2016 SGMS Meeting

Program Plenary Lectures Short Communications Posters Registration & Deadlines

The 34th meeting of the SGMS has taken place at the Dorint Resort Blüemlisalp Beatenberg, 27-28 October 2016 high above Lake Thun in the Bernese Oberland, with a scenic view of the Swiss Alps!

Program

2016 SGMS Meeting Registration & Deadlines

Deadlines

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

A surcharge of CHF 25 will be enforced to all payments submitted after the meeting.

Registration

Closed

Plenary Lectures

Masses and Moments of Antimatter Particles and Atoms

Gerald Gabrielse Harvard University Leverett Professor of Physics 17 Oxford street Cambridge (MA) USA

Mass spectroscopy typically compares the charge-to-mass ratios of interesting ions to calibrated reference ions. Charged masses are often distinguished using differences in trajectory, transit time or orbit frequency. This lecture is the tale of a kindred quest to measure the charge-to-mass ratios and moments of matter and antimatter particles, and of antimatter atoms, to test the most fundamental symmetries and theories of nature. The quest requires one-particle sensitivity, incredible vacuum (e.g < 10^{-16} Torr), and no contact between the matter apparatus and any antimatter particles under study. Nonetheless, precisions as high as 3 parts in 10^{13} have been achieved, well beyond the precision of any available calibration, and higher precision is expected in comparisons of antihydrogen and hydrogen.

Protein structural changes in health and disease

Paola Picotti Department of Biology -Institute of Biochemistry ETH Zurich Otto-Stern Weg 3 8093 Zurich Switzerland

Protein aggregation diseases are associated with the intracellular accumulation of aggregates of specific misfolded proteins. Aggregation-prone proteins (APPs) are characterized by a variety of seemingly unrelated sequences, functions and localizations, but upon misfolding they adopt very similar structures, that share the so- called amyloid fold. These structures have been associated to cellular toxicity and cell degeneration. Recent genetic screens uncovered sets of genes that significantly reduce the toxicity of each APP and are conserved from yeast to higher organisms. These modulators are very promising for therapeutic purposes, but their mechanisms of action is currently unknown. We applied a strategy based on unbiased and targeted proteomic analyses to a set of yeast models of APP toxicity with the purpose of characterizing cellular responses to different APPs and identifying mechanism of action of known toxicity modulators. We selected a set of markers for the activation state of ~200 pathways and biological processes in yeast and designed a targeted proteomic assay that measures such markers in about 1 hour. We used the assay to analyze how cells respond to APP toxicity and determine which cellular processes or pathways each genetic modulator rewires or activates to compensate for the toxic insult. Such an assay can probe multiple pathological features at the same time and has potential for drug screening and for the elucidation of enigmatic mechanisms of action. In order to relate the observed cellular effects to the specific structural states produced along the aggregation process of each APP, we developed a novel mass spectrometric tool to quantitatively analyze the conformational changes of amyloid forming proteins directly in their cellular environment. In general, the approach can be used to analyze protein conformational changes, such as those associated to allosteric regulation, receptor activation or amyloid formation, in vivo, on a large scale, and without the need for protein purification or enrichment and can thus find a variety of applications in biological and biomedical research.

Recent Advances of Orbitrap Mass Spectrometry

Alexander Makarov Thermo Fisher Scientific Hanna-Kunathstrasse 11 Bremen Germany

The widespread adoption and use of Orbitrap mass spectrometry has been driven by its characteristic ability to acquire high-resolution, high mass accuracy data for qualitative and quantitative analysis. Increasing speed of chromatographic separations imposes ever increasing requirements on throughput of mass spectrometric analysis and demand higher spectral acquisition rates, improved spectral quality and better control of different ion optical devices within mass spectrometers.

Recent improvements in these directions are exemplified for Q Exactive and Orbitrap Fusion families of instruments, with numerous new modes of operation enabled by parallelization of detection and ion processing and concerted operation of different ion- optical devices. A special emphasis is made on technical solutions that allow quantitative analysis in these instruments, despite Orbitrap analyzer being of an ion trapping type. New modes of data-independent, targeted and top-down acquisition are overviewed.

In conclusion, future trends and perspectives of Orbitrap mass spectrometry are discussed, including its inroads into emerging areas of mass spectrometric analysis. It is shown that Orbitrap-based mass spectrometers possess compelling potential as an (ultra-) high resolution platform not only for high-end proteomic applications but also for screening, trace and targeted analysis by LC/ and GC/MS.

Short Communications

UniSpray: An Aerodynamic Perspective

Steve Bajic

Waters Corporation, Wilmslow

High flow rate ESI remains the preferred ionisation technique for LC/MS analyses since it offers the most facile coupling for a wide range of analytical flow rates. However, when compared to nanospray ionisation, ESI is known to suffer from poor ionisation efficiency at high flow rates. Here we present a novel impactor spray source, named UniSpray, which demonstrates significant enhancements in ionisation efficiency and compound coverage when compared to conventional, nebuliser-assisted ESI sources. We will examine the unique aerodynamic features that result from the “cylinder in cross-flow” geometry and give a perspective on how these features may enhance API sensitivity. Particular emphasis will be given to the phenomena of high-We number droplet impacts, the Coanda effect, turbulent vortex shedding and surface microvorticity

A Multipurpose Ambient Ionization Platform (MAIP) for Mass Spectrometry by solid-state driven RF-Energy

Ralf D. Dumler¹, Marketa Smidkova¹, Arash Sadeghfam², Holger Heuermann², Peter C. Hauser¹

¹ Analytical Sciences & Instrumentation, Department of Chemistry, University of Basel, Spitalstrasse 51, 4056 Basel, Switzerland
² Fachhochschule Aachen - University of Applied Sciences, Institute of Microwave and Plasma Technology (IMP), Germany

Fast analysis in real time and the direct ionization of small molecules are ongoing challenges in modern mass spectrometry instrumentation and methods. Numerous publications about ambient ionization techniques such as DESI, DART and many other acronyms have been introduced in the last decade [1]. This shows the constant need for such direct ionization techniques. However, all the current methods have their unique advantages and disadvantages and are still not considered as standard equipment in mass spectrometry laboratories. In order to provide a complementary ionization technology, the multipurpose ambient ionization platform (MAIP) is introduced.

This new ion source configuration is based on recent outstanding developments in the RF semiconductor industry [2] in the microwave frequency range of 2.45 GHz, growing in the slipstream of the global mobile growth. Solid-state RF energy delivers the outstanding benefits of frequency-, phase- and power-agility combined with superb accuracy. Besides replacing the bulky magnetrons and vacuum tubes, these solid-state electronic furthermore offer a smaller form factor and low voltage drive. Analytical instrumentation in the field of ambient ionization mass spectrometry techniques has the great chance to benefit from of the solid-state driven RF-Energy.

The modular platform consisting of a solid-state microwave generator and various interchangeable ion sources for ambient ionization is the basis of the newly introduced MAIP. Miscellaneous ion sources can be chosen and quickly changed in front of the atmospheric pressure interface of the mass spectrometer. The MAIP provides desorption atmospheric pressure photoionization (DAPI), low temperature and high temperature microwave plasma torches (MPT), and a contactless electrode low temperature dielectric barrier discharge plasma (DBDI), generated in a fused silica capillary. The interchangeable microwave plasma torches can be used with helium, argon, nitrogen or air as an ionization gas. Hard and soft ionization of the microwave plasma torches can be achieved by exact controlling of power density of the plasma.

The sample introduced may either be solid, liquid or in a gas phase. Preliminary experiments show an excellent range of detection for active pharmaceutical ingredients, narcotics, pesticides, herbicides, and precursors of chemical warfare agents. The high temperature microwave plasma torch could find a potential application in atomic mass spectrometry as a promising alternative ionization source to ICP-MS. MAIP therefore enables unforeseen applications as a new ionization technique and enhances various new methods in mass spectrometry in one platform.

[1] Ambient Ionization Mass Spectrometry (New Developments in Mass Spectrometry), ISBN-13: 978-1849739269

[2] K. Werner, H. Heuermann and A. Sadeghfam, “The Potential of RF Energy for the Ignition of Microplasmas”, High Frequency Electronics, November 2012, pp. 38243

Native Protein Mass spectrometry applied to Fragment Based Drug Discovery: Investigating noncovalent interactions.

Agni Faviola Mika Gavriilidou¹, Finn P. Holding², Renato Zenobi¹

¹ ETH Zurich, Department of Chemistry and Applied Biosciences, CH-8093 Zurich, Switzerland
² Astex Pharmaceuticals, 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, United Kingdom

Native nano-ESI-MS has successfully been applied to investigate noncovalent interactions of biomolecules that are potential drug targets. Fragment-based drug discovery (FBDD) is an established approach for generating hit compounds but despite the advantages of native nano-ESI-MS, this approach has not been widely used in FBDD.

In this study, the noncovalent interactions of cyclin dependent kinase 2 (CDK2) with small molecule inhibitors that were generated with FBDD approach were investigated. The dissociation constants (Kd) of compounds of different molecular weight and binding affinity to CDK2 were determined by native protein mass spectrometry. Factors such as compound solubility, ESI compatible buffer, collisional dissociation energy and minimisation of nonspecific interactions by varying the compound concentration were studied. Optimisation of these factors is key to enable successful application of native ESI-MS for the analysis of protein-ligand interactions in a FBDD environment.

ESI-MS experiments were carried out with a hybrid quadrupole time-of-flight mass spectrometer (Q-TOF ULTIMA, Waters/Micromass, Manchester, UK). Very gentle MS source conditions were used to ensure minimum in-source dissociation of analyte comlpexes, and MS parameters were also carefully optimized in order to reduce nonspecific binding and to minimize adduct formation.

In a FBDD campaign many techniques are used (X-ray crystallography, NMR spectroscopy, fluorescence based methods, SPR, ITC) consequently it is a time consuming, laborious and expensive process. Native MS is a rapid and sensitive technique and can provide a direct measure of ligand binding stoichiometry and relative affinity and can therefore act as a complementary analytical tool to support the FBDD process. Based on this study it is suggested that mass spectrometry can be routinely applied to FBDD as a rapid complementary approach for the detection and quantification of protein-ligand binding.

Analysis of peptide and protein conformers by high resolution ion mobility spectrometry-mass spectrometry

Michael Groessl, Benoit Plet

TOFWERK AG, Uttigenstrasse 22, Thun, 3600, Switzerland

Over the last decade, nanoelectrospray ionisation coupled with ion mobility spectrometry-mass spectrometry (IMS-MS) has been extensively used for the structural characterisation of biomolecules, in particular proteins. IMS-MS data can be used to investigate tertiary and quaternary structures and to monitor conformational changes due to ligand binding. We present data obtained on a novel high-resolution low field, drift tube IMS-MS instrument with high duty cycle and superior ion mobility resolving power. The IMS cell is kept at atmospheric pressure which is believed to better preserve native-like protein structures in the gas phase.

We have investigated the conformations of several proteins commonly used in for structural studies of proteins (including ubiquitin, cytochrome C, myoglobin, calmodulin and albumin). Whereas collision cross sections averaged over all conformations of a single charge states are in good agreement with values report in literature, we show that these single charge states consist of an ensemble of multiple conformers. This allows to gain unprecedented insight into the substructures of these biomolecules.

Additionally, we utilize the instrument to analyse conformations of the two prolyl-peptide bonds in the bradykinin 1-5 fragment (either cis or trans oriented). By applying a temperature ramp to the IMS cell, the temperature-dependent conversion of the geometry of the proly-peptide bonds can be monitored and even allows estimation of the energy barrier for the cis-trans isomerization.

Native MS provides novel structural insights into the HerA-NurA complex

Zainab Ahdash, Argyris Politis

Department of Chemistry, King’s College London, 7 Trinity Street, London, SE1 1DB, United Kingdom.

The helicase–nuclease complex (HerA-NurA) is implicated in repairing double-stranded DNA breaks. The HerA ATPase cooperates with the NurA nuclease generating of a channel that transverses the HerA-NurA complex where the DNA is translocated and processed. We used mass spectrometry (MS)-based approaches to study the assembly structure and dynamics of the HerA-NurA complex and in response to DNA and ATP binding. Using native MS, we established a 6:2 composition of the HerA-NurA complex consistent with previous findings (Byrne et al., 2014). Utilising tandem-MS and in-solution disruption, we revealed a possible mechanism of cooperatively between the two proteins.

Coupling native MS with ion mobility (IM-MS), a method that allows separation of ions based on their overall shape (Zhou, Politis et al, 2014), we calculated the orientationally averaged collision cross section (CCS) of the (sub)complexes, enabling us to model the gas-phase HerA-NurA complex structure, in agreement with an electron microscopy map. We have unravelled a novel mechanism of synergistic binding initiated by the recruitment of dsDNA and the sequential binding of ATP to the HerA-NurA+dsDNA complex. We observe for the first time the mechanism of ATP binding associated with different stability and conformational changes. Our findings propose a novel mechanism of cooperativity between the archaeal HerA6 and NurA2 which build the helicase–nuclease machine necessary for performing dsDNA-end processing.

Biomarker search and validation of environmental synthetic glucocorticoid exposure in zebrafish (Danio rerio) embryos by targeted proteomics

Anita O Hidasi^1,2, Ksenia J Groh^1,3, Kristin Schirmer^1,2,4, Marc J-F Suter^1,4

¹ Eawag, 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

Synthetic glucocorticoids (GCs) are human and veterinary pharmaceuticals that mimic the natural stress hormone cortisol and are used for their anti-inflammatory effects. Endogenous cortisol is produced in fish when the hypothalamus-pituitary-interrenal (HPI) axis is activated. GCs bind and activate the glucocorticoid receptor (GR) and thus regulate gene expression patterns of e.g. development, immune response, osmoregulation, and glucose metabolism. They can reach the aquatic environment due to incomplete elimination in wastewater treatment plants. In our work, zebrafish embryos were used to investigate whether exposure to environmentally relevant concentrations of a model GC, clobetasol propionate (CP), results in regulation of GC-action related proteins.

GC-action related genes from literature evidence and already examined in zebrafish at the mRNA level earlier in this project were selected to monitor their protein products. The 41 targeted proteins have different physiological roles, e.g. myogenesis, vitamin D metabolism, immune function, and glucose metabolism. In order to test for GC-specificity of the selected proteins, the environmentally relevant non-steroidal anti- inflammatory drug (NSAID) diclofenac (DCF) was also used for zebrafish embryo exposure. The GC- sensitivity of the potential biomarkers in a complex matrix was tested by GC- and DCF-containing environmental water samples, identified using an LC-MS/MS method developed earlier in our research group.

Targeted proteomics analyses were carried out on tryptic peptide digests of embryos exposed to ≤ 100 nM CP. The proteotypic peptide (PTP) targets were monitored using LC-MS and selected reaction monitoring (SRM). The SRM assays were optimized using synthetized PTPs in order to find the most intense transitions (i.e. m/z of the peptide and m/z of one of its fragment) to monitor in the embryo extracts. 12 out of the selected 41 proteins were detected in control zebrafish embryo digests . The 12 detected proteins were monitored simultaneously after exposing zebrafish embryos to ≤100 nM CP or ≤10 nM DCF from 0 to 4 days post fertilization (dpf). We found that Myhz2, a muscle protein was significantly up-regulated after DCF, but not CP exposure. IκBα, an inflammatory response-related protein was significantly increased after CP exposure, but was not regulated in DCF-exposed embryos. Furthermore, IκBα showed the same regulation on the mRNA level. Thus, this gene appears to be regulated by nanomolar concentrations of a GC, but not a NSAID. Unfortunately, the response of these two proteins were not robust enough after exposure to the environmental water samples. However, we did see that an other inflammatory response related gene included in our protein target list, Anxa1b, was similarly regulated on the mRNA level after CP, DCF and environmental water exposures too. Thus, the potential of Anxa1b to be used as a biomarker for anti-inflammatory drugs need to be further investigated.

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

Mass Spectrometry Imaging of Human Brain Tumors Resected by Fluorescence-Guided Surgery

Ann-Christin Niehoff^1,2, Sabrina Kröger², Astrid Jeibmann³, Walter Stummer⁴, Uwe Karst²

¹ Shimadzu Europa GmbH, Duisburg, Germany
² Institute of Inorganic and Analytical Chemistry, University of Münster, Münster, Germany
³ Institute of Neuropathology, University Hospital of Münster, Münster, Germany
⁴ Department of Neurosurgery, University Hospital of Münster, Münster, Germany

Introduction: Glioblastoma is the most common and aggressive malignant brain tumor. The survival rate can be significantly enhanced by total surgical resection of malignant glioma cells. However, this can only be achieved in less than 20 % of all cases. Resection can be improved by contrast enhancement using fluorescence-guided surgery (FGS). The major proagent for FGS is 5- aminolevulinic acid (5-ALA), a natural biochemical precursor of heme b, with fluorescent protoporphyrin IX (PPIX) as intermediate product. Based on the fluorescence of formed PPIX, tumor tissue can be differentiated from healthy brain tissue. To extent the knowledge about biochemical processes related to FGS and examine possible limitations by false positive and false negative fluorescence, the development of suitable imaging methods is of great interest. In this study, a complementary molecular and elemental imaging method combining spatially resolved information from matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is developed and applied to human brain tumor tissue.

Methods: After resection of the human cerebral tumor, 10 μm cryosections were prepared. The distribution of 5-ALA, PPIX and heme b in the tumor tissue was investigated by MALDI-MS imaging. Four positive ion mode experiments in MS and MS/MS mode were performed using a focal laser spot of 20-25 μm and a step size of 50 μm. The matrix α-cyano-4-hydroxycinnamic acid (CHCA) was sublimated and recrystallized before analysis.

The Fe distribution was examined by LA-ICP-MS. Cryosections were ablated linewise with a 50 μm spot diameter and 100 μm/s scan speed. The LA system was equipped with a low volume custom-built ablation cell providing a laminar gas flow. Quantitative data were obtained by external calibration with matrix-matched standards.

Results: MALDI-MS analysis of the tumor tissue was successively performed in four different modes. MS/MS imaging experiments of PPIX and heme b improved limit of detection and validated MS data. The developed MALDI-MS and MALDI-MS/MS imaging method was applied to several human brain tumor samples. Next to 5-ALA detection, the correlation between PPIX accumulation and high tumor cell density was shown for glioblastomas. Heme b accumulation correlated with blood vessel distribution. Necrotic tissue showed no detection of 5-ALA or PPIX, which is in accordance with the absence of fluorescence during FGS. Furthermore, a gliosarcoma, one rare type of glioma, which appears non-fluorescent during FGS, was investigated. The imaging results verified the penetration of 5-ALA through the blood-brain barrier. PPIX was not detected in gliosarcoma tissue which correlates with the non-fluorescent appearance of this kind of tumor during FGS. An explanation for this limited fluorescence has not yet been reported. Next to molecular analysis, elemental bioimaging of Fe was performed by LA-ICP-MS. Fe accumulations of over 200 μg/g could be detected in the tumor tissue correlating well with heme b and blood vessel distribution.

Analysis of peptide and protein conformers by high resolution ion mobility spectrometry-mass spectrometry

Olga Borovinskaya, Martin Tanner

TOFWERK AG, Uttigenstrasse 22, Thun, 3600, Switzerland

Fast and high resolution elemental imaging by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is of growing interest for many geological and biological applications but poses lots of challenges. The speed and lateral resolution of LA is mostly limited by aerosol dispersion within the system and slow hardware. Currently applied quadrupole and sector-field sequential mass spectrometers are often to slow for the multi-element detection of rapidly changing LA signals. Therefore, in most of the cases the user has to compromise between sensitivity, resolution, element coverage, imaging speed, image quality or a combination of these parameters, when using currently available technologies.

We present a new LA-ICP-MS method, which combines latest LA technology for fast and high resolution imaging [1,2] with icpTOF MS for simultaneous detection of the complete mass spectra. Every pixel is represented by an individual laser pulse, providing improved image quality. Precise synchronization of the LA system and icpTOF simplifies image reconstruction.

This combination provides a x10 increase in imaging speed, higher pixel signal/noise ratio, and, therefore, the possibility to apply smaller ablation spots, and the sensitivity independent of the mass coverage.

We demonstrate the performance of the system on two applications. High resolution multi-element imaging of Ge-rich sphalerite, used for Ge mining, revealed a more detailed correlation of Ge with other trace elements than the bulk analysis [3]. This correlation is used to study Ge-enrichment mechanisms. 2D biomaging of ceriodaphnia (500 x 500 µm thin section) at the resolution of 5 µm enabled a detailed investigation of the contaminant distribution within the body after short-term exposure.

1. http://www.teledynecetac.com/product/laser-ablation/aris (accessed on 31.08.2016)
2. D. N. Douglas et.al. Anal. Chem., 2015, 87, 11285-11294
3. R. Belissont et.al. Geochimica et Cosmochimica Acta, 2014, 126, 518-540.

Ion mobility-HRMS based screening of residues in complex matrices without the use of physical reference substances

Anton Kaufmann

Kantonales Labor Zurich

Modern multiresidue methods analyse several hundreds of compounds within a single chromatographic run (e.g. pesticides in fruits). Such methods rely on the use of physical reference compounds in order to confirm suspected peaks, based on their retention times, precursor and product ions. It is very challenging to produce and maintain reference compound solutions containing hundreds of anaytes. Furthermore, there are analytical tasks where no commercial reference substances are available (e.g. pyrrolizidin alcaloids, marine toxins or metabolites of veterinary drugs). Hence, there is a strong motivation to move away from relying on physical reference substances.

Data independent acquisition (DIA) can be used to produce product ion information for each observed chromatographic peak in a complex sample. Such product ion data can be used to tentatively confirm suspected analytes by in-silico fragmentation data. To be successful, two aspects are important. First: The used in-silico algorithm has to postulate at least some of the actually observed analyte product ions. Second: DIA based product ion spectra should contain the least possible number of signals not directly related to the studied precursor ion (e.g. not originating from endogenous matrix compounds).

The DIA approach investigated in this paper, was based on ion mobility Q-TOF technology (Vion). This instrument monitors a comprehensive 4-dimensional space consisting of retention time, drift time, m/z and ion abundance. The continuous switching between an unfragmented and a fragmented (all ion fragmentation) trace permits the drift time based assignment of product ions to the responsible precursor ion. A “chopping” in-silico fragmentation algorithm (MassFragment) is fed with the chemical structures of the targeted compounds (mol file). Each chromatographic peak is tested for the presence of the accurate mass of the precursor and any of the accurate masses of the postulated product ions.

This approach produced encouraging results even when analysing residues in complex matrices (e.g. veterinary drugs in bovine liver extracts). The high number of false positive findings was successfully reduced with a novel concept: There is high correlation among drift times and m/z of equally charged ions. Hence candidates showing a drift time not corresponding to the m/z of the analyte precursor ion, can be deleted. Hence, interfering, non analyte related ions (e.g. derived from multiple charged peptides, column bleed or noise) are successfully removed. Important, this ion mobility based filtration hardly increased the rate of false negative findings.

Posters

(Size: 146 cm high x 118 cm wide)

P01: Advanced data acquisition electronics and signal processing for FTMS

Anton N. Kozhinov, Konstantin O. Nagornov, Yury O. Tsybin

Spectroswiss Sàrl, EPFL Innovation Park, 1015 Lausanne, Switzerland

Owing to the recent developments in the field-programmable gate array (FPGA) technology, sophisticated digital signal processing (DSP) as required for high-performance FTMS can be executed in real time as analog transient signals are digitized. This enables new architectures of data acquisition systems for FTMS, allowing time-domain data acquisition without certain limitations such as the necessity of data-correction post-processing, as well as those in sensitivity and transient duty cycles. Here, we present an advanced data acquisition system based on the state-of-the-art FPGA technology, and its integration with contemporary FTMS instruments and standard analytical workflows, with a specific focus on quantitative bottom-up proteomics, petroleomics, and top-down proteomics.

Experiments were conducted on customized and standard instruments including LTQ FT-ICR MS and Orbitrap FTMS (Thermo Scientific). The work on the data acquisition (DAQ) electronics included the development of a stand-alone, high-performance DAQ system and its interfacing to the FTMS instruments of interest. The DAQ system includes an embedded host computer and a hybrid digitizer with a high-throughput FPGA chip onboard. A custom firmware was developed to implement real-time DSP on the FPGA chip. System control, user interface, and other DSP, e.g. FT methods, were implemented via custom host-side software. The system’s analytical values are in improved sensitivity and S/N ratio, optimized resolving power, data averaging efficiency (e.g., compared to measurements with reduced-profile spectral mode), and facilitated implementation of on-line absorption-mode FT processing. We will also discuss a custom Python wrapper of the XRawfileCtrl library (Thermo Scientific), developed for improved integration with metadata from standard RAW files. For the applications of the latter, we will present the advantages of the recently developed capabilities of spectral and transient averaging of data acquired from multiple LC-MS/MS runs or long direct infusion experiments for petroleomics and top-down mass spectrometry.

We will also discuss recent optimizations in the least-squares fitting (LSF) algorithm’s computation times, a phase calibration procedure introduced for ease of applications using various FTMS instruments, and data integration with the standard workflow (MS data and experimental parameters in RAW files). Analytical advantages will be evaluated in applications of quantitative bottom-up proteomics with 10-plex TMT reagents

P02: Reengineering Alkaloid Biosyntheses via Metabolic Shifting and Molecular Refining

Nadine Bohni, Ellen Piel, Karl Gademann

Department of Chemistry, University of Zurich, Zurich, Switzerland

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 shown to be difficult. In contrast, organic synthesis is unrivalled in its power to unravel new reactions and reagents. In this project we are connecting biosynthesis with organic synthesis for the generation of novel alkaloid derivatives. We are using the wild-type cyanobacterium Nostoc 78-12A that is naturally producing the chlorinated -carboline nostocarboline[1]. A biocompatible variant of the Suzuki-Miyaura cross-coupling reaction is executed directly on the biosynthetically derived substrate to obtain novel nostocarboline derivatives. As the reactivity of this cross-coupling is increased for brominated substrates, the biosynthesis of the cyanobacterium is chemically modified to produce N-methyl eudistomin N.

The Suzuki transformation – challenging on this substrate due to its ionic nature and its ability to bind to the catalyst – was investigated using synthetic starting material. Several water-soluble catalysts were tested and boundary conditions for the organic reaction as well as the conditions’ tolerability to the biological organism were evaluated. The surveillance of the reaction proceeding was done using sensitive UHPLC-UV-MS analysis whereas quantification by UV (with external calibration) has proven superior over MS (with internal standard) in terms of precision.

[1] Becher, P., Beuchat, J., Gademann, K., Jüttner, F. Nostocarboline: Isolation and Synthesis of a New Cholinesterase Inhibitor from Nostoc 78-12A. J. Nat. Prod. 2005, 68, 1793-1795.

The work was financially supported by the ERC Starting Grant 280271 and the ERC Proof-of- Concept Grant 680873.

P03: Characterization of a quadrupolar-detection NADEL ICR cell for (bio)molecular FT-ICR MS at the cyclotron frequency

Konstantin O. Nagornov, Anton N. Kozhinov, Yury O. Tsybin

Spectroswiss Sàrl, EPFL Innovation Park, 1015 Lausanne, Switzerland

The analytical performance of FT-ICR MS is known to be influenced by space charge effects. In the first approximation, the precession frequency of an ion depends only on its mass to charge ratio and the applied magnetic field. Frequency measurements at the true (unperturbed) cyclotron frequency instead of the reduced cyclotron frequency would result in a significantly reduced measured frequency dependence on space charge, providing improved mass accuracy. So far, useful measurements at (or near) true cyclotron frequency had only been reported for light ions (m/z<100) over a narrow mass range using quadrupolar ion detection schemes [1, 2]. Recently, we introduced FT-ICR MS at (an experimental estimate of) the true cyclotron frequency using narrow aperture detection electrodes (NADEL) ICR cells operated in the broadband mass range [3]. Extensive SIMION simulations of the NADEL ICR cells revealed that a collective motion of a group of ions (>20 ions) with a certain initial energy spread is responsible for generation of a new frequency resonance, which is located close to the theoretical cyclotron frequency. Here, we present experimental and simulation studies of this new cyclotron frequency resonance generation using NADEL ICR cells. Experiments were performed using standard commercial 7 T LTQ FT-ICR MS equipped with standard Ultra ICR cell (Thermo Scientific) and a 9.4 T SolariX FT-ICR MS (Bruker Daltonics) equipped with dynamically harmonized ParaCell, as well as a hybrid 10 T LTQ FT-ICR MS (Thermo Scientific) and 9.4 T SolariX FT-ICR MS (Bruker Daltonics) equipped with 2xNADEL ICR cell with four flat detection electrodes based on the design of an open-ended cylindrical cell (Ultra ICR cell, Thermo Scientific).

In comparison with the standard reduced cyclotron frequency operation of the ICR cells studied here, the cyclotron frequency mode displayed dramatically diminished dependences of the detected frequency on the variation of trapping potential and the number of charges. Overall, the 2xNADEL ICR cell operated in this mode provided highly-accurate mass measurements for the analyses of peptides, proteins, and petroleum fractions. Mass accuracy was evaluated with peak abundances spreading a range of >3 orders and using a classical two-parameter calibration equation for all experiments. The impact of space charge was also investigated with the 2xNADEL ICR cell for coalescence onset. For two isobaric peptides with mass difference of 18 mTh the coalescence onset (total number of charges immediately prior to coalescence) was at least twice higher in the cyclotron frequency mode than for standard ICR operation at the reduced cyclotron frequency for the same magnetic field. Analytical advantages will be evaluated for the (bio)molecular analysis in comparison with conventional ICR cells.

The work was financially supported by the ERC Starting Grant 280271.

1. L. Schweikhard, et al. Rev. Sci. Instrum., 60 (8), 2631-2634 (1989).
2. M. Heck, et al. Hyperfine Interact, 199, 347-355 (2011).
3. K.O. Nagornov, et al. JASMS, 26, 741-751 (2015).

P04:Accelerated determination of amphetamine enantiomers in human urine and hair using chiral liquid chromatography and on-line column-switching coupled with tandem mass spectrometry.

Marianne Hädener¹, Pia Bruni^1,2, Wolfgang Weinmann¹, Stefan Schürch², Stefan König¹

¹ Institute of Forensic Medicine, Forensic Toxicology and Chemistry, University of Bern, Bühlstrasse 20, CH-3012 Bern
² Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern

Amphetamine (AM) is a potent stimulant of the central nervous system existing in two optically active forms. Determination of the enantiomeric composition of AM in biological specimens can assist clinicians and forensic experts in differentiating between abuse of illicitly synthesized racemic AM and ingestion of pharmaceutical AM formulations containing either S-AM or different proportions of the S- and R-enantiomers. Therefore, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying AM enantiomers in urine and hair, matrices of choice for detecting recent drug use and assessing long-term drug exposure, respectively.

Diluted urine samples and methanolic extracts of hair samples were directly injected onto an achiral C18 trapping column for on-line cleanup and concentration. Subsequent backflush elution of the enriched AM enantiomers onto a chiral Lux 3 μm AMP column for enantioselective separation and detection was achieved by the use of a switching valve and an isocratic mobile phase consisting of 25% acetonitrile in 0.1 M aqueous ammonia. Injection, cleanup and backflush of the next sample performed before the previous sample had eluted from the chiral analytical column, thus allowing for simultaneous enantiomeric separation of up to three samples within the analytical column. Analyte detection was accomplished with a triple-stage quadrupole mass spectrometer operated in positive electrospray ionization and selected reaction monitoring mode. The method was fully validated and all predefined criteria were met. Linearity ranged from 0.05 – 25 mg/L for both enantiomers in urine and from 50 – 2500 pg/mg in hair. The method was successfully used on urine and hair specimens obtained from AM abusers and patients treated with an S-AM prodrug, demonstrating its applicability in the forensic and clinical setting.

The newly developed chromatographic concept allows for accelerated sample preparation and LC analysis without compromising the analytical quality, thus overcoming the drawbacks of time-consuming sample preparation procedures and long analytical run times from which previously reported methods for enantioselective analysis of AM have suffered.

P05: Enzyme–Substrate Complexes Studied by Native Mass Spectrometry: First Steps Towards Gas-Phase Enzymology

Martin F. Czar and Renato Zenobi

ETH Zurich, Department of Chemistry and Applied Biosciences, Vladimir-Prelog-Weg 3, ETH Zurich, 8093 Zurich, Switzerland

In the field of biological mass spectrometry (MS), understanding the degree to which desolvated biological ions, such as those produced by electrospray ionization (ESI) resemble their native forms, remains an important goal. While many structural probes have been able to answer specific questions on this point, the level of structural detail is quite limited. Furthermore, soft-landing experiments have successfully shown that biological molecules can at least regain their biological function after passage through vacuum as multiply charged ions. However, it remains unclear whether functionality is preserved in the vacuum itself.

In this work, steps towards observing enzyme reactions in the gas phase using MS are described. As a model enzyme, we chose the tightly folded serine protease trypsin (~23 kDa), which in solution specifically hydrolyzes amide bonds on the C-terminal side of arginine (R) and lysine (K) residues. Model substrates were small (< 8 amino acid) benzoyl-capped peptides, each containing a single R or K residue. Nano-ESI mass spectra of trypsin–substrate mixtures were acquired using a Waters Synapt G2-S mass spectrometer. Mass spectra of co-dissolved trypsin/substrate were measured from aqueous solutions containing 0.5 M ammonium acetate buffer. Experiments at low pH were done in 1% aqueous acetic acid. To enable studies on short-lived trypsin–substrate complexes, a simple rapid-mixing scheme was necessary: here, dual channel (theta-glass) ESI capillaries were used.

We first established that trypsin–substrate complexes can be delivered to the gas phase under native solution conditions. Chiefly, the survival yield of these complexes in the gas phase appears to mirror the solution-phase specificity of trypsin. Furthermore, acid denaturation of the enzyme dramatically reduces the survival yield of these complexes, while also slowing down the reaction rate. Second, we sought to see whether collisional activation of these complexes would produce fragment ions consistent with the occurrence of specific enzymatic cleavage. This was found to be true for one substrate: Bz-D-Pro-Phe-Lys-Gly-Gly-Gly. In this case, collisional activation of the 8+ charge state of the trypsin–substrate complex produces the hydrolyzed product peptide Bz-D-Pro-Phe-Lys, which can be unambiguously identified in the mass spectrum. These observations are consistent with the idea that hydrolysis can take place in the absence of bulk water, and suggest that the active site of trypsin can remain correctly organized in the gas phase. Interestingly, these results further implicate the transfer of water from the enzyme to the substrate as a driving force for the reaction to occur in the gas phase. Experiments using other model substrates and serine proteases are currently underway to assess the generality of these observations.

P06: First steps in platform development for toxicology trials with Danio rerio

T. Hettich, A. Jejina, A. Weston, A. Zenker, C. Berchtold, E. Kübler, G. Schlotterbeck

University of Applied Science and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, 4132 Muttenz, Switzerland

Introduction: To understand toxic effects of chemicals at different organization levels (cellular level, organ or phenotype), an in depth understanding of the complex mechanisms of the cellular network is needed. In the last decades, technologies in modern biology get more and more robust and sensitive: (i) automated data processing is now straightforward, (ii) various tools and workflows are available and, (iii) much faster processing times are possible in comparison as ten years ago. Now, there is an opportunity to correlate a snapshot of pathway activation from mRNA, with proteins/enzymes and metabolites. This allows generating a bigger picture of molecular networking. Hence, to study adverse effects of toxic compounds on living organisms, there is a need to bring different "omics"- technologies together.

Method: Bside obesity and diabetes the herbicide Pendimethalin is a suggested risk factor for pancreatic cancer [1]. In this study Pendimethalin was used as model compound with cancer-causing potential to induce molecular effects. A dose of 0.32 µmol/L over 144 hours after post fertilization was applied following OECD guideline 236. For acute fish toxicity 50 - 70 embryos were preserved for each sample in liquid nitrogen, followed by homogenization. About two milligrams of homogenate for non-targeted metabolite analysis by GC-MS and two milligrams homogenate for targeted mRNA analysis with RT-PCR method were used, respectively.

Results: Differences between treated and untreated control group were examined by PCA with an explained variance of 57 % of the first and 31 % on the second principle component, respectively. By the inspection of the PCA loading plot it was possible to distinguish entities which are responsible for high diversity between the groups. A univariate T test analysis was applied, significance levels were adjusted according to Benjamini-Hochberg false discovery rate. After data review interesting compounds are identified: Phosphoric acid (adjusted p value; 0.00289, FC; 2.3, PC1 loading; - 0.6365) and cholesterol (adjusted p value; 0.00688, FC; 1.9, PC1 loading; -0.6370).

Conclusion: Even within a short treatment of six days with Pendimethalin, an effect on primary metabolite regulation was observed. Although phosphoric acid and cholesterol are involved in many pathways, beside that phosphoric acid is a common functional group for endogenous metabolites as phosphate. However, an enhancement in pathway related to energy consumption for the treated group could be assumed due to stress induced by Pendimethalin. To achieve more detailed information on affected pathways, further research using different omics technologies is currently ongoing.

1. Gabriella Andreotti et al., International Journal of Cancer 2009 124, 2495-2500

P07:Time-Dependent Frequency of Ion Motion, Transient Modulation, and Spectral Composition in FT-ICR MS

Oleg Yu. Tsybin¹, Konstantin O. Nagornov², Anton N. Kozhinov², Yury O. Tsybin²

¹ Peter the Great St. Petersburg Polytechnic University, 195251 Saint-Petersburg, Russia
² Spectroswiss Sàrl, EPFL Innovation Park, 1015 Lausanne, Switzerland

Spectral composition of FTMS mass spectra is determined by trajectories of ion motion in ion traps, e.g., ICR or Orbitrap, generation and detection of the resulting time-domain signals (transients), and their corresponding signal processing. The detailed understanding of a transient generation process is indispensable for a comprehensive interpretation of mass spectra and further technique development. Constant in time frequencies of ion motion, including cyclotron and magnetron components in ICR, have been widely described in the FTMS literature using well- known algebraic identities that follow from linear differential equations of ion motion. These equations are applicable when ions are trapped in a quadratic potential and their Coulombic interaction is negligible. However, as we have recently shown, in a real-life FTMS the instantaneous frequency of ion motion is normally time-dependent [1]. This time-dependent instantaneous frequency of ion motion in FTMS is determined by thus introduced differential equation, where Φ(t) is a non-linear time-dependent phase and ω (t) = dΦ (t) / dt is its time-dependent instantaneous frequency (TDIF). Frequencies of 2D ion oscillations in FT-ICR MS are thus given simultaneously in the azimuthal direction as ωφ (t)=−dφ/dt, and in the radial direction as ωr (t) = dr / rdt . The radius-vector of an ion is given in a complex form as r(~) = r ⋅exp[−iΦ(t)], r = const. Therefore, the TDIF is defined by ω(~)(t) =ω +iω . Here, we apply the developed theory of time-dependent instantaneous frequency (TDIF) to establish a relationship between the asymmetric trajectories of ion oscillations, transient modulation, detection electrode dimensions, and composition of resulting Fourier spectra.

Transient waveforms at single and multiple cyclotron periods were measured experimentally and calculated theoretically by taking into account: (i) the trajectories of ion- oscillators with TDIF, and (ii) different angular size of detection electrodes, including traditional wide (e.g., 90-120 degrees) and narrow (e.g., as in NADEL ICR cells) configurations. All experiments were carried out using hybrid linear ion trap FT-ICR mass spectrometers (LTQ FT Ultra, Thermo Scientific, Bremen, Germany) equipped with actively shielded 7 T or 10 T superconducting magnets. Two ICR cells have been employed: standard Ultra ICR cell (Thermo Scientific) and an open-ended cylindrical narrow aperture detection electrode (NADEL) ICR trap (Spectroswiss) [2]. Transients analytically calculated using improved algorithms were further compared with the experimental and SIMION-simulated ones. Transient generation based on SIMION modeling was performed in accordance with an algorithm based on the Green’s reciprocity theorem [3].

The work was financially supported by the ERC Starting Grant 280271.

1. Tsybin O.Y. and Tsybin Y.O. IJMS 376, 75–84, (2015)
2. Nagornov K.O., Kozhinov A.N., Tsybin O.Y., Tsybin Y.O. JASMS 26, 741–751 (2015)
3. Hendrickson C.L., Beu S.C., Blakney G.T., Marshall A.G., IJMS 283, 100–104 (2009)

P08: Characterization of durable anti-fungal resistance processes in cereals by combined GC- and UHPLC-HR-MS based metabolomics

Rahel Bucher¹, Daniel Veyel², Lothar Willmitzer², Simon Krattinger³, Beat Keller³, Laurent Bigler¹

¹ Institute of Chemistry, University of Zurich, CH
² Max Planck Institute of Molecular Plant Physiology, Golm-Potsdam, DE
³ Institute of Plant Biology, University of Zurich, CH

Introduction of durable resistance genes in crops is an important strategy to prevent yield loss caused by fungal pathogens and to maintain food security. The resistance gene Lr34 of wheat (Triticum aestivum) durably confers resistance to four major fungal pathogens leaf rust, stripe rust, stem rust and powdery mildew. Lr34 is functionally transferable to barley (Hordeum vulgare) [1] and rice (Oryza sativa). The molecular resistance mechanism of Lr34, encoding for an ATP-binding cassette transporter [2], is not known yet. The overall aim of this multi-disciplinary project was to increase the understanding of the molecular function and defense response of durable disease resistance in cereals.

To characterize Lr34 functionality on metabolite level, a metabolomics approach based on combined UHPLC-HR-MS and GC-MS technology was applied. Comprehensive metabolic profiles of Lr34 barley, rice and wheat grown under different conditions were investigated and a broad range of structurally diverse primary metabolites (e.g. amino- and organic acids, sugars), lipids and secondary metabolites (e.g. flavonoids) were identified. UHPLC-HR-MS/MS allowed the annotation of a variety of defensive secondary metabolites [1] contributing to the understanding of the durable, multi-pathogen resistance Lr34 in different crop species.

1. Chauhan, H., Boni, R., Bucher, R., Kuhn, B., Buchmann, G., Sucher, J., Selter, L. L., Hensel, G., Kumlehn, J., Bigler, L., Glauser, G., Wicker, T., Krattinger, S. G., Keller, B., Plant J., 2015, 84, 1, 202-215
2. Krattinger, S. G., Lagudah, E. S., Spielmeyer, W., Singh, R. P., Huerta-Espino, J., McFadden, H., Bossolini, E., Selter, L. L., Keller, B., Science, 2009, 323, 1360-1363

P09: Members of GST family are differently expressed in early life stages of zebrafish

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

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

Zebrafish embryos are increasingly employed as alternative to the conventional acute fish toxicity test. One important requirement for the establishment of an alternative test model is that both models should be comparable in terms of uptake and biotransformation of xenobiotics. However, little is known about the expression of xenobiotic metabolizing enzymes in the developing zebrafish embryo.

In this study, we have developed a targeted proteomic approach to characterize the expression of Glutathione S-transferases. Selected members of the family were monitored in zebrafish embryos by mass spectrometry on the basis of proteotypic peptides and peptides characteristic for the enzyme groups of interest.

The basal expression of GSTs on the protein level was investigated at 4, 8, 24, 48, 72, 96, 120 hpf and compared to adult liver samples. Samples collected at 4 and 8 hpf were used to estimate the relevance of maternal mRNA transfer in zebrafish eggs - samples from the later time points give an insight into the dynamic of GST expression throughout the development. The expression pattern of selected GST enzymes is presented including members of the alpha and pi class – candidates that have been suggested to be involved in xenobiotic metabolism.

This study will help to fill existing knowledge gaps regarding the comparability of metabolic capacity of fish at different life stages

P10: Shedding light on electrospray ionization mechanisms using laser-induced fluorescence

Prince Tiwari, Martin Czar, Renato Zenobi

Swiss Federal Institute of Technology Zurich (ETH Zurich), Department of Chemistry and Applied Bioscience, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland

Electrospray ionization (ESI) is one of the most common ways to generate gaseous ions for mass spectrometry (MS) analysis. However, the mechanisms which govern ion production remain difficult to study. Mechanistic insights promise to not only further our understanding of the ESI process, but will facilitate optimization of ion formation as well. Towards these ends, several groups have devised different experimental and theoretical schemes. In particular, it has been shown [1, 2] that laser-induced fluorescence experiments in an electrospray plume, at different distances along the electrospray axis, provide snapshots of the conformations, electronic structures, and dynamics of electrosprayed analytes both within charged droplets, and also once completely desolvated.

Here, we present steps towards the development of an improved set-up for performing laser-induced fluorescence measurements in an electrospray plume. As our light source, we use a pulsed (~100 fs pulse duration), tunable (690-1040 nm) titanium sapphire laser, which we further frequency-double to access the UV-Vis wavelength range (345-520 nm). This flexibility in tuning range will be key to probing the spectroscopic properties of an array of biologically relevant chromophores. Further, the ultra-short pulses enable time-resolved fluorescence measurements, which are key to disentangling complex (i.e., heterogeneous) fluorescence signals.

In our experiments, we plan to study mechanistic aspects of the electrospray process using this novel apparatus. In particular, since there remain three generally accepted mechanisms for ion formation [3, 4, 5], all of which occur on different timescales, fluorescence measurements of carefully designed fluorescent conjugates of various biopolymers should enable us to track their evolution from solution to the gas phase as they traverse the electrospray plume. As an extension of this, time-resolved fluorescence anisotropy measurements of biopolymers are planned, to assess how their dynamics are perturbed when they are entrained in small, charged droplets. In the future, it is envisioned that the distance- sensitive fluorescence resonance energy transfer (FRET) method could be used to track conformational changes of electrosprayed proteins, as they are transferred from solution to the gas phase.

References:

1. S. E. Rodriguez-Cruz, J. T. Khoury, and J. H. Parks, “Protein Fluorescence measurements within electrospray droplets,” J. Am. Soc. Mass Spectrom., 12 (2001) 716.
2. Chingin, K., Frankevich, V., Balabin, Roman M., Barylyuk, K., Chen, H., Wang, R. and Zenobi, R., “Direct access to isolated biomolecules under ambient conditions.” Angew. Chem. Int. Ed., 49 (2010) 2358.
3. L. Konermann, E. Ahadi, A. D. Rodriguez, and S. Vahidi, “Unraveling the mechanism of electrospray ionization,” 85 (2012) 2.
4. J. V Iribarne, B. A. Thomson, J. V Iribarne, and B. A. Thomson, “On the evaporation of small ions from charged droplets” J. Chem. Phys., 64 (1976) 2287.
5. M. Dole, L. L. Mack, R. L. Hines, R. C. Mobley, L. D. Ferguson, and M. B. Alice, “Molecular beams of macroions a macroion electrokinetics algorithm,” 49 (1968) 2240.

P12: Evidence for laser-induced redox reactions in matrix-assisted laser desorption/ionization between cationizing agents and target plate material

Guido Paul Zeegers¹, R. F. Steinhoff¹, S. M. Weidner², R. Zenobi¹

¹ Swiss Federal Institute of Technology Zurich (ETH Zurich), Department of Chemistry and Applied Bioscience, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland; email: guido(dot)zeegers(at)org(dot)chem(dot)ethz(dot)ch, robert(dot)steinhoff(at)org(dot)chem(dot)ethz(dot)ch, renato(dot)zenobi(at)org(dot)chem(dot)ethz(dot)ch, respectively
² Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Strasse 11, 12489 Berlin, Germany; email: steffen(dot)weidner(at)bam(dot)de

Introduction: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is often applied to assess the dispersity and the end groups of synthetic polymers through the addition of cationizing agents. Here we address how these cation adducts are formed in a redox reaction context using polystyrene (PS) as a model polymer. The addition of several trifluoroacetate (TFA) salts as cationizing agents to a mixture of PS and matrix on a range of different target plate materials was systematically investigated, revealing the existence of laser-induced redox reactions between cationizing agents and target plate material during MALDI.

Methods: PS (M_w 1,920 Da) was mixed with a range of TFA salts (Li, Na, K, Cs, Ba, Cr, Pd, Cu, Ag, Zn, Al and In, as well as trifluoroacetic acid) and analyzed with MALDI using 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB) as matrix on different target plate materials (copper, Ti90/Al6/V4, Inconel^® 625, stainless steel, as well as chrome-, silver- and gold-plated stainless steel) to evaluate the occurrence of redox reactions. Spectra, obtained on a Bruker Autoflex I MALDI-Time-of-Flight mass spectrometer, were processed with MATLAB to obtain PS- and DCTB-adduct ion signal intensities for direct comparison between chosen conditions.

Results: It was found that on a stainless steel substrate the metal cations Al⁺, Li⁺, Na⁺, Cu⁺ and Ag⁺ formed polystyrene adducts, whereas K⁺, Cs⁺, Ba²⁺, Cr³⁺, Pd²⁺, In³⁺, or their lower oxidation states, did not. For the copper and silver substrates, PS and DCTB adduct formation with cations liberated from these target plate materials was observed upon addition of a cationizing agent (unless TFA salt cluster ions were preferably formed), which indicates the occurrence of redox reactions between the added TFA salts and the target plate material. To understand at what point these ions were liberated from the substrate surface, control experiments employing salt solutions incubated with copper granules before mixing with matrix and polymer solutions were carried out. Judging from their standard electrode potentials, the redox reactions, which would not normally occur, leading to the observed Cu⁺ complexation behavior when using a copper substrate in combination with a copper-free TFA salt, require an additional energy input, strongly suggesting that the observed redox reactions are laser-induced. Furthermore, copper granules were found to successfully sequester PS from a tetrahydrofuran (THF) solution, consistent with the view that adduct formation is preceded by complex formation with the copper target plate prior to the MALDI-MS measurement, facilitating the laser-induced redox reactions.

Novel aspect: Reversed redox reactions between added salts and target plate material are enabled by laser photon energy input during MALDI experiments.

P12: Elucidation of the Binding Sites in Antitumor Metallocenes-Dinucleoside Monophosphates Adducts

Rahel P. Eberle and Stefan Schürch

Department of Chemistry and Biochemistry, University of Bern

Most of the organometallic compounds used in chemotherapy today are based on cisplatin and its derivatives. However, because cisplatin shows severe side effects and some types of cancer are resistant against cisplatin, much effort is put into the development of more potent and less toxic alternatives. The antitumor activity of various metallocenes (Cp₂M²⁺, M=Ti, V, Nb, Mo) was discovered in the late 1970s and they represent a promising substitute for platinum-based drugs, as they are effective against cisplatin resistant cancer cell lines. Cell distribution studies revealed that metallocenes accumulate in the nucleus, thus, pointing towards DNA as a major target.

High-resolution tandem mass spectrometry was used for the comprehensive comparision of the binding preferences of four metallocenes and cisplatin to dinucleoside monophosphates. Dinucleoside monophosphates serve as the smallest nucleic acid model compounds that are able to provide information on the targeted functional groups and sequences. Data reveal that the extent of adduct formation and the binding pattern are primarily influenced by the type of transition metal. Moreover, the transition metal coordination center was found to considerably influence the gas-phase dissociation of the adducts, resulting in specific fragment ions that enable the elucidation of the binding patterns and specificities. The interaction of metallocenes with DNA strongly contrasts the binding of cisplatin.

P13: Mechanistic studies of electron transfer dissociation of natural and sugar-modified DNA

Yvonne Hari and Stefan Schürch

Department of Chemistry and Biochemistry, University of Bern

There is a dual interest in short nucleic acids in medical research: They present potential targets of, for instance, platinum-based anti-tumour drugs or function as active agents themselves in antisense therapy. This potential motivates the development of reliable analytical techniques like tandem mass spectrometry for the structure elucidation of natural and modified nucleic acid. Most published studies on tandem mass spectrometric characterisation and sequencing of nucleic acids rely on collisional activation. While collision-induced dissociation (CID) gives rise to a series of sequence-specific fragment ions, unspecific loss of water and nucleobases is observed as well. The contribution of these alternative channels is reduced when radical activation methods, such as electron transfer dissociation (ETD), are applied. However, the fragmentation of the oligonucleotide radical ions in the gas-phase is not yet fully understood. Our work on the ETD fragmentation of DNA and sugar-modified analogues aims to shed light on the dissociation mechanism. ETD experiments in the positive ion mode demonstrate that the preferred cleavage site depends on the nucleobase sequence. Moreover, the efficiency of backbone cleavage is higher in sugar-modified analogues, in which the distance between adjacent nucleobases is almost 1.5 times higher than in natural DNA. This difference indicates that nucleobase stacking may stabilize the radical ion and reduce the yield of backbone cleavage.

P14: A Comparative Study of the Formation of Apurinic/apyrimidnic Sites in DNA and RNA by Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry

Tin Yan Wong and Wan Chan

Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

Apyrinic/apyrimidnic (AP) site is one of the major DNA lesions that is formed by spontaneous hydrolysis as well as by exposing it to different physical/chemical treatments. It is therefore depurination and depyrimidnation of DNA followed by the generation of AP site are considered to be the crucial prior to the base excision repair (BER) mechanism. However, the studies of AP site in RNA are limited comparing to that of DNA. Recent studies showed that AP site in long-lived RNA can be repaired after exposure of oxidative or alkylating agents and thus the comparative study of abasic DNA and RNA is of great importance. Therefore, we have developed in this study a sensitive method to quantitate the amount of AP site in DNA and RNA by combining pentafluorophenylhydraine (PFPH) derivatization and isotope dilution high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques. In our studies, we used wild-type S. cerevisiae as a model for in vivo studies in order to compare the amount of AP site generated in both DNA and RNA. Our preliminary results suggested that AP site can also be generated in RNA by physically/chemically induced treatments. Besides, AP site in RNA is generated in a higher abundance than that in DNA. It is therefore we believe AP site in both DNA and RNA may act as a biomarker for assessing the risk of age-related human diseases and for evaluating the biological effects of environmental mutagens.

Reference
Li, J., Leung, E. M., Choi, M. M., & Chan, W. (2013). Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA. Analytical and bioanalytical chemistry, 405(12), 4059-4066.