Alena Tierbach^1,3, Ksenia J Groh⁴, Kristin Schirmer^1,2,3, Marc J-F Suter^1,2

¹Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland
²ETH Zürich, Swiss Federal Institute of Technology, Department of Environmental Systems Science, 8092 Zürich, Switzerland
³EPF Lausanne, School of Architecture, Civil and Environmental Engineering, 1015 Lausanne, Switzerland
⁴Food Packaging Forum Foundation, 8045 Zürich, Switzerland

Zebrafish has gained growing interest from the scientific community due to its multifaceted applications in biomedical sciences and toxicology. Yet, the development of defense systems, such as phase II biotransformation pathways, during zebrafish early life stages and in adulthood is largely unexplored.

Glutathione S-transferases (GSTs) is one enzyme family that plays a major role in phase II biotransformation processes. GST catalyzed conjugation reactions are considered a critical contributor to detoxification and clearance of various intracellular metabolites, but also natural toxins and xenobiotic compounds.

Given the important role of GSTs in xenobiotic metabolism, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The MRM assays developed within the study enable the monitoring of specific GST isoenzymes and GST classes through a combination of proteotypic peptides and peptides shared within the same class.

We could show that some GST classes (alpha, mu, pi and rho) are present in zebrafish embryos as early as 4 hours post fertilization (hpf). The majority of GST enzymes, however, were expressed at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ-dependent, with most of the GST classes showing the highest expression in the liver.

The early expression of GSTs during zebrafish embryogenesis and the wide range of cytosolic GST classes expressed in adult fish supports the use of zebrafish as a model organism in chemicals-related investigations.

O7: Proteins in food matrices: from multiple reaction monitoring to intact protein analysis

Christophe Fuerer, Sandrine Wagnière, Guillermo Julián Moreno, and Michael Affolter

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

Protein analyses are routinely performed in the food industry to assess the composition, integrity, and authenticity of finished products and raw materials. Protein quantification by trypsin digestion and Multiple Reaction Monitoring (MRM) can be applied to food matrices such as raw milk, but finished products are more challenging to analyze because proteins can become modified during the manufacturing process. Thermal processing and drying favor Maillard reactions and lead to the formation of sugar adducts. These adducts are mostly found on lysine residues and thus interfere with trypsin proteolysis and subsequent MRM analyses. To address this issue, we developed an LC-MS assay based on intact proteins that allows a precise fingerprinting of the major milk proteins and their proteoforms. Examples will be presented to highlight the potential and challenges of this method for food composition, integrity, and authenticity analyses.

O8: Sönke Szidat: Radiocarbon analysis with accelerator mass spectrometry: recent developments and applications

 Sönke Szidat

Department for Chemistry and Biochemisty
Oeschger Centre for Climate Change Research

University of Bern

Freiestrasse 3

CH-3012 Bern

Switzerland

The mass spectrometric analysis of radiocarbon (¹⁴C) and other long-lived radioisotopes is challenged by very low abundances (ambient ¹⁴C/¹²C ratios are <10^-12) and interferences from stable isobars or molecular fragments of the same mass. Accelerator mass spectrometry (AMS) is a powerful tool for radiocarbon measurement due to high ion currents, the complete suppression of the stable isobar ¹⁴N and the efficient breakup of molecular ions such as ¹³CH⁺ in a collision cell within the accelerator (the so-called stripper). However, any kind of structural information of organic compounds is lost. Recent improvements of the stripping process and the gas-filled particle detectors paved the way for AMS systems with substantially smaller accelerators and, thus, for smaller and more robust devices.

At the University of Bern, the AMS system MICADAS (MIni CArbon DAting System) was installed in 2013. It is equipped with a 200 kV tandem accelerator and requires a floor space of (only) 2.5 × 3 m². The hybrid ion source allows the analysis of both, graphite and gas targets. Whereas graphite is often used for routine dating applications of samples containing >0.1 mg carbon, gaseous CO₂ is introduced into the ion source for smaller samples down to 1 µg carbon. Furthermore, it opens up the possibility of hyphenated systems employing CO₂-producing analytical instruments, such as an elemental analyser, temperature-ramped combustion devices or even chromatography coupled with oxidation. This presentation will summarize these technical developments and give examples of applications from atmospheric sciences, archaeological dating and pharmacokinetics.

O9: Investigation of the suitability of high-resolution ion mobility spectrometry-mass spectrometry for the rapid differentiation of legal and illegal marijuana

Marianne Hädener¹, Michael Kamrath², Wolfgang Weinmann¹, Michael Groessl³

¹ University of Bern, Institute of Forensic Medicine, Bühlstrasse 20, CH-3012 Bern
² Tofwerk, Uttigenstrasse 22, CH-3600 Thun
³ University Hospital Bern, Department of Nephrology and Hypertension, Freiburgstrasse 15, CH-3010 Bern

The isomers Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the two most abundant cannabinoids present in marijuana, mainly in their carboxylic acid forms (“-acid-A”). In Switzerland, only plant material with a total THC content of more than 1% is subject to the Narcotics Act. CBD-rich/THC-poor marijuana products (THC < 1%) can be legally sold and their consumption has become increasingly popular in Switzerland. Legal and illegal marijuana cannot be reliably distinguished by appearance or smell, but only by forensic chemical analysis of organic extracts thereof. This is usually achieved by quantitative GC- or LC-based techniques. The present study aimed to investigate the suitability of high-resolution ion mobility spectrometry-mass spectrometry (IMS-MS) as an alternative, faster method for the analysis of the isomeric compounds THC and CBD as well as their biogenetic precursors CBD-acid A and THC-acid A.

Measurements of methanolic plant extracts were performed on a TOFWERK IMS-TOF instrument comprising a nanoESI source and a pressure- and temperature-controlled IMS cell interfaced to a high-speed TOF mass spectrometer. Acquisition time was 1 min. Operating the IMS drift tube at atmospheric pressure allowed the use of high electric field strength, thus achieving high IMS resolution (> 150 with post-processing) and baseline separation of the isomeric pairs CBD/THC (positive ion mode) and CBD-acid A/THC-acid A (negative ion mode) which only differ by about 1% in their collision cross sections. Drift times were 38.5, 39.0, 43.5 and 44.0 ms, respectively. Quantification was accomplished using six-point calibration curves (20 – 1000 ng/mL for CBD/THC; 5 – 500 ng/mL for the acids) and deuterated internal standards. IMS-MS analysis of marijuana extracts showed good agreement with a validated HPLC-DAD method except for low concentrations of THC-acid A. This issue is currently being investigated further.

Our preliminary results suggest that high-resolution IMS-MS is a promising approach for the differentiation and quantification of isomeric cannabinoids, requiring a shorter analytical run time than currently used methods.

O10: Chiral Liquid Chromatography to Quantify Enantiomeric Fractionation of Selected Organic (Micro) Pollutants in the Aquatic Environment: Method Development and Validation

Dominique Rust

Eawag, Environmental Chemistry, Überlandstrasse 133, 8600 Dübendorf, Switzerland

Nowadays an increasingly large number of commercially available pharmaceuticals and pesticides feature stereogenic (chiral) structural elements. Released to the (aqueous) environment, these chemicals might exhibit highly stereoselective chemical (transformation) behaviour and ecotoxicity, for which reason the determination of enantiomeric compositions of organic (micro) pollutants is crucial.

In environmental analytical chemistry, progress on chiral multi-residue methods has been achieved in recent years. However, the range of substances that can enantiomerically be resolved is still limited, also due to the great selectivity of the chiral stationary phases employed.

In the presented project, we tried to develop one or more chiral liquid chromatography-electrospray ionization-high resolution mass spectrometric (LC-ESI-HRMS) multi-residue methods in order to enantiomerically separate as many of the targeted analytes as possible. For this purpose, 39 pharmaceuticals of various therapeutic applications and five pesticides were selected, all compounds containing one stereogenic center, however covering a comprehensive range of structural differences, polarity, molecular weight and pKa values.

After extensive tests of MS-compatible mobile phase compositions under diverse elution conditions, and by employment of a vancomycin-based ChirobioticTM V chiral stationary phase, four final chromatographic methods were selected to enantiomerically resolve 20 compounds of different pharmaceutical and pesticide classes, some of which have not been reported in a chiral multi-residue LC-MS method yet.

For method validation, two sample preparation workflows, i.e. direct injection and evaporative pre-concentration prior to analysis, were applied. Validation of the most promising method was conducted with reference compounds dissolved in nanopure water and in three environmental matrices, i.e. surface water, effluent wastewater and influent wastewater.

O11: Kevin Pagel: Gas-Phase Structural Analysis of Complex Carbohydrates

 Kevin Pagel

Institute of Chemistry and Biochemistry
Freie Universität Berlin
Takustraße 3
14195 Berlin
Germany
 

Currently, the vast majority of glycans are characterized using mass spectrometry-based techniques (MS). Measuring the molecular weight of a sugar, however, immediately poses a fundamental problem: entire classes of monosaccharide building blocks exhibit an identical atomic composition and, consequently, an identical mass. Therefore, glycan MS data can be highly ambiguous and often it is not possible to clearly assign a particular structure.

A promising approach to overcome this limitation is to implement an additional gas-phase separation step using ion mobility-mass spectrometry (IM‑MS). Here, ions travel through a gas-filled cell aided by an electric field and are separated according to their collision cross section (CCS). Here, we demonstrate the potential of IM-MS to be used as a tool for the separation and identification of isomeric glycans and glycopeptides. First, six synthetic oligosaccharide isomers that differ with respect to their composition, connectivity, or configuration were analysed. Our data reveal that linkage- and stereoisomers, which are difficult to distinguish using established techniques, can be separated and unambiguously identified on basis of their CCS.¹ Second, we extended our investigations to glycopeptides. Our data show that glycopeptides, which merely differ in the regiochemistry of the attached glycan can be distinguished using fragmentation and subsequent IM-MS analysis.²

Finally, we recently assessed the potential of cold-ion spectroscopy for oligosaccharide analysis.³ Gas-phase IR spectra of a series of synthetically derived mono-, di- and trisaccharide standards were recorded. For each of these oligosaccharides, unique and highly diagnostic absorption patterns with a variety of well-resolved bands was obtained. In some cases, the line-width in the spectra was equal to the corresponding bandwidth of the laser radiation (FWHM ≈ 4 cm^-1). This unprecedented resolution reveals remarkable differences in the overall IR signatures and allows a simple, fingerprint-based discrimination between isomers, even if they merely differ in the stereochemistry of a single OH group.

References

1.              J. Hofmann, H. S. Hahm, P. H. Seeberger, K. Pagel, Nature 2015, 256, 241-244.

2.              H. Hinneburg, J. Hofmann, W.B. Struwe, A. Thader, F. Altmann, D. Varón Silva, P.H. Seeberger, K. Pagel, D. Kolarich, Chem. Commun. 2016, 52, 4381-4384.

3.              A.I. González Flórez, E. Mucha, D.-S. Ahn, S. Gewinner, W. Schöllkopf, K. Pagel, G. von Helden  Angew. Chem. Int. Ed. 2016, 55, 3295-3299

O12: Structure elucidation of small neurobiological active com-pounds from the venom of the spider Parawixia bistriata

Yvonne Forster¹, Wagner F. dos Santos², Laurent Bigler¹

¹University of Zurich, Department of Chemistry, 8057 Zurich, Switzerland
²University of Saõ Paulo, Department of Biology, Ribeirão Preto - SP, 14040-900, Brazil

The venom of spiders is a rich cocktail of proteins, peptides and small molecules such as acylpolyamines. However, the venom of less than 1% of all spider species have been analysed to date.

Bioactivity studies of the venom of the spider Parawixia bistriata showed that it stimulates the uptake of the neurotransmitter glutamate and inhibits GABA uptake in cortical synaptosomes and COS cell expressing transporters for these neurotransmitters.[1,2] The malfunction of neurotransmitter transporter is closely associated with sever pathological conditions such as epilepsy, Alzheimer’s disease and ischemia. Therefore, the venom is considered as interesting source of probes for novel therapeutic strategies.

The aim of our study was the structure elucidation of the small molecules in the venom of Parawixia bistriata. This was done by UPLC-MS, MS/MS and H/D-exchange experiments. Where the identification of most small molecules needs comparison with reference material, the characteristic fragmentation pattern of the acylpolyamines allow their structure elucidation just based on their MS/MS spectra.[3]

We found several small molecules such as amino acids, nucleotides and betaines. Beyond, the venom contains more than 30 acylpolyamines. To our knowledge, only two of them were described before and for the first time acylpolyamines with and without an amino acid linker were found within the same venom.

1. H.A. Fachim, A.O.S. Cunha, A.C. Pereira, R.O. Beleboni, L. Gobbo-Neto, N.P. Lopes, J. Coutinho-Netto, W.F. dos Santos, Epilepsy Behav. 22 (2011) 158–164.
2. E.A. Gelfuso, J.L. Liberato, A.O.S. Cunha, M.R. Mortari, R.O. Beleboni, N.P. Lopes, W.F. dos Santos, Neurosci. Lett. 543 (2013) 12–16.
3. M. Tzouros, S. Chesnov, L. Bigler, S. Bienz, Eur. J. Mass Spectrom. 19 (2013) 57–69.

O13: Comprehensive Steroid Analysis by GCxGC-TOFMS

Andrea Bileck*, Sofia Verouti, Genevieve Escher, Michael Groessl

Division of Nephrology and Hypertension, Department of Clinical Research, University Hospital Bern, Switzerland

*contact person: andrea(dot)bileck(at)dkf(dot)unibe(dot)ch

Introduction: Steroid metabolites are typically analyzed in biological samples for the screening of various hormonal disorders, such as cortisone reductase deficiency, congenital adrenal hyperplasia, hyper- and hypo-aldosteronism, Cushing’s disease etc. Yet, many of these metabolites are closely related isomers and cannot be separated by LC or one dimensional GC due to their chemical and structural similarity which complicates or even impedes analysis and impacts sensitivity. GCxGC in combination with a fast TOFMS with its superior separation power overcomes this problem.

Experimental:  Experiments were carried out on a GCxGC-TOFMS comprising an electron ionization TOFMS (EI-TOF, TOFWERK), a GC oven (7890A, Agilent) and a thermal modulator (ZX2, Zoex). An HP-1MS column (15 m x 0.250 mm i.d., 0.25 μm film thickness) was used in the 1st, a BPX-50 (1 m x 0.1 mm i.d., 0.1 μm) in the 2nd dimension GC. Steroids standards were obtained from Steraloids (USA). Samples were derivatized using methyloxime and trimethylsilylimidazole.

Results: We present a validated method that enables the absolute quantitation of 40 steroids from different classes (progestogens, androgens, estrogens, glucocorticoids, mineralocorticoids) in biological and clinical samples, e.g. urine and plasma from patients and animal models. The use of GCxGC-TOFMS allows baseline separation and highly sensitive detection of steroid metabolites that cannot be resolved by conventional techniques. Clean mass spectra of single analytes can be recorded with high mass accuracy, enabling the identification of previously undetected steroids and making the comparison to libraries more reliable. We also discuss the wider applicability of the approach for the untargeted analysis of steroids and related metabolites and compare the results to other techniques such as LC-MS/MS and GC-MS.

O14: David R. Goodlett: Lipid A as a Therapeutic and Diagnostic Target

 David R. Goodlett

Professor of Pharmaceutical Sciences
University of Maryland School of Pharmacy
20 North Pine Street, Room N707
Baltimore, MD 21201

USA
 

Lipid A is the membrane anchor for Gram-negative bacteria that holds the much larger lipopolysaccharide (LPS) molecule in place in the outer membrane. Importantly in mammals, Toll receptor 4 (TLR4) recognizes lipid A the result of which can be activation of a cytokine cascade that can aid the host in clearing the infection. Depending of the configuration of the lipid A structure (i.e. the presence or absence of specific length fatty acids or phosphate groups), altered endotoxic activity is observed. Through an effort with the Ernst laboratory, with which we have collaborated for nearly twenty years, we have characterized numerous lipid A structures by mass spectrometry for the purpose of developing it both as diagnostic and therapeutic agents. As a model organism, we have focused on the Gram-negative bacterium, Francisella tularensis subspecies novicida (Fn), which is a murine pathogen with lipid A structures that poorly activated the TLR4 complex thereby delaying activation of the innate immune cascade resulting in lethality.

In order to better understand the structure function relationship of lipid A we are currently developing a lipid A structure-activity relationship (SAR) library (Scott). Having a better understanding of the lipid A SAR will allow us to design novel lipid A structures/functions that are antagonistic or agonistic toward the MD2/TLR4 receptor complex. At the heart of this work is structure determination by mass spectrometry. This is made difficult as lipid A extracts consist of complex mixtures of molecules closely related in structure that are not water soluble. For example, over 100 structural variants of Fn lipid A were characterized using electrospray ionization (ESI) with a linear ion trap (IT) Fourier transform ion cyclotron resonance (FT-ICR) hybrid mass spectrometer (Shaffer; Ting). These results were generated using hierarchical tandem mass spectrometry (HiTMS) by combination of manual and automated data analysis (Ting). Recently, we investigated more efficient ways to characterize lipid A species using quadrupole-CID (q-CID) on a SYNAPT G2 Q-TOF-MS (Yoon), as opposed to our traditional ion trap method (Li), as well as using surface acoustic wave nebulization (SAWN) that does not suffer from the clogging issues of ESI and is less energetic than ESI or MALDI (Huang). Recently, we have begun to explore top-down fragmentation of LPS to characterize simultaneously the lipid A moiety as well as the immunogenic carbohydrate moiety that can encompass as much as 80% of the mass of LPS. Finally, we will review development of lipid A and related compounds from Gram-positive bacteria and fungi to identify microbes in a manner analogous to protein extracts on the Bruker Biotyper (Leung), but with a number of advantages.

References

• Huang Y, Yoon SH, Heron SR, Masselon CD, Edgar JS, Tureček F, Goodlett DR. J Am Soc Mass Spectrom. 2012. 
• Li Y, Yoon SH, Wang X, Ernst RK, Goodlett DR. Rapid Commun Mass Spectrom. 2016.
• Leung LM, Fondrie WE, Doi Y, Johnson JK, Strickland DK, Ernst RK, Goodlett DR..Scientific Reports, 2017, in review.
• Scott AJ, Oyler BL, Goodlett DR, Ernst RK. Biochim Biophys Acta. 2017.
• Shaffer SA, Harvey MD, Goodlett DR, Ernst RK. J Am Soc Mass Spectrom. 2007
• Ting YS, Shaffer SA, Jones JW, Ng WV, Ernst RK, Goodlett DR. J Am Soc Mass Spectrom. 2011.
• Yoon SH, Liang T, Schneider T, Oyler BL, Chandler CE, Ernst RK, Yen GS, Huang Y, Nilsson E, Goodlett DR. Rapid Commun Mass Spectrom. 2016.

O15: DynaStI: an Expert-Curated Dynamic Retention Time Database for Steroidomics

G. M. Randazzo¹, F. Lehmann², J. Boccard¹, R. Liechti², A. J. Bridge³, I. Xenarios², S. Rudaz¹

¹School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, 1211 Geneva 4, Switzerland
²Vital-IT Group, SIB Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland
³Swiss-Prot Group, SIB Swiss Institute of Bioinformatics, CMU, 1211 Geneva 4, Switzerland

Introduction: Hundreds to thousands of metabolites can be simultaneously monitored in biological matrices using untargeted LC-MS experiments. Unambiguous compound identification remains mandatory to draw relevant biological conclusions from the data. As several hits can match a molecular formula, unique molecular identity can be difficult to obtain. Retention time constitutes an essential information to complement HRMS and MSn spectra for positional and constitutional steroid isomers identification [1]. Thus, an automatic steroid annotation based on retention time and HRMS is proposed.

Aims: A web application was developed for automatic steroid annotation using the annotation levels established by the MSI/COSMOS initiatives [3]. DynaStI(Dynamic Steroid Identification) is an expert-curated endogenous steroid database designed for LC-MS steroidomic studies.

Methods: DynaStI collects experimental and in silico (Quantitative Structure Retention Relationship) linear solvent strength (LSS) parameters to predict dynamically the retention time of steroid in any gradient conditions. Experimental LSS measures were obtained from a previous work [1]. The database runs on a MySQL server and a web application front-end/back-end was developed for steroid annotation. To date, the database contains 198 endogenous molecules and each steroid entry includes key chemical information (IUPAC name, CAS number, human curated SMILES, the most abundant ion detected in HRMS) as well as links to major databases, i.e. HMDB, LipidMaps and SwissLipids. DynaStI was validated using different biological samples: the OECD reference H295R cell line [2], mouse testis tissue and human seminal fluid.

Results: DynaStI is a web application developed using a JavaScript/HTML front-end and a PHP/C++ back-end. The front-end allows to access the database, browse/edit/add steroids and upload LC-HRMS features. Compound annotation is managed by a C++ software, DynMetId, which predicts retention times using LSS parameters (log kW and S), compares monoisotopic masses, molecular adducts, isotopic pattern and assigns annotation levels according to COSMOS/MSI standards [3]. DynaStI can be applied after LC-MS raw data processing with Progenesis, Compound Discoverer, MZmine or XCMS. DynaStI thus converts unknown feature lists into a subset of identified steroids giving the possibility to get sound biological information from the data. DynaStI allowed a deeper understanding of mechanistic effects involved in steroidogenesis in cell lines, animal tissue and human biofluids.

Conclusions: A dynamic retention time prediction database was implemented using a user-friendly web application. The database was developed to be publicly available and durable overtime. DynaStI is supported by the Swiss National Science Foundation “Grant 31003A_166658”.

Keywords: Database, steroidomics, Retention Time Prediction, QSRR, UHPLC-HRMS.

References:

1. Randazzo GM et al. Anal Chim Acta 2016; 916: 8-16.
2.  RandazzoGM et al. J Chromatogr B Analyt Technol Biomed Life Sci 2017; in press.
3.  Salek RM et al. GigaScience 2013; 2: 13.

O16: Quantitative analysis of terpene hydroperoxides by LC-MS and GC-MS 

Authors: Susanne Kern, Tina Haupt, Andreas Natsch

Fragrances S&T, Givaudan Suisse SA, Ueberlandstrasse 138, 8600 Dübendorf, Switzerland

Many fragrance ingredients derived from terpenes can undergo oxidation if saturated with air or oxygen and if stored under these conditions as neat oils, not diluted in a product matrix. However, limited information is available what happens if the ingredients are diluted into a product matrix and stored under typical oxygen availability of a formulated product. To address these questions, we especially studied the hydroperoxide products of oxidized limonene and linalool which are known to be skin sensitizing in animal tests. An analytical method had to be developed which is able to detect the hydroperoxides of limonene or linalool in low ppm concentrations in commercial perfumes [1, 2], creams, body lotions, and deodorants selected from the market.

At quality control of raw materials iodometric titration methods are in use which detect different (hydro)peroxides and potentially other oxidants without being selective. However, to characterise and quantify terpene hydroperoxides in fragrance products directly, more selectivity and sensitivity had to be gained using two techniques: a HPLC HR-MS method to analyse linalool hydroperoxides directly, and a GC-MS method detecting the specific alcohols formed upon derivatisation of hydroperoxides by triphenylphosphine reduction. Both analytical methods have some advantages and drawbacks which were evaluated in a ring study [3] and which will be discussed in detail.

Clinical data on over 10’000 patients tested in dermatological centers had indicated that a significant fraction of patients react to oxidized linalool and limonene. However, our methods applied to aged fragrances could only detect trace levels of hydroperoxides which cannot explain the clinical reactions. These analytical methods will now further be applied to solve the question of potential consumer exposure to these sensitizing hydroperoxides and to better understand the clinical reports.

References:

1. S. Kern, T. Granier, H. Dkhil, T. Haupt, G. Ellis, A. Natsch; Stability of limonene and monitoring of a hydroperoxide in fragranced products. Flavour and Fragrance Journal. 2014, 29, 277–286
2. S. Kern, H. Dkhil, P. Hendarsa, G. Ellis, A. Natsch; Detection of potentially skin sensitizing hydroperoxides of linalool in fragranced products. Analytical and Bioanalytical Chemistry. 2014, 406, 6165–6178
3. A. Natsch, B. F. Günthardt, E. Corbi, C. Pérès, A. Düsterloh, H. Leijs, M. van Strien, U. Nilsson, M. J. Calandra, Y. Wang; Interlaboratory evaluation of methods to quantify skin sensitizing hydroperoxides potentially formed from linalool and limonene in perfumes. Flavour and Fragrance Journal. 2017, 32, 277-285.

O17: From Spatially Coherent Ion Clouds to Ion Slabs:  FT-ICR MS at the true cyclotron frequency

Konstantin Nagornov¹, Anton Kozhinov¹, Edith Nicol², Yury Tsybin¹

¹ Spectroswiss, EPFL Innovation Park, 1015 Lausanne, Switzerland
² Ecole Polytechnique, Palaiseau, France

Fourier transform mass spectrometry (FTMS) is the leader in mass accuracy and resolution for mass measurements. However, in many cases its performance is limited by the space charge - the strong Coulombic interaction of ions in the mass spectrometer. Space charge is responsible for peak interferences in the mass spectra resulting in peak shifts and, ultimately, coalescence. Examples include applications involving fluctuating ion sources, such as MALDI, as well as the need to resolve isobaric compounds while keeping the appropriate ion abundance accuracy. The former is pronounced in both, ion cyclotron resonance (FT-ICR MS) and Orbitrap FTMS. The latter is more limiting for Orbitrap FTMS and can be important in pharma industry where isotopic fine structure or doublets of isotopically-labelled metabolites are to be analyzed quantitatively. There, statistics requires reaching a certain number of ions per measurement, whereas ion coalescence puts a too low threshold for the maximum allowed number of ions, rendering isotopic abundance measurements inaccurate even when sufficient resolving power is provided. Therefore, developing FTMS technologies tolerant to the space charge is important.

Recently, we introduced FT-ICR MS at the cyclotron frequency instead of the reduced cyclotron frequency using the narrow aperture detection (NADEL) ICR cells applicable for biomolecular applications [1]. In this presentation, we will describe our current understanding of the underlying principles of ion motion, excitation and detection in the NADEL ICR cells. Interestingly, SIMION simulations demonstrated that ion spatial re-distribution in the NADEL ICR cells during ion detection in the non-quadratic trapping potential is not random, but forms fundamentally novel types of ordered ion structures – elliptic cylinders and ion slabs. These ion structures rotate about their axes with a half-cyclotron (Larmor) frequency as a whole, whereas each of the ions contributing to this motion appears as oscillating along these structures. Consideration of these ion structures provides an alternative and visually-enhanced explanation for the occurrence of FT-ICR MS at the cyclotron frequency. It should also aid in the design and implementation of novel mass analyzers for high-performance FT-ICR MS, e.g., for imaging applications.

1.              Nagornov, K.O., Kozhinov, A.N. & Tsybin, Y.O. J. Am. Soc. Mass Spectrom. (2017) doi: 10.1007/s13361-017-1598-y

O18: High-Throughput Trace Analysis in Solid and Liquid Matrices Using an Active Capillary Plasma Ionization Source

Anna Katarina Huba ¹, Mario Francesco Mirabelli ¹, and Renato Zenobi ¹

¹ ETH Zürich, Department of Chemistry and Applied Biosciences, 8093 Zürich, Switzerland

The ability to perform a fast and simple trace analysis has become a necessity in numerous fields, such as biomedical, forensic, food, and environmental analysis. Our approach to address this demand involves the direct coupling of solid-phase microextraction (SPME) to a very efficient active capillary plasma ionization source based on a dielectric barrier discharge (DBD) [1]. This coupling enables an automated, quick, sensitive, and robust detection of compounds of interest. The versatility and sensitivity of our source and approach were previously demonstrated, for example for the sub-ng/L detection of pesticides [2]. Nonetheless, poorer ionization efficiencies for low-polarity compounds (such as polycyclic aromatic hydrocarbons, PAHs) precluded their detection at comparably low levels. By studying the effect of solvents on the ionization, both from a mechanistic as well as a practical point of view, we were able to boost the ionization efficiency for PAHs and lower the detection limits into the ng/L range.

This allowed us to develop a variety of different high-throughput trace level screening methods in both liquid and solid matrices. For example, a robust method for the detection of organic contaminants in water matrices was developed, requiring a total analysis time of less than 10 minutes per sample and achieving ng/L detection limits both for polar as well as non-polar substances. We also addressed the area of sports doping analysis, a field in which a large amount of samples needs to be screened at various events (e.g., Olympics) and thus enormously benefits from the remarkable speed of our approach. Finally, the applicability of our set-up for the analysis of solid matrices was also investigated, and a successful detection of μg/L levels of pesticides from soils and fruits was achieved with minimal sample preparation. The analysis of solid matrices usually requires lengthy and sample preparation intensive methods, the simplicity and speed of our approach thus provides a very interesting alternative.

References:

1. Maryia M. Nudnova, Liang Zhu, Renato Zenobi, Rapid Communications in Mass Spectrometry, 2012, 26, 1447-1452.
2. Mario F. Mirabelli, Jan-Christoph Wolf, and Renato Zenobi, Analytical Chemistry, 2016, 88, 7252-7258.

O19: Establishment of a quality control mixture for benchmarking LC-MS based dereplication protocols in natural product research

Miwa Dounoue-Kubo^1,2, Pierre-Marie Allard¹, Jean-Luc Wolfender¹

¹School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, Geneva, CMU - 1, rue Michel Servet, CH-1211 Geneva 4, Switzerland
²Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan

Contact: Jean-Luc(dot)Wolfender(at)unige(dot)ch (JL), Miwa(dot)Dounoue(at)unige(dot)ch(MK)

In natural products (NPs) research, recent dereplication workflows are usually based on the treatment of data obtained from generic untargeted metabolite profiling by UHPLC-HRMS/MS of crude extracts [1]. When applied on large collection of complex NPs extracts, the amount of information generated by such approaches is extremely important and requires to finely tune the acquisition parameters. Additionally, various computational solutions, each presenting a great numbers of parametrical options, exists to mine the generated data. In order to assess the overall quality of the NPs profiling and dereplication workflows we propose to use a well-defined Quality Control (QC) sample mixture prepared from the combination of 5 extensively described plants of the European Pharmacopeia for which extensive phytochemical studies have been performed. Plants were selected by mapping the chemical space of their constituents against the space occupied by all NPs known to date for the best coverage and extended polarity range. This mixture was then analysed in different LC gradient conditions and MS/MS acquisition modes on different MS platforms. By using MS-DIAL [2], for example, it could be observed that the number of identified features increased by a factor ranging from 2 to 15% when the measurement time increased by 2 min steps starting from a 5 min gradient. The quality of automated annotations was also assessed in various chromatographic regions and over a large dynamic range in data dependent or data independent modes.

The results suggested that this plant mixture easily generable in any laboratory could become a universal QC mix for the quality evaluation of NPs profiling and annotation workflows. Evaluation of various dereplication strategies in terms of number of annotations and their quality and acquisition modes will be discussed.

1. Wolfender J-L, et al. J Chromatogr A 2015; 1382:136-164.
2. Tsugawa H, et al. Nature Methods 2015; 12: 523–52.

Poster Presentations 

P1: Development of a LC-MS/MS Method for the Quantification of 16 Steroids in Childrens Plasma

L. Stoob, A. Cremonesi and M. Hersberger

Steroids are biological compounds playing an essential role in a multitude of processes ranging from water and salt homeostasis, stress response, development and maintenance of male and female secondary sex characteristics as well as maintenance of pregnancy. To date, many medical conditions are known to alter the steroid homeostasis. Such disorders can be often diagnosed and monitored by measuring the steroid concentrations in either blood or urine. From the clinical point of view, it is more informative to determine their concentrations in blood. However, the analysis of steroids in blood is very challenging due to (a) their structural similarity, (b) their broad dynamic range (from few pmol/L to several umol/L) and (c) their relatively low concentration, especially in newborns and children. Several methods exist for the quantification of steroids in blood, which are mainly based on immunological assays. While these methods are usually very fast, they are often suffering from low specificity due to the structural similarity of the recognized epitopes. Furthermore, such methods can only detect one single steroid at a time and usually require large amounts of sample. Finally, they might lack the sensitivity required to measure steroids in newborns or children samples. It is therefore crucial to develop analytical methods, which enable the simultaneous measurement of many different steroids in a small amount of blood with good precision, accuracy and sensitivity.

The present method is based on solid-phase extraction and subsequent LC-MS/MS analysis in positive polarity and enables the measurement of 16 steroids in just 100 uL of plasma. No chemical derivatization is required. The reported method is linear (r2 > 0.99), sensitive, precise (intra-assay CV = 0.3% – 7.0%; inter-assay CV = 1.5% – 12.4%) and has a broad dynamic range. The method accuracy was assessed by analysing several external quality control samples with established consensus concentrations: >95% of the measurements were within ±2σ and none of them was outside the ±3σ. The recovery ranged between 83% and 120%. A comparison between the results obtained with this method and those obtained with alternative immunological methods revealed good correlations for all tested analytes. However, the concentrations measured by LC-MS/MS were in general slightly lower, probably due to lack of specificity of the immunological assays.

P2: General Unknown Screening – Peak Extraction and Hit Optimization Using a Design of Experiment Approach

Marco P. Elmiger, Michael Poetzsch, Andrea E. Steuer, Thomas Kraemer

Zurich Institute of Forensic Medicine, Department of Forensic Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland

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

Introduction: General unknown screening (GUS) in biological matrices without reference standards available becomes more and more crucial in forensic toxicology. High resolution tandem mass spectrometry combined with data independent acquisition provides the possibility to dare GUA applications. However, peak extraction, automated fragmentation and compound identification needs to be evaluated and algorithm parameters carefully optimized. The aim of this study was to optimize the algorithm parameters of the PeakView® software coupled to the Chemspider database using a design of experiment (DOE) approach.

Method: Blank whole blood samples were spiked with low and high concentration mixes of 22 DUID (Driving Under the Influence of Drugs) core substances covering authentic blood concentration ranges. Screening was performed on a Sciex TripleTOF® 6600 in SWATH mode coupled to a Thermo UltiMate 3000 HPLC. Cycle time was 1.6 sec including survey scan (100 to 1000 m/z) and 27 SWATH windows (Width: 25 Da; 140-800 m/z). Measuring was in positive ion mode and total run time was 25 minutes. Data analysis was performed with PeakView® 2.2. Optimization of parameters for the peak finding process was made using the DOE software MODDE Go. To assess applicability of the optimization of the PeakView® parameters, 62 authentic blood samples were analyzed and the results were compared with results using validated routine methods.

Results and Discussion: Before optimization, 68.2% of the 22 compounds were found on average in the large number of possible peaks provided by the non-targeted peak finding option in PeakView®. After a simultaneous optimization of parameters (e.g. peak detection sensitivity, mass tolerance, signal width etc.) with MODDE Go, 86.4% compounds were found on average. For the 62 authentic samples, optimization lead to an increase to 88.1% of compounds found compared to only 68.8% before optimization.

Conclusion: The DOE approach allowed the optimization of algorithm parameters of the PeakView software using only a minimum of resources with optimal results.

Keywords: General unknown screening, high resolution mass spectrometry, design of experiment approach, whole blood

P3: Decrease of ethyl glucuronide concentrations in hair after exposure to chlorinated swimming pool water.

Marc Luginbühl1, Susanne Nussbaumer1, Wolfgang Weinmann

Institute of Forensic Medicine, University of Bern, Bühlstrasse 20, 3012 Bern, Switzerland

The direct alcohol marker ethyl glucuronide (EtG) is widely used for the assessment of alcohol consumption behavior. In this study, we investigated the influence of chlorinated swimming pool water on EtG concentrations in hair in comparison to deionized water (Milli-Q) containing no chlorine. For this purpose, EtG concentrations were measured with a validated online-SPE-LC-MS/MS method. By incubating EtG positive hair samples obtained from three regular drinkers for 0, 2, 4, 6, 8, and 10 hours, respectively, at room-temperature, significant washout effects of EtG in chlorinated water and deionized water were observed. EtG concentrations in hair were reduced after two hours of incubation in chlorinated water by 20±12% (range: 4-33%), in deionized water by 24± 5% (range: 18-29%). Incubation for 10 hours resulted in a decrease in EtG concentrations of 57±6% (range: 52-65%) for chlorinated water and 47±11% (range: 32-60%) for deionized water. To demonstrate washout in forensic hair samples, 20 samples from subjects with known alcohol consumption behavior were investigated additionally. The samples were divided into two strands and analyzed with incubation in chlorinated water for 10 hours and for comparison without any incubation. A mean decrease of 53±18% (range: 26-88%) was observable. These results clearly demonstrate that washout effects are caused by water itself and have a significant impact on EtG concentrations in hair. For people with hair that are regularly exposed to water for a longer period of time (e.g. swimmers) this can lead to false negative results.

Keywords

ethyl glucuronide, EtG, hair analysis, alcohol marker, drug test adulteration

P4: Electrochemical Reduction for Enhanced Characterization of Bio-pharmaceuticals by HDX-MS

Jean-Pierre Chervet¹, Hendrik-Jan Brouwer¹, Pablo Sanz de la Torre¹, Rasmus U. Jakobsen², Kasper D. Rand²

¹Antec Scientific, Zoeterwoude, The Netherlands

²Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark

jp(dot)chervet(at)AntecScientific(dot)com

Conformational changes and protein structural dynamics play an important role in the activity of proteins. Hydrogen-deuterium exchange (HDX) coupled to mass spectrometry (MS) is used to study the changes in conformation and dynamics of proteins.

In solution, labile hydrogens bound of the protein backbone undergo exchange with protons from the surrounding solvent. Hydrogen atoms are exchanged with deuterium atoms when the protein is dissolved in D2O. However, all hydrogens do not exchange at the same rate. In very dynamic regions, exchange reactions take place in the millisecond-to-second timescale while other hydrogens exchange more slowly (minutes to hours). The degree of exchange is indicative of the proteins local structure and dynamics. The deuteration pattern is “frozen” by quenching the HDX reaction by lowering the pH down to 2.5. The quenching solution also contains a reducing agent - e.g. tris(2-carboxyethyl)phosphine (TCEP) - present in large concentration in an attempt to compensate for the poor reducing activity at low pH. Immediately after quenching and reduction of the disulfide bonds, the protein sample is digested using immobilized pepsin followed by chromatographic separation and mass spectrometric analysis. Speed is essential as reversion of deuterated positions (referred to as back-exchange) after the quench step takes place.

Because TCEP reduction is greatly ineffective at the low pH necessary to limit back-exchange, a faster and more efficient reducing protocol is necessary to maximize the performance of HDX workflows. We demonstrate here the advantages electrochemical (EC) reduction presents over the traditional chemical approach for the analysis of proteins.

All experiments were performed on a ROXY EC system (Antec Scientific, The Netherlands) consisting of a ROXY potentiostat equipped with a new type of electrochemical flow cell – µ-PrepCell2.0. The

reaction cell was integrated into a HDX system consisting of a pepsin column, a trap column and an analytical column, all cooled to 0°C and connected to a Synapt QTOF mass spectrometer (Waters, USA).

Data will be shown using this new electrochemical cell for reduction of Insulin, a cystine knot: Nerve Growth Factor-β (NGF) and different monoclonal antibodies (MAbs). Faster and more efficient EC reduction at low pH results in greatly improves sequence coverage, especially in the case of TCEP-resistant proteins such as cystine knots (99% vs. 46%).

P05: RMassScreening: an open-source workflow for smart biotransformation product elucidation using LC-HRMS data

Michael A. Stravs^1,2, Juliane Hollender^1,2

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

² Institute of Biogeochemistry and Pollutant Dynamics, ETH, Zürich, Switzerland

Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) allows the acquisition of comprehensive information about organic molecules in a sample with one or few complementary measurements, and structure elucidation of unknown compounds using fragment information. This allows to study transformation reactions of organic molecules, such as micropollutant biotransformation in the environment or drug/xenobiotic metabolism. However, LC-HRMS/MS data necessitates complex data processing. Commercial software is available for the elucidation of transformation products (TPs), but is usually vendor-specific, has limited flexibility, and limited possibilities for interactions with other software. Alternatively, open-source tools can be used, but often require tedious manual handling. Currently available complete open-source workflows for HRMS/MS processing mostly target metabolomics, or environmental non-target screening.

RMassScreening (https://github.com/RMassScreening) is a new open-source workflow for TP elucidation, comprised of a script-based processing stage and a graphical data analysis and exploration stage (Figure 1). The user supplies a set of raw files (mzXML), parent compounds and candidate transformation reactions (csv), and a tabular description of the experimental design (csv). Peak picking on raw files is performed using enviPick (https://github.com/blosloos/enviPick) and peaks are aligned across samples to form profiles using enviMass (http://github.com/blosloos/enviMass). To associate isotopes, adducts and in-source fragment peaks to components, a custom implementation of the RAMClustR algorithm (Broeckling et al., 2014) was developed which is able to handle large datasets using the largeVis algorithm (https://github.com/elbamos/largeVis). The workflow provides functions to predict TP candidates from parent compounds and candidate reactions, which can be applied recursively, generating large suspect lists. The profiles can subsequently be screened for TP candidates, and the results compiled into time series by experimental groups and conditions. Screening results are finally loaded in an interactive viewer application, which allows to filter and sort results according to custom criteria based on ratios between experimental groups, conditions and/or timepoints. Because simple and open data formats are used, alternative suspect lists (e.g. from other TP prediction systems), or result lists (e.g. generated through PCA or clustering) can be easily imported.

References:

1. C. D. Broeckling, F. A. Afsar, S. Neumann, A. Ben-Hur and J. E. Prenni, Anal. Chem., 2014, 86, 6812–7.

Figure 1. Top: Schematic processing workflow. Bottom: Exemplary commented screenshots: interactive filter generation (left), data analysis and exploration (right)

P6: Top-down protein structural analysis benefits from a multiplexing FTMS

Natalia Gasilova,^a Konstantin O. Nagornov,^b Yury O. Tsybin^b, Luc Patiny^a, and Laure Menin^a

^a École Polytechnique Fédérale de Lausanne, Service de Spectrométrie de Masse de l’ISIC, Rue de l’Industrie 17, 1951 Sion, Switzerland

^b Spectroswiss Sàrl, EPFL Innovation Park, Building I, 1015 Lausanne, Switzerland

Contact email: natalia(dot)gasilova(at)epfl(dot)ch

Top-down analysis of proteins greatly benefits from the use of high-resolution FTMS/MS-based techniques. The commercial software for FTMS transient signal processing and mass spectral data analysis allows operating within a single LC-MS/MS experiment, providing excellent performance for peptide and small protein analysis but insufficient sensitivity and dynamic range for top-down analysis of larger (>20 kDa) proteins. Therefore, increasing the number of MS/MS scans per precursor ion is an attractive approach to improve the top-down analysis performance for large proteins. For example, combined processing of several consecutive LC-MS/MS experiments (technical replicates) could be beneficial for protein structural biology applications or monoclonal antibody characterization. Herein, we demonstrate the implementation and the application advantages of such multiplexing top-down data analysis workflow for several large, 20-150 kDa, proteins.

All experiments were carried out using HCD-enabled QExactive HF Orbitrap FTMS instrument. Typically, ten consecutive LC-MS/MS experiments were acquired for each sample. In parallel with standard mass spectra (reduced profile), the time-domain signals (transients) were acquired independently using both a built-in and external data acquisition systems. The mass spectra and transients were processed using Peak-by-Peak software (Spectroswiss). The low resolution isotopically-unresolved mass spectra of intact antibodies were deconvoluted and analyzed with the “Intact” software package (Protein Metrics). High-resolution HCD mass spectra of the proteins and Fc/Fab/Lc/Fd subunits of antibodies, obtained by digestion with enzymes Gingiskhan and IdeS (Genovis), were analyzed with MASHsuite Pro and Byonic (Protein Metrics) software tools, as well as in-house developed software.

The developed and benchmarked here workflow for FTMS-based multiplexing top-down protein analysis includes the following steps: summation of mass spectra or transients for an unlimited number of separate LC-MS/MS experiments, baseline correction, data-dependent noise thresholding, peak picking, and mass spectra recalibration. The described workflow increases the sensitivity and confidence of the MS/MS product ion identification for top-down analysis of large proteins, especially their low intensity modifications, demonstrating improved mass accuracy and isotopic envelope correlation score.

P7: Characterization of Kinase Activity by Phosphoproteomics in My-eloid Cell Lines for Identification of Driving Oncogenic Pathways

Mahmoud Hallal^1,2, Manfred Heller², Rémy Bruggmann³, Ramanjaneyulu Allam^1,2, Raphael Joncourt¹, Elisabeth Oppliger Leibundgut^1,2, Cedric Simillion^2,3, Nicolas Bonadies^1,2

(1) Department of Haematology and Central Haematology Laboratory, Inselspital Bern, University Hospital, University of Bern

(2) Department for BioMedical Research, Inselspital Bern, University Hospital, University of Bern

(3) Interfaculty Bioinformatics Unit and Swiss Institute of Bioinformatics, University of Bern

Introduction:

Myelodysplastic syndromes (MDS) are heterogeneous disorders caused by sequential accumulation of genetic lesions in haematopoietic stem cells (HSC). MDS are characterized by dysplasia, ineffective haematopoiesis and a propensity to evolve towards acute myeloid leukemia (AML). Clonal heterogeneity, genetic interactions, and clonal evolution remain enigmatic phenomena of oncogenesis and important investigational challenges in the post-genomic era.

Aim and objectives: The aim of this project was to build a bioinformatics pipeline to integrate phosphoproteomic data with genetic information to characterize targettable kinase-activities of involved oncogenic pathways. Our objective was to establish a network analysis of the kinase-substrate relationship in myeloid cell lines, before moving into primary cells. Here, we present data on the ongoing phosphoproteome characterization and the inferred kinase-activity from five myeloid cell lines.

Methods: The five myeloid cell lines K562 (erythroid), NB4 (promyelocytic), THP1 (monocytic), OCI-AML3 (myelomonocytic) and MOLM13 (monocytic) were used. Cytogenetic analysis and ionTorrent panel sequencing were performed (Table 1). Phosphoproteomes were enriched using Titanium-dioxide (TiO2) and samples were analyzed in triplicates by reversed-phase nano liquid chromatography coupled to tandem-mass spectrometry (nanoLC-MS2). Raw data was analyzed using MaxQuant software and further processed with R. For kinase-activity enrichment analysis (KAEA), we integrated all substrate- kinase datasets from five different databases. The SetRank R-package was

employed for KAEA.

Results: We identified 15’698, 14’087, 13’969, 13’993 and 14’201 unique phosphopeptides corresponding to 3’536, 3’363, 3’411, 3’410 and 3’403 unique phosphoproteins, respectively. KAEA identified 77 different kinases across the five cell lines (Table 2). In the heat-map of the KAEA, phenotypically related cell lines clustered together and unique kinase-activity patterns emerged for each cell line (Figure 1). Two cell lines are driven by oncogenic kinases; K562 by BCR-ABL1 and MOLM13 by FLT3-ITD. An ABL1- kinase signal was detectable from 2 different databases in K562 as well as additional downstream kinases of ABL1. We could not enrich for the FLT3- kinase in MOLM-13, due to lack of representation in the currently available databases. However, our pipeline was able to identify the activity of downstream kinases of FLT3.

Conclusion: Our bioinformatics pipeline was able to detect unique phosphoproteins and enrich for kinase-activities in distinct myeloid cell lines. Moreover, it reproduced an expected pattern of kinase-activities for two relevant driving oncogenic kinases. Further improvement on quantification and annotation are expected by using heavy labeled cell lines (SILAC) as well as kinase-motif analysis, respectively.

P8: LESA^PLUS enables an improved in-depth analysis of the lipidome in comparison to the traditional LESA (Liquid Extraction Surface Analysis) approach.

Reinaldo Almeida¹, Andreas Wiesner¹, Daniel Eikel²

¹Advion Ltd., Harlow, UK, awiesner(at)advion(dot)com

²Advion Inc., Ithaca, NY, USA

Spatial lipid composition, distribution and regulation are very important factors for mediating lipid functionality and, when disrupted, can cause pathophysiological processes leading to cancer, obesity, atherosclerosis, and neurodegeneration.

The LESA^PLUS (LESA = Liquid Extraction Surface Analysis) approach combines the standard liquid extraction surface analysis with an additional step of a nano liquid chromatography separation. This combination is ideally suited to investigate lipids, small molecule drugs or metabolites from thin tissue sections and here, we make a comparison between LESA^PLUS and LESA in the analysis of lipids from mouse brain.

Standard liquid extraction surface analysis allows for a rapid sample analysis from a multitude of locations across tissue sections. For a shotgun lipidomics analysis approach, a 5-minute infusion experiment provides sufficient time to investigate lipid composition in detail. However, due to the sample complexity and matrix involved, some minor components may show a signal intensity insufficient for detailed MS analysis. In those cases, LESA^PLUS adds an additional dimension of analyte separation and allows an improvement in both spatial resolution as well as analyte sensitivity. Furthermore, LESA^PLUS can separate isobaric lipid species and therefore, allows a more in-depth analysis of the lipidome.

The TriVersa NanoMate offers both modes of operation within the same automated nanoelectrospray source and enables rapid analyte screening for surface profiling as well as in- depth lipid analysis.

P9: Rapid Evaporative Ionisation Mass Spectrometry (REIMS) and Live-ID™ for Fodd Authenticity

Sara Stead¹, Renata Jandova¹,Olivier Chevallier²,Connor Black² Chris Elliott² and Zoltan Takats³

¹Waters Corporation, Wilmslow, SK9 4AX, UK
²Queens University, Belfast, BT7 1NN, UK
³Imperial College, London, SW7 2AZ, UK

Rapid Evaporative Ionisation Mass Spectrometry (REIMS) is an emerging technique that allows rapid characterization of biological tissues [1,2]. We demonstrate here that REIMS is able to differentiate meat and fish samples originating from different species, regardless of which tissue is chosen.

Commercial and authenticated samples of different types of meat and fish were procured and supplied by collaborators. All samples were analyzed using the i-Knife to cut the tissue surface. Latest results using this novel hyphenated technique will be presented.

P10: Evaluation of a Novel Micro-Flow Electrospray Ion Source for MS/MS

Dr. Christoph Siethoff¹ and Werner Döbelin²

1. Swiss BioQuant AG, Kägenstrasse 18, 4153 Reinach, Switzerland
2. Prolab Instruments GmbH, Christoph-Merian Ring 31, 4153 Reinach, Switzerland

Abstract

Responding to the increasing sensitivity requirements for protein and peptide biomarker analysis as well as for mbA and therapeutic protein quantitation by LC-MS/MS or LC-HRMS in biological matrices, a newly developed electrospray micro-flow ion source for the Sciex 6500/6500+ tandem mass spectrometers was evaluated. Besides the sensitivity, attention was paid to robustness and ease of use compared to the original manufacturer source, as well as to the attainable duty cycle times which should ideally be less than 10 minutes. This is usually difficult to achieve with nano-flow electrospray sources or nano-HPLC systems.

We demonstrate excellent sensitivity and performance in a micro-flow HPLC application. The quantitation of a therapeutic protein was performed in the low ng/mL range using immunocapturing, a special digestion with cyanogen bromide and the direct injection onto a two-dimensional HPLC system. A trapping column of 1 mm ID was used with a loading flow rate of 200 µL/min and a 0.3 mm ID analytical column with an analytical flow rate of 4 µL/min.

P11: The Interaction of Transition Metal-Based Anticancer Compounds with Nucleic Acids

Rahel P. Eberle and Stefan Schürch

Department of Chemistry and Biochemistry, University of Bern

The discovery of the antitumor activity of cisplatin represented a milestone in the transition metal-based chemotherapy. Despite the great success of cisplatin against a variety of cancer types, severe side effects and resistances (acquired or intrinsic) necessitated the development of alternative anticancer agents. Metallocene dichlorides (Cp2MCl2, with Cp=cyclopentadienyl, M=Ti, V, Nb, Mo) and their derivatives are a class of metallodrugs with promising anticancer characteristics. Their activity against cisplatin-resistant cell lines combined with their general low toxicity motivated the synthesis of novel Cp-functionalized metallocenes. The antitumor activity of certain metallocenes (e.g., titanocene) is reported to arise from the interaction with nuclear DNA. However, the precise mechanisms of action are not fully elucidated yet and appear to be rather diverse.

Building on previous ESI-MS/MS experiments with dinucleoside monophosphates, this study investigates the binding preferences and binding sites of titanocene with DNA and RNA hexanucleotides. The influence of the sugar moiety, the types of nucleobases, and the nucleobase sequences on the adduct formation was elucidated by means of competition experiments. To localize the exact binding sites in MS/MS experiments, the precursors were dissociated by different activation techniques (CID, HCD, and ETD). The effect of the titanium coordination center on the gas-phase dissociation of the nucleic acids is discussed.

P12: Column switching techniques for rapid quantification by LC-MS/MS

Stefan König, Thomas Wüthrich, Wolfgang Weinmann, Susanne Nussbaumer, Marianne Hädener 

Institute of Forensic Medicine, Forensic Toxicology and Chemistry, University of Bern, Switzerland

Run times for LC-MS/MS analysis are typically between a few up to thirty minutes depending on the analytical challenges. Before LC-MS/MS analysis the samples can be prepared in vials or in 96-deep-well plates and about 100 μL of body fluids are often used for these assays. In addition, a high degree of automation for the sample preparation can be implemented. Still, the demand for further decrease of the overall turnover time is always imminent. In this presentation different assays are illustrated which take advantage of column switching in order to accelerate the HPLC run times. In addition, the choice of appropriate trapping columns and analytical columns will further increase the selectivity. Among these examples are newly developed assays for the analysis of THC and its metabolites, benzodiazepines as well as for the enantiomeric separation of L- and D-amphetamine.

P13: Extending Targeted Mass Spectrometry to study Disease Related Signaling

Thierry Schmidlin^a, Luc Garrigues^a, Albert J. R. Heck^a, A. F. Maarten Altelaar^a

^aBiomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands

Selected Reaction Monitoring (SRM) is the method of choice for accurate and reproducible quantification of multiple peptides or proteins across numerous conditions. So far mainly used to measure protein abundance based on tryptic peptides, we extended the technology to fields such as peptidomics and phosphoproteomics.

In peptidomics, especially the analysis of neuropeptides has recently moved into focus, as their regulation is key in understanding a variety of functional processes, such as energy balance, drug addiction, etc. The biosynthesis of neuropeptides however involves multiple trimming steps and PTMs, impeding the prediction of the active peptide from the genome sequence or the precursor protein. Hence MS-driven analyses of undigested endogenous peptides are vital to determine the biologically active compounds. For SRM however, the chemical characteristics of endogenous peptides pose a substantial challenge to the MS analysis, as they do not always adhere to optimal ionization and fragmentation considerations for triple quadrupole instruments. By performing a proof of principle study on 15 neuropeptides we were able to demonstrate the potential of SRM to also quantify long and highly basic endogenous peptides. Using this approach to quantify neuropeptide levels in hypothalami of rats subjected to different diets revealed several significant changes, most notably an upregulation of the VGF-derived signaling peptide AQEE-30 upon high caloric feeding.

Likewise the analysis of protein phosphorylation has gained momentum in recent years, as phosphorylation events can be linked to multiple vital cellular processes such as enzyme activation, cell-cell signaling, etc. The combination of phosphopeptide enrichment and SRM enables hypothesis-driven quantification of numerous phosphorylation events across large populations. We demonstrated the power of this approach by performing comprehensive analyses of functionally relevant phosphosites of the PI3K-mTOR/MAPK signaling network in drug sensitive and resistant cancer cell lines. The targets include activity-inducing sites of several protein kinases (e. g. PDK1, BRAF), phosphorylation sites affecting the protein’s binding partners (4EBP1, AKT1S1) and phosphorylation sites promoting the protein’s degradation (PIK3C2A). This enabled us to gain new functional insights into drug-dependent signaling dynamics in cancer.
 

Contact Person:
Thierry Schmidlin
Biomolecular Mass Spectrometry and Proteomics Group
Z602, H. R. Kruytgebouw
Padualaan 8
3584 CH Utrecht, The Netherlands
t(dot)t(dot)schmidlin(at)uu(dot)nl

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