2023 SGMS Meeting
 

The 40th SGMS Meeting will take place in Beatenberg to celebrate our anniversary on 25-27 October 2023 high above Lake Thun in the Bernese Oberland, with a scenic view of the Swiss Alps!

Invited speakersRegistration, Info, Deadlines, FeesProgramPlenary lecturesOral presentationsPoster presentations, session 1Poster presentations, session 2Sponsors

Invited speakers for meeting 2023

• Erin S. Baker, University of North Carolina (abstract)
• Brian Chait, Rockefeller University New York (abstract)
• Sarah Cianferani, CNRS - University of Strasbourg (abstract)
• Thierry Delatour, Nestlé Research (abstract)
• Neil Kelleher, Northwestern University (abstract)
• Jaroslav Minář (replacing Sascha Nowak), University of Münster (abstract)

2023 SGMS Meeting Registration, Deadlines & Fees

Registration is closed

Registeration is closed as the maximum number of participants has been reached already. (contact registration(at)sgms(dot)ch to be on the waiting list)

Abstract submission is closed. 

Deadlines

• Early abstract submission: August 1st (Poster/Oral acceptance will be notified by September 1st).
• Abstract submission: September 1st (Poster/Oral acceptance will be notified by September 21st).
• Standard registration: August 31st
• Late registration: October 1st (the maximum number of participants has been reached already, contact registration(at)sgms(dot)ch to be on the waiting list)

General Information

All lectures and workshops will take place at the main lecture hall of the Dorint Hotel in Beatenberg, Switzerland.

There is no need to register personally at the Dorint Resort Blüemlisalp! The SGMS committee will manage the hotel reservation and payments. Only the “extras” will have to be paid directly at the hotel reception (e.g. extra nights, phone-calls, muh-bar, mini-bar).

We will follow a first come first serve policy for the distribution of the hotel rooms (~110 rooms available!). Students that want to make use of the student price will be asked to share a room. A roommate can be indicated on the registration form.

Registration procedure

The registration is via online form. (registration is closed already) After registration you will receive a confirmation email with attached bill for the meeting and where appropriate SGMS membership fees. You can opt for becoming a member and benefit of a reduced registration fee.

A surcharge of CHF 50 will be for payments submitted after or during the meeting. Please pay in advance. 

Only full board can be chosen! There is no attending the meeting without hotel accommodation. 

Cancellations

Cancellation requests, received by e-mail before October 1st, will qualify for a refund. There will be a handling fee of 25%. After October 1st, no refunds will be made; however, substitute participant can attend.

Notification of all such changes must be sent by e-mail before October 15th. 

No show: No refund will be made. Fees remain due.

Family-friendliness

The rooms and facilities at the Dorint hotel in Beatenberg are well suited if you need or want to bring family members of all ages. Contact us by e-mail to find the best arrangement for you. For the registration of accompanying people, please write an e-mail. 

Abstract submission

Abstract submission is now closed. 

If you have submitted an abstract, don't forget to register for the attendance at the meeting as well (registration). 

Short Oral Contributions: Early deadline for abstract submission for both talks and posters is August 1st. NEW! The extended deadline is September 8th, Friday. Abstracts submitted before August 1st will have priority. 

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

Meeting fees

SGMS Annual Meeting (registration includes two nights with breakfast at the Dorint hotel, Wednesday and Thursday lunch, Wednesday dinner, Apéro and SGMS dinner on Thursday, and coffee breaks). Tuesday night accommodation and dinner on request.

Student support program

All students are hosted at a special rate of CHF 200; double room occupancy. If no roommate is indicated on registration form, it will be allocated by the organizers.

Meeting program

inspiration for Friday hikes: https://niederhorn.ch/summer-experience/hiking/?lang=en

Plenary lectures

Erin S. Baker: Linking chemical exposure to human health with novel multidimensional analyses and informatics approaches

University of North Carolina, Chapel Hill

Upon completion of the human genome project, it was determined that greater than 90% of human diseases are due not solely to a person’s genetics but a combination of genetic factors and non-genetic environmental influences. While genetic factors can be readily assessed using rapid genome sequencing technologies, measuring environmental factors is much more challenging. To date either direct or indirect measurements of exposure are often employed for their analysis. In direct measurements, specific xenobiotic compounds are analyzed in environmental samples or in biofluids and tissues, however, many xenobiotics are often excreted before responses even occur. Indirect analyses evaluate how biological processes change due to chemical exposure using one or more complementary omic techniques such as transcriptomics, proteomics, metabolomics or lipidomics. This presentation will demonstrate how combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) separations for direct xenobiotic measurements and indirect multi-omic evaluations enables an in-depth understanding of molecular responses occurring due to chemical exposures. 

Brian Chait: Mass spectrometry as a tool for throwing light on the cellular machinery

Laboratory for Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York

Although mass spectrometry-based proteomics is facilitating many new avenues of biological research, this remarkable set of tools is still limited in its applications. I will discuss some of these limitations and our efforts to address them. In particular, I will address methods for accurately modeling large macromolecular assemblies that may be flexible and undergo conformational changes; capturing specific interactions that are weak and/or transient; elucidating dynamic macromolecular processes; as well as addressing limitations in the signal-to-noise ratio and dynamic range of current mass spectrometric technologies. 

Sarah Cianferani: A journey into native structural mass spectrometry methods to dissect therapeutic antibody structure, function and interactions: from the Moon to Mars?

CNRS, University of Strasbourg, France

Structural mass spectrometry (MS) has emerged as a versatile and powerful tool for investigating the intricate structures of biological complexes and therapeutic proteins, effectively bridging the gap between molecular and structural characterization. This strategy encompasses a comprehensive set of MS-based methods, such as native MS, cross-linking, hydrogen-deuterium exchange, and top-down proteomics, with the primary goal of collecting extensive structural information on biomolecular complexes. By combining high resolution and sensitivity of MS with various biophysical and analytical techniques, structural MS enables the exploration of the three-dimensional architecture, conformational dynamics, and interactions of large biomolecules.

Until recently, these techniques were primarily the playground of a handful of experts. However, significant progress in sample preparation, MS instrumentation, and data analysis have sparked increasing interest in structural MS. It now overcomes challenges related to size, heterogeneity, and solubility of a wide range of protein complexes, and integrated most structural biology programs. 
In addition, as crucial information regarding the higher-order structure, post-translational modifications, and heterogeneity of biologics can be obtained with structural MS, it has also made its way into the biopharmaceutical industry for the characterization of biologics, including therapeutic antibodies, recombinant proteins, and nucleic acid-based drugs.

Here different applications of structural MS for the characterization of mutiprotein complexes or therapeutic proteins will be presented, with a special emphasis on their complementarities and integration to other analytical and biophysical techniques.

Thierry Delatour: The pivotal role of mass spectrometry in chemical food safety and quality: An industry perspective

Société des Produits Nestlé S.A., Nestlé Research, Lausanne, Switzerland

Today, the food system that consists of production, transportation and distribution is global. It is intended to respond to the demand that is increasing constantly with the world population expected to reach 9 billion people by 2050. This leads to an obvious complexity in the food supply chain with risks that need to be assessed and managed. Regulations defined by countries or Codex Alimentarius provide foundations for setting adequate controls in contaminants of chemical origin. Additionally, new consumer expectations, economic pressure, climate change or spread of pollutants are trends that need special attention as regard to the presence of contaminants in food. 

In the past twenty years, mass spectrometry has undergone tremendous technological advances and it is now versatile enough for being used in several applications related to the monitoring and understanding of contaminants in food. Specifically, new avenues of exploration are possible with high-resolution mass spectrometry platforms that combine adequate sensitivity and excellent identification capabilities for compounds warranting stringent surveillance. A selection of cases will be presented for showing how mass spectrometry is an essential technology for food safety and quality. On purpose, examples from research and operations environments will be selected to emphasis the capability of mass spectrometry to address various challenges related to chemical contaminants in food. 

Neil Kelleher: Exploiting individual ions in FTMS to enable proteoform systems biology & medicine

Northwestern University

Since the completion of the Human Genome Project, much has been made of the need to bridge the gap from genes and traits. As a key nexus for the many interacting ‘-omes’ (genome, transcriptome, proteome, metabolome, exposome, interactome, etc.), the proteome should offer a tight link between genotype and phenotype. Why then is proteomics challenged in this connection after 20 years of intense development? The proteoform concept offers a potential solution by considering protein composition with complete molecular specificity. Proteoforms, or all the precise molecular forms of a protein, capture all sources of variability in protein composition (i.e., SNPs, isoforms, post-translational modifications, etc.). Our group’s studies of disease-related genes have demonstrated the importance of proteoform measurement in clinically relevant targets like ApoA-I and ApoA-II (Wilkins et al., JAHA, 2021), KRAS (Ntai et al., PNAS, 2018), and immunoproteoforms in liver transplant (Melani et al., Science, 2022). More recently, “single molecule” mass spectrometry is poised to convert genes to proteoform signatures at a far faster rate. Recent advances which use individual ion mass spectrometry (I2MS) like Immunoglobulin-MS (Melani et al., J. Proteome Res., 2022) and proteoform imaging MS in tissues (Su et al., Sci. Adv., 2022) will be discussed. 

Jaroslav Minář: Aging investigations in lithium ion battery electrolytes with different mass spectrometric approaches

MEET Battery Research Center, University of Münster, Germany

For most commercial available batteries lithium hexafluorophosphate (LiPF₆) is established as the conducting salt. Based on the high hygroscopicity of LiPF₆, such systems are always contaminated with a certain amount of water that accelerates the decomposition of the conducting salt to LiF and PF₅, which subsequently may release hydrofluoric acid (HF) and other potential toxic compounds during interaction with the organic carbonated which are used as the organic solvent in this system.  

Due to the complexity of the system (organic solvents, inorganic salt, various additives, metal migration from the electrodes and both the direct influence of temperature and electrochemical energy) there is not the one method to investigate this kind of sample. In fact to obtain a complete picture of all decomposition products and reaction mechanisms, different methods with miscellaneous ionization principles have to be applied. This work presents these combined efforts of different mass spectrometric methods for the investigation of lithium ion battery electrolytes.  

Oral presentations

Electrospray ionization: How many different mechanisms?

Valérie Gabelica

Institut Européen de Chimie et Biologie (IECB) & Laboratoire Acides Nucléiques, Régulations Naturelles et Artificielles (ARNA), France

Here I will present thoughts on electrospray mechanisms. The historical ones are the charged residue mechanism (CRM) and the ion evaporation mechanism (IEM). But some hallmarks of IEM can also be accounted for by droplet fission, if one imagines a small enough droplet. Besides, flexible (bio)polymers can undergo the chain ejection mechanism (CEM). But this is a limiting case, and ion mobility spectrometry reveals that not all intermediate charge states are ejected as an extended chain. For example, we recently studied therapeutic proteins consisting of folded domains tethered by intrinsically disordered linkers, and propose that intermediate charge states are ionized by a bead ejection mechanism (BEM, Khristenko et al., JACS 2023), which is a hybrid between the CRM and the CEM. So how many mechanisms are there? Shouldn't all they obey to the same laws of physics? In this talk we will discuss the possibility of unifying the description of electrospray ion desolvation scenarios.

Gas-phase structures of biomolecular ions

Ri Wu, Lukas R. Benzenberg, Despoina Svingou, Renato Zenobi

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

Mass spectrometry is a powerful technique for the structural and functional characterization of biomolecules. However, it remains challenging to accurately gauge the gas-phase structure of biomolecular ions and assess to what extent native-like structures are maintained. Here we propose a synergistic approach which utilizes Förster resonance energy transfer (FRET) and two types of ion mobility spectrometry (i.e., traveling wave and differential) to provide multiple constraints (i.e., shape and intramolecular distance) for structure-refinement of gas-phase ions. FRET is a highly sensitive distance-dependent method, which depends on nonradiative energy transfer from an excited donor to an acceptor chromophore. The experimental collision cross-section (CCS) and donor-acceptor distance (rDA) values were used as constraints for computational studies to reveal the structure of biomolecular ions in the gas phase. Multiple systems are being studied with some or all of the methods mentioned above, including polyalanine-based ? helical peptides [1, 2], a peptide forming a β-hairpin, “stapled” peptides [3], cyclic neuropeptides with disulfide bonds [4], neuropeptide-amyloid beta complexes, and metalloprotein. In some cases, seemingly contradictory results were obtained that highlight the problem of relying on only a single method for deriving gas-phase structures. Our work allows more stringent structural characterization of biologically relevant molecules (e.g., peptide drugs) and large biomolecular ions than using only a single structural methodology in the gas phase. 

Ultra-fast evaluation tool to assess mass spectra of chlorinated paraffins and their transformation products in plastic consumer goods

Oscar Mendo Diaz^1,3, Luc Patiny², Adriana Tell^1,4, Urs Stalder³, Laurent Bigler³, Susanne Kern⁴, Davide Bleiner^1,3, Norbert V. Heeb¹

1. Swiss Federal Laboratories for Materials Science and Technology Empa, Überlandstrasse 129, 8600 Dübendorf, Switzerland
2. Zakodium Sàrl, Chemin des Plantaz 10, 1440 Montagny-Chamard, Switzerland
3. Department of Chemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland
4. Zürich University of Applied Sciences ZHAW, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland

Chlorinated paraffins (CPs) are commonly used as plasticizers, flame retardants and coolant fluids [1]. Technical CP mixtures can contain hundreds of different carbon- (C, nC=9–30) and chlorine- (Cl, nCl=2–20) homologues with millions of constitutional isomers and stereoisomers. Exposing CPs to heat leads to the formation of unsaturated compounds such as chlorinated mono- (COs), di- (CdiOs) and tri-olefins (CtriOs) [2]. This produces even more complex mixtures of CPs and their transformation products, which may contain more than 1000 C- and Cl-homologues.
 
The mass spectrometric (MS) analysis of such homologue mixtures is preferred. Polychlorinated compounds show characteristic isotopic patterns in MS based on the isotopic abundances of ¹²C, ¹³C, ³⁵Cl and ³⁷Cl. Due to that, mass spectra contain several thousands of ions, which interfere with the isotopic patterns and hinders an efficient data evaluation. Advances were made when the R-based automatic spectra evaluation routine (RAS-ER) was introduced [3]. With it, MS signals of chlorinated isotopic clusters could be compared with theoretical ones from libraries. However, a last step of tedious manual validation was required, which could take up to 2 days per mass spectrum and was prone to human error. 

To overcome this issue and further accelerate data processing, we have adapted ChemInfo for the automatic evaluation of CPs. It is a browser-based and open access platform, where mass spectra are evaluated locally and therefore, safely. A CP-containing plastic item obtained from the Swiss Market was measured by a liquid chromatographic system coupled with an atmospheric pressure chemical ionization source and an Orbitrap mass analyzer (LC-APCI-Orbitrap-MS). The formation of [M+Cl]- adduct ions by the soft ionization technique, the high resolution of the Orbitrap-MS (R = 120000) and the data analysis with ChemInfo led to the detection of CPs and their olefinic transformation products. In total, 3816 ions were assigned to 134 CP-, 101 CO-, 55 CdiO- and 31 CtriO-homologues in 30 seconds.

[1] Glüge, J. et al., Sci. Total Environ., 89 (2016) 1123–1146. 
[2] Schinkel, L. et al., Chemosphere, 194 (2018) 803–811. 
[3] Knobloch, M. et al., Anal. Chem., 94 (2022) 13777–13784.

Advances and challenges in quantitation of volatile organic chemicals with secondary electrospray ionization-mass spectrometry

Cedric Wüthrich, Stamatios Giannoukos, Renato Zenobi

Department of Chemistry and Applied Biosciences ETH Zürich

Analytical techniques used in human-related research often require the accurate quantitation of metabolites. In this context, the non-invasive on-line breath analysis, using secondary-electrospray ionization (SESI) coupled to mass spectrometry (MS), presents a promising alternative to conventional blood analysis. To ensure the reliability of SESI, a gas standard generation system was developed based on the controlled evaporation of liquid analytes and their dilution in a carrier gas stream. This system can produce low-concentration (part-per-trillion) standards [1] and operates effectively at flow rates and humidity levels relevant to human exhaled breath. To assess the system’s capabilities, short-chain fatty acids were used as test compounds to determine the limits of detection, quantification, and linearity. Additionally, this system was employed to investigate the matrix effect of ion suppression which had been previously postulated as a potential concern in SESI [2]. With the use of the evaporation-based system, the phenomenon of ion suppression was thoroughly studied. Various experiments were conducted to highlight the significance of compounds present in the gas phase in relation to ion suppression within SESI. Specifically, gas standards of D6-acetone, D3-acetic acid and pyridine were generated to assess their impact on each other’s signal. Notably, D3-acetic acid seemed to be most affected by rising levels of D6-acetone and pyridine, whereas pyridine was the least affected by increasing the concentrations of the other two compounds. This indicated a mechanistic rationale for signal suppression within SESI related to gas-phase acid-base chemistry. Pyridine with the highest gas-phase basicity was not affected by increasing concentrations of D3-acetic acid and D6-acetone, with the other two with lower basicity being more affected when the concentration of pyridine was increased. Additionally, the study underscored the significant role of concentration as a contributing factor alongside basicity. When quantificating breath metabolites with SESI-MS, external calibration is sometimes not feasible and internal standards must therefore be employed. To mitigate the suppression effect, dilution of exhaled samples is an option. The findings emphasize the importance of employing internal standard addition as the most accurate method for quantification. 

[1] Wüthrich, C.; Fan, Z.; Vergères, G.; Wahl, F.; Zenobi, R.; Giannoukos, S. Analysis of Volatile Short-Chain Fatty Acids in the Gas Phase Using Secondary Electrospray Ionization Coupled to Mass Spectrometry. Analytical Methods 2023, 15 (5), 553–561. https://doi.org/10.1039/D2AY01778D. 
[2] Bruderer, T.; Gaugg, M. T.; Cappellin, L.; Lopez-Hilfiker, F.; Hutterli, M.; Perkins, N.; Zenobi, R.; Moeller, A. Detection of Volatile Organic Compounds with Secondary Electrospray Ionization and Proton Transfer Reaction High-Resolution Mass Spectrometry: A Feature Comparison. J Am Soc Mass Spectrom 2020, 31 (8), 1632–1640. https://doi.org/10.1021/jasms.0c00059.

Characterization of new biologics modalities: A big challenge for mass spectrometry

Patrick Schindler

Novartis

At the Novartis institutes of Biomedical Research Biotherapeutics Center, the biologics have moved from the well-known antibodies, to bispecifics, and then to a zoo of modalities such as ADC, multispecifics, Radio-ligand therapies molecules, therapeutics proteins, AAVs, etc. 
The initially developed methods based on reverse phase chromatography coupled to electrospray mass spectrometry have reached their limits when these new modalities must be characterized. In our laboratory we have developed a series of mass spectrometric methods to tackle these new modalities. Different types of chromatography were set-up, e.g., denaturing, or native SEC-MS, and capillary electrophoresis chromatography MS (CE-MS). Also, native MS based technologies to characterize large entities such as non-covalent multimeric protein complexes, e.g., AAVs. And inductively coupled plasma (ICP) for the characterization of radio-ligand therapy (RLT) molecules.
 
Native MS has been introduced over the last few years in the laboratory to characterize noncovalent complexes. Especially, native SEC MS has shown its unique ability to characterize noncovalent cysteine-based ADC’s and siRNA conjugates. Data will be presented for the latter molecules which proves the preservation of the noncovalent siRNA duplex during the measurements. A new emerging technology charged detection MS (CD-MS: Pierson et al, Anal. Chem. 2016; Orbitrap CD-MS/DMT: Wörner et al and Kafader et al, Nat. Methods, 2020) is also currently explored in the context of the AAV field, especially its application for the determination of empty/partial/full capsid/DNA ratio’s will be discussed. 
For radio ligand therapy molecules, a mass spectrometry technology, ICP-MS which is normally not used for biologics characterization was evaluated and integrated in the characterization workflow of these molecules. Some data will be shown from our recent “proof-of-concept” study that ICP-MS can be used as a method for the in vitro and in vivo characterization of non-radioactive metal conjugates to predict the properties of analogous radiopharmaceuticals (Wallimann at al., Molecular Pharmaceutics 2023 20 (4), 2150-2158). 

With this presentation, we will give an insight on the diversity of mass spectrometry technologies and the hyphenated chromatography needed to be able to characterize the new biologicals modalities which will become tomorrow’s drugs.

Fast photochemical oxidation of nucleic acids coupled to high-resolution MS analysis

Marek Polák^1,2, Jiří Černý³, Michael Volný^1,2, Petr Novák^1,2

1. BioCeV – Institute of Microbiology, The Czech Academy of Sciences, Prague, Czechia
2. Charles University, Faculty of Science, Prague, Czechia
3. BioCev – Institute of Biotechnology, The Czech Academy of Sciences, Prague, Czechia

Recent years have seen significant growth in the methods of structural proteomics, which have had had a significant impact in the field of structural and molecular biology. These methods may address questions related to structure and dynamics of protein and protein complexes, making them favorable for studying protein-DNA interactions. One specific method, radical covalent labelling, has emerged as an effective analytical tool for characterization of biomolecules. Fast Photochemical Oxidation of Proteins (FPOP), the most common radical labelling method, is now exclusively employed for mapping the structure and interaction of proteins. However, in the early stages of radical chemistry, it was primarily utilized for studying structure and interaction of nucleic acid with transcription factor. 
In this study, we applied FPOP oxidation to investigate the interaction between a double-stranded nucleic acid known as an Insulin Response Element (IRE) and DNA binding domain of FOXO4 transcription factor. To study such complex, FOXO4 in the absence or presence of dsIRE was oxidized by FPOP in a quench-flow capillary reactor. Oxidized protein samples were analyzed by classical bottom-up approach – a Trypsin digestion followed by an LC-MS/MS analysis. Modified residues were localized and quantified from the LC-MS. 
To study DNA damage, IRE in the absence and the presence of FOXO4 was subjected to fragmentation/oxidation in the FPOP platform. Residual protein was digested using Proteinase K, and the resulting DNA fragments were analyzed using high-resolution 15T-FT-ICR mass spectrometry operated in negative ion mode. The study also emphasizes the analytical aspects and highlights the benefits and drawbacks associated with analyzing DNA fragments throughout the experimental process, including FPOP oxidation, LC-MS analysis, and data analysis. 

Analysis of separated IRE fragments revealed that hydroxyl radicals cleave the DNA nonspecifically, creating a set of all possible 3’OH, 3’P, 5’OH and 5’P terminal fragment ions. Complementary fragment ions were found in the LC-MS trace and subsequently quantified. Comparison of IRE fragment ions revealed a significant protective effect around the binding sequence in the major groove of DNA, also lower protection in the minor groove. 
Obtaining detailed information about solvent accessibility of IRE might enable ab initio design of FOXO4/IRE structural model. This is potentially valuable because the corresponding crystal structure is currently unclear.

Acquisition and processing of reference MS2 spectra of non-standard materials – Example of secondary metabolites from cyanobacteria (CyanoMetDB)

Anne Dax, Michael Stravs, Elisabeth Janssen

Department of Environmental Chemistry, Swiss Federal Institute of Aquatic Science and Technology (EAWAG), Dübendorf, Switzerland

Conveying the confidence of the level for compound identification is essential in mass spectrometry. When no reference standard materials are available, a match with reference MS2 spectra still allows to gain high confidence in the identification. The help of reference spectra matching is limited to available compounds/spectra in accessible databases. MassBank Europe is an open-source mass spectral library of more than 15k compounds with more than 93k spectra. However, certain compound classes are not well represented in spectral libraries, one of them being toxins and secondary metabolites from cyanobacteria. CyanoMetDB is a comprehensive, publically accessible structural database of currently 2425 cyanobacterial secondary metabolites (2023). However, reference spectra are available for less than 1% of these compounds - only for those where commercial reference standards exist. To improve spectral library coverage of cyanobacterial metabolites, we collected extracts for previously analyzed compounds across CyanoMetDB collaborators from 8 institutes, and acquired and compiled reference MS2 spectra on an Orbitrap Exploris 240 mass spectrometer. 
Here we explain the workflow and challenges of generating high-quality reference spectra, exemplified for our work on cyanobacterial toxins. The workflow includes compound-specific instrument methods to record 36 spectra types, with two scan range settings, for each in positive and negative ionization mode with a range of 9 collision energies per mode (200-500 total spectra, 10-30 replicate spectra per spectrum type). The data is processed using the RMassBank workflow, an open-source R package developed by Stravs et al. (2013) originally optimized to be used for reference materials. The workflow allows automatic formula annotation for recalibration and cleanup, and molecular formula assignment. To improve quality control of spectra from non-reference material, further quality control is implemented by extracted ion chromatogram correlation of fragments with the precursor, statistical evaluation and visual inspection. If required quality criteria are not met, spectra or whole metabolites are dismissed. Compound information is retrieved via internet services and cross-checked with the CyanoMetDB database. One of the main challenges working with (semi)purified or crude extracts are increased interferences compared to standard material, resulting in a higher workload for checking spectra manually, and ultimately less spectra qualifying for upload. 
Finally, we compiled >2000 spectra for >150 cyanobacterial metabolites for the MassBank library. This new dataset will greatly improve access to toxin reference spectra, facilitate automated data processing, and enable researchers to reach a higher level of confidence for the identification of cyanobacterial metabolites when no in-house reference materials are available.

High concentrations of nicotine vs. low concentrations of tobacco impurities, the challenge of e-liquid analysis: A validated LC-MS/MS method for nicotine and 10 nicotine-related impurities and tobacco specific nitrosamines

Vera van der Velpen^1,2, Mats B. Hirt^1,2, Kristína Žajdlíková^1,2, Fereshteh Rouholahnejad^1,2, Samuel E. Christen^1,3, Evangelia Liakoni¹, Manuel Haschke^1,2

1. Clinical Pharmacology and Toxicology, Department of General Internal Medicine, Inselspital, Bern University Hospital, University of Bern, Switzerland 
2. Institute of Pharmacology, University of Bern, Switzerland 
3. Graduate School for Health Sciences, University of Bern, Switzerland
 
E-liquids for Electronic Nicotine Delivery Systems (ENDS) can contain nicotine at high concentrations intended to deliver nicotine concentrations comparable to tobacco cigarettes. Nicotine concentrations in these products are regulated up to 20mg/ml in Switzerland and the EU whereas in other countries, such as the US, the maximum concentration is unrestricted. At the same time, e-liquids may contain low concentrations of nicotine-related impurities (e.g. anabasine, anatabine, beta-nicotyrine, cotinine, myosmine, nicotine-n-oxide and nornicotine) and tobacco specific nitrosamines (TSNAs, e.g. NNN, NNK), which generally should not exceed 1% of the products’ nicotine content. Covering this wide concentration range with a single method is challenging, as high nicotine concentrations can lead to ion suppression, detector saturation, carry-over, equipment contamination and thus interfere with very low concentrated analytes. On the other hand, at the high dilution factors required for the quantification of nicotine, the impurities and TSNAs may become undetectable. 
We aimed to develop and validate an LC-MS/MS method to quantify high concentrations of nicotine as well as low concentrations of 10 nicotine-related impurities and TSNAs in e-liquids in the range of regulatory interest. The e-liquids were diluted in 70% MeOH in three subsequent 50-fold dilution steps. The 10 impurities were measured after the second dilution step (2.5e3x) and nicotine concentrations were quantified after the third dilution step (1.25e5x). The samples underwent another 10-fold dilution with the internal standard mix before injection into the LC-MS/MS system. The LC method started at 5% B for 0.5 minutes, followed by an elution gradient of 30 to 70% over 2 minutes and a 1.5min wash phase at 100% B, before re-equilibration until 6 minutes. The mobile phases were A: 0.01% NH4OH in H2O and B: 0.01% NH4OH in MeOH. A pH-stable reversed phase C18 column (Waters) was used with a delay column (same type RP C18) to separate environmentally abundant nicotine and cotinine present in the solvents from the sample peaks. To quantify the impurities whilst avoiding MS contamination with nicotine, the nicotine peak was diverted from the MS to waste after the analytical column. This LC-MS/MS method achieved LLOQs of 0.25ng/ml for anabasine, nornicotine and myosmine and 0.1ng/ml for all other impurities, whilst nicotine had an LLOQ of 0.5ng/ml. The standard curves for the impurities, as well as for nicotine, had a range up to 100ng/ml (all with R>0.997). For the impurities, the overall within-run accuracy ranged from 94.2-109.6% with a precision of 1.8-5.1% and the between-run accuracy range was 95.7-109.8% with a precision of 0.03-9.9%. For nicotine, we report a within-run accuracy of 94.3-103.2 % with a precision of 2.1-5.1% and a between-run accuracy range of 92.2-101.3% with a precision of 3.2-8.3%. In summary, we have prepared and validated an LC-MS/MS method that can consecutively measure e-liquid nicotine content as well as individual nicotine-related impurities and TSNAs with LLOQs relative to 0.01-0.03% of the nicotine content of a 20mg/ml nicotine-containing e-liquid. This method enables the evaluation of nicotine and impurity content of e-liquids in the range of regulatory interest as well as stability of these products over time, whilst limiting nicotine contamination in the MS. This is particularly useful for labs that measure nicotine and impurity content of e-liquids, whilst specializing in quantifying nicotine and metabolites at low concentrations in biological fluids after use of these products.

Methods and instrumentation of cold ion spectroscopy for identification of isomeric biomolecules

Oleg Boyarkine, Andrei Zviagin, Vladimir Kopysov, Vyacheslav Kozlovskii

SCI-SB-RB, ISIC, EPFL

Identification of isomeric biomolecules is of great importance in many fields of life science. IR/UV photo fragmentation spectroscopy of cold ions, when combined with MS detection of the charged fragments is one of the recent approaches that allows identification and quantification of biomolecules in complex mixtures of their isomers. We will present the examples of such identifications and the newly developed prototype add-on module, which is integrated with a Q-Exactive Orbitrap-based commercial high resolution mass spectrometer. The module fully conserves its functionality, while enables 2D IR/UV-MS spectroscopy of cold ions.

“mabritecCentral”, a new web application for bacterial typing using MALDI-TOF MS protein profiles

Guido Vogel, Samuel Lüdin, Daniel Vogel and Valentin Pflüger 

Mabritec AG, Lörracherstrasse 50, 4125 Riehen, Switzerland

Mabritec AG this year went life with its new web based fully automated database for identifying bacteria with MALDI-TOF MS profiles. MALDI-TOF MS based profiling of cellular proteins has become a standard procedure for the typing of microorganisms in microbiology because of the ease of use, speed, and unmatched cost efficiency. “mabritecCentral” is by far the largest online MALDI-TOF MS database for bacterial species identification and is based on whole genome sequence data. Unlike other commercial databases and software solutions, the mabritecCentral classification is based on “marker mass” detection rather than a “pattern recognition” approach. In short, it combines genomics and MALDI-TOF analyses: marker masses are in silico predicted from publicly available whole genome data and validated by MALDI-TOF MS. Such public data are quality controlled and taxonomic classification is curated. In addition to the high species coverage (>16’000 valid species and >8000 genomospecies), mabritecCentral allows an improved discrimination of closely related species and a typing at subspecies level.

MALDI-TOF MS users worldwide are now just a few clicks away from accessing the biggest knowledge base for bacterial typing. The numerous advantages resulting from additionally questioning the mabritecCentral database will be discussed.

Poster presentations, session 1

Chemoselective approach for natural products isolation

Simon Sieber, Michelle Jessy Müller, Andrea Dorst, Constanze Paulus, Imran Khan

Department of Chemistry, University of Zurich, 8057 Zurich, Switzerland
 
The discovery of novel bioactive natural products (NPs) has been crucial for the development of the human health system [1]. However, the field suffers from the high cost of the isolation and extraction process and the rediscovery of known compounds [2]. To overcome those issues, several technologies have emerged, such as using well-designed derivatization agents to target a class of NPs specifically [3,4]. This approach led to a fast identification of a target class of compounds while using only a small amount of sample. A downside of this methodology stands in obtaining a derivatized NP. An ideal protocol would use a chemical reaction that can be reversed at the end of the process to obtain the NP as an underivatized compound. 

Herein, we present a chemoselective approach that catches and enriches amine-containing NPs and releases them as underivatized compounds [5]. The amine-containing NPs react with the chemoselective probe using a standard amide coupling procedure (catch). Next, the matrix composed of unreacted compounds is removed by exploiting the high affinity of streptavidin for biotin (enrich). In this case, a derivative of biotin, biotin sulfone, was used to ease the removal of the modified probe from the resin. Finally, the underivatized amine-containing NPs are obtained by selective cleavage of the amide bond by the protease legumain (release). 

[1] A. G. Atanasov, B. Waltenberger, E.-M. Pferschy-Wenzig, T. Linder, C. Wawrosch, P. Uhrin, V. Temml, L. Wang, S. Schwaiger, E. H. Heiss, J. M. Rollinger, D. Schuster, J. M. Breuss, V. Bochkov, M. D. Mihovilovic, B. Kopp, R. Bauer, V. M. Dirsch, H. Stuppner, Biotechnol. Adv. 2015, 33, 1582–1614.
[2] S. Bernardini, A. Tiezzi, V. Laghezza Masci, E. Ovidi, Nat. Prod. Res. 2017, 32, 1926–1950. 
[3] W. Lin, Z. Yang, A. Kaur, A. Block, M. Vujasinovic, J.-M. Löhr, D. Globisch, RSC Chem. Biol. 2021, 2, 1479–1483. 
[4] C. L. Cox, J. I. Tietz, K. Sokolowski, J. O. Melby, J. R. Doroghazi, D. A. Mitchell, ACS Chem. Biol. 2014, 9, 2014–2022. 
[5] M. J. Müller, A. Dorst, C. Paulus, I. Khan, S. Sieber, Chem. Commun. 2022, 58, 12560–12563.

Investigating the effect of microsolvation on the structure of peptides in the gas phase

Elena Giaretta

ETH Zürich, Zurich, Switzerland

Native mass spectrometry aims to find the experimental conditions to conserve the properties and topology of biomolecular ions throughout the desolvation process and analysis stages. However, in the absence of bulk solvent charged sites act stronger, which can induce protein unfolding. One potential solution to counteract the alteration of these interactions is microsolvation in the gas phase. By using additives that emulate the role of the solvent in vacuo, it has been suggested to sustain a more native-like structure of biomolecular ions. Hence, an investigation can be undertaken to assess whether specific complexing agents maintain this structural integrity and to what degree. 

The current study focuses on a -helix peptide presenting charged lysine residues. Gas-phase interactions such as Coulomb repulsion and interaction of charged side chains with the peptidic backbone can notably impact protein structure, resulting in the elongation of the helical structure. Crown ethers, specifically 18-crown-6, have recently shown to shield the charged residues via non-covalent attachment in the gas phase.[1-2] The present research employs gas- and solution-phase fluorescence spectroscopy, specifically Förster Resonance Energy Transfer, and Ion Mobility Mass Spectrometry to probe the extent of structural compaction following the charge capping achieved by crown ethers. 18-crown-6 has demonstrated sufficient binding affinity to the charged lysine residues, enabling the manipulation of biomolecular structures for a more native-like investigation in the gas phase. 

[1] Warnke, S., von Helden, G., & Pagel, K. (2013). Protein structure in the gas phase: the influence of side-chain microsolvation. Journal of the American Chemical Society, 135(4), 1177-1180.
[2] Zhou, L., Liu, Z., Guo, Y., Liu, S., Zhao, H., Zhao, S., ... & Wang, F. (2022). Ultraviolet photodissociation reveals the molecular mechanism of crown ether microsolvation effect on the gas-phase native-like protein structure. Journal of the American Chemical Society, 145(2), 1285-1291.

Elevating on-DNA hit validation of DNA-encoded libraries using native mass spectrometry

Philipp Bittner, Andreas Gloger, Michelle Keller, Jörg Scheuermann, Renato Zenobi

Department of Chemistry and Applied Biosciences, ETH Zürich, Zurich, Switzerland

The discovery and development of novel drugs against targets of pharmacological interest is still a highly challenging and expensive process. Recently, DNA-encoded chemical libraries (DELs) have been proven to be a fast and cost-effective platform for the discovery of novel small molecules. [1] These libraries consist of thousands to billions of diverse chemical compounds produced by combinatorial chemistry, each encoded by a unique DNA sequence, which serves as a "barcode" to be later read out by next-generation DNA sequencing methods. DELs are used in affinity-based selections against a target protein where only a few binders are separated from the millions of non-binding molecules. These few lead candidates are then usually re-synthesized for a detailed characterization.

However, it remains unclear how binding constants are influenced by the molecule’s DNA-tags, as binding of small molecules could be altered by steric or electrostatic effects induced by the presence of the DNA label. We have therefore set out to investigate the binding properties of small molecules with various DNA tags. We apply native electrospray ionization mass spectrometry (ESI-MS), which is capable of preserving non-covalent protein-ligand interactions and of determining the dissociation constant (Kd) of DNA-tagged ligands using a titration approach. It has been shown that Kd values determined by native ESI-MS titration agree with solution-phase binding properties. In a comparative study we investigate the effect of different DNA tags such as single- or double stranded DNA tags versus no DNA attached to the ligand. Preliminary results have already shown significant differences in the binding affinities of human carbonic anhydrase II (hCAII) against various binders with Kd values in the nM to µM range, depending on the type of DNA tag. A combination of ion mobility spectrometry and fragmentation techniques such as surface-induced, collision-induced, and temperature-induced dissociation will further help us to understand possible DNA interactions with the protein happening in solution and/or in the gas phase. 

[1] Adrián Gironda-Martínez, Etienne J. Donckele, Florent Samain, and Dario Neri, ACS Pharmacology & Translational Science 2021 4 (4), 1265-1279.

Solvent optimization in electrospray-based swab spray mass spectrometry 

Thomas Muggli, Stefan Schürch

Department of Chemistry, Biochemistry, and Pharmaceutical Sciences, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland 

Fundamentals of electrospray were already described in 1913 by the essential work of John Zeleny addressing electrical discharge from liquid points [1]. Sir Geoffrey Ingram Taylor continued to describe the behavior of liquids in strong electric fields in his important work “disintegration of water drops in an electric field” in 1964 [2]. From then on, the solvent deformation induced through an electric field became known as the Taylor cone. In 1984 the electrospray ion source was presented for the first time by Masamichi Yamashita and John B. Fenn, which marked the beginning of modern electrospray ionization coupled to mass spectrometry [3]. After the advent of desorption electrospray ionization in 2004, the benefits of numerous electrospray-based ambient ionization techniques including swab spray ionization were demonstrated.

Swab spray ionization allows the direct generation of ions from the swab head by application of solvent flow and electric potential. The swab head not only allows effective sample collection on manifold surfaces, but it also acts as a depleting analyte reservoir during analysis. The solvent optimization in swab spray ionization mass spectrometry is a crucial step at the beginning of an analysis as the characteristic of the Taylor cone is influenced by numerous solvent properties. To achieve maximum ionization efficacy and high Taylor cone stability the effects of solvent combinations were investigated. 

[1] Zeleny, J. The Electrical Discharge from Liquid Points, and a Hydrostatic Method of Measuring the Electric Intensity at Their Surfaces. Physical Review 1914, 3, 69–91, doi:10.1103/PhysRev.3.69. 
[2] Geoffrey Ingram Taylor Disintegration of Water Drops in an Electric Field. Proc R Soc Lond A Math Phys Sci 1964, 280, 383–397, doi:10.1098/rspa.1964.0151. 
[3] Yamashita, M.; Fenn, J.B. Electrospray Ion Source. Another Variation on the Free-Jet Theme. J Phys Chem 1984, 88, 4451–4459, doi:10.1021/j150664a002.

Ex-vivo mass spectrometry-based method for the biodistribution of tumor-targeting small-molecule drug-conjugates and released payload

Lucrezia Principi¹, Domenico Ravazza¹, Aureliano Zana¹, Matilde Bocci¹, Andrea Galbiati¹, Samuele Cazzamalli¹, Dario Neri^2,3, Ettore Gilardoni¹

1. Philochem AG, Libernstrasse 3, CH-8112, Otelfingen, ZH, Switzerland
2. Philogen S.P.A. Via Bellaria 35, I-53018 Sovicille, SI, Italy
3. Swiss Federal Institute of Technology, Department of Chemistry and Applied Biosciences, CH-8093 Zurich, Switzerland

Small-Molecule Drug-Conjugates (SMDCs) are new promising tumour-targeting drug prototypes to selectively deliver highly potent cytotoxic payloads to cancer lesions. Determining the tumour accumulation of novel targeting products and the released payload is paramount in predicting the therapeutic potential of preclinical candidates. Here we present a novel MS-based quantification workflow as a fast and flexible method for determining the ex-vivo biodistribution of an SMDC and free payload.

Plasma, tumour, and healthy organs were collected from tumour-bearing mice at different time points after intravenous administration with test compounds. To correct for errors of detection and for sample preparation variations, an internal standard was added to the thawed plasma and mouse tissues. Isotopically stable labeled (SIL) compounds have been used as internal standards for payloads (Deuterium) and prodrugs (Carbon 13). Samples were homogenized, deproteinized, and finally injected into the UHPLC-MS system. Chromatographic separation was carried out on a Hypersil Gold C18 column at 50 °C, with a flow rate of 700 µL/min and a gradient program. The LC system was coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific) via an Ion Max-S API ESI Source (Thermo Fisher Scientific). The detector was working in a positive ion mode and the mass spectrometer was operating in targeted Single Ion Monitoring (t-SIM) scanning mode to improve the sensitivity of the analysis. In Skyline software, peak areas of analytes and internal standards were integrated, and corresponding ratios were calculated. The ratios were then transformed into pmol/g of wet tissue using a single-concentration external calibration point and corrected by the total weight of the sample analyzed. The percentage of injected dose per gram (%ID/g) was finally calculated. 

This flexible and easy-to-implement quantitative workflow for the evaluation of SMDCs ex-vivo biodistribution has been applied to a significant number of different tumour-targeting compounds in biological matrices, allowing us the accurate assessment of drug accumulation at tumours and organs of interest. 

Quantitative Breath Analysis: Full Scan, SIM, or MRM with SESI-Q-Orbitrap? 

Timon Käser, Stamatios Giannoukos, Renato Zenobi

Department of Chemistry and Applied Biosciences, ETHZ, Zurich, Switzerland

Until today, most studies involving secondary electrospray ionization (SESI) to analyze breath have primarily relied on MS1 measurements, utilizing accurate mass measurements, but without additional confirmation. Several studies have employed secondary measurements using exhaled breath condensate or exhaled breath particles to identify substances. However, even after confirmation, they continue to depend on MS1 features with SESI to monitor changes between measurements and individuals. Nevertheless, the potential for overlapping features of isomeric compounds cannot be ruled out.

The gold standard for quantitative analysis using LC-MS is selected reaction monitoring (SRM) or multiple reaction monitoring (MRM). These techniques separate specific MS1 ions of interest and apply an optimized fragmentation energy to generate MS2 ions. Distinct MS1 to MS2 transitions are selected for each substance, and their intensity serves for quantification. 

Adopting this approach in breath analysis would help to mitigate the risk of interferences from isomeric compounds. However, in direct injection mass spectrometry using a Q-Orbitrap, substances that are nearly isobaric and cannot be separated by the quadrupole will occupy the C-trap and elevate the detection limit. This prompts the question of whether there is still some increase in sensitivity with MS2 measurements instead of MS1 measurements for breath analysis. To explore this, we mix different gas standards to compare the sensitivity and selectivity of the different measurement methods full scan (FS), SIM, and MRM for breath analysis with SESI.

Preliminary results indicate that all three methods can be employed for quantification. SIM exhibits greater sensitivity compared to FS, and MRM facilitates the separation of the isomeric compounds and their quantification with SESI. Nevertheless, meticulous selection of specific transitions is essential to maintain selectivity.

Ex-vivo mass spectrometry-based biodistribution of total antibody and released payload of an ADC

Domenico Ravazza¹, Lucrezia Principi¹, Lydia Bisbal Lopez², Andrea Galbiati¹, Aureliano Zana¹, Matilde Bocci¹, Sheila Dakhel Plaza¹, Samuele Cazzamalli¹, Dario Neri^3,4, Alberto Dal Corso², Ettore Gilardoni¹

1. Philochem AG, Libernstrasse 3, CH-8112, Otelfingen, ZH, Switzerland
2. Università degli Studi di Milano, Chemistry Department, Via C. Golgi 19, I-20133, Milan, Italy
3. Philogen S.P.A. Via Bellaria 35, I-53018 Sovicille, SI, Italy
4. Swiss Federal Institute of Technology, Department of Chemistry and Applied Biosciences, CH-8093 Zurich, Switzerland 

Antibody-Drug Conjugates (ADCs) are a class of biopharmaceutics consisting of monoclonal antibodies bearing small molecule drugs (payloads) covalently bound via chemical linkers. Most ADCs are designed to selectively deliver cytotoxic payload to tumoral lesions, using the targeting ability of the antibody moiety.

Quantitative biodistribution studies are becoming crucial during the development of novel ADCs as they allow to accurately evaluate the accumulation of the pro-drug and of the drug payload at the site of disease and in healthy tissues. In this study, we describe the development of a hybrid platform based on affinity enrichment-LC-MS and an LC-MS analysis, respectively for the quantification of the total antibody and the released payload of tumor-targeted ADC in different organs. To quantify the monoclonal antibody, an enrichment step was performed to reduce sample complexity and concentrate the analyte. Protein A was chosen to develop a method transferrable to different antibodies. Enriched proteins were digested with trypsin and a SIM-dda method analysis was carried out. The three most abundant peptides were selected for the antibody identification and quantification. A commercially available deuterated R848 and a mAb with a different light chain isotype were respectively selected as IS for the free payload and for total antibody quantification. Finally, spiked-in samples were prepared to compensate organ specific matrix effects and used as single concentration calibration point. As case study we chose a fully human monoclonal antibody targeting hCAIX, site-specifically coupled with the TLR7/8-agonist Resiquimod through the Val-Cit protease-cleavable linker. The quantitative biodistribution of the released payload revealed low %ID/g (<0.02) values and no specific tumor accumulation both at 6 and 24h post injection. Biodistribution analysis of the total ADC was carried out at the same time points and a Tumor uptake of ~5 %ID/g was observed already 6h after systemic administration. The antibody tumor uptake increased at the 24h time point (~14 %ID/g), when tumor-to-organ ratios higher than 3 were observed for all organs except plasma. The latter resulted to be the organ with the highest ADC accumulation at all time points (as expected for a conjugate based on a full IgG). Our data show that even though the ADC failed to selectively deposit free Resiquimod in target lesions (likely due to labile linker-payload connections), the product efficiently accumulated in CAIX-positive tumors.

Extending the capabilities of orbitrap-based mass spectrometers to cold ion spectroscopy

Andrei Zviagin, Vladimir Kopysov, Viacheslav Kozlovskii, Oleg V. Boyarkin

SCI SB RB Group, EPFL, CH-1015 Lausanne, Switzerland

Identification of isomeric biomolecules remains a significant challenge in analytical chemistry. Although such analytical techniques as mass spectrometry (MS), liquid chromatography (LC), ion mobility (IM) and their combinations can separate most isomers, they do not solve the problem completely. A recently demonstrated approach, which is based on cold ion spectroscopy and MS detection of photofragment ions, provides high selectivity and allows quantification of isomers of different classes of molecules such as saccharides, lipids and drugs. In addition, this method is quite fast and can be coupled with LC to further increase selectivity and versatility. To date, the method has only been used in an academic-level laboratories on a specialized instrument. To spread and commercialize this method, we develop a new add-on instrument that is interfaced with an Orbitrap-based MS. The poster will present the design and give examples of applications of this prototype add-on instrument.

Metal pollution in freshwater sediments as a driver for microbial biodiversity loss

Xin Hu¹, Ahmed Tlili^1,4, Kristin Schirmer^1,3,4, Bürgmann, Helmut²

1. Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland
2. Eawag, Swiss Federal Institute of Aquatic Science and Technology, Kastanienbaum, 6047, Switzerland
3. EPF Lausanne, School of Architecture, Civil and Environmental Engineering, 1015 Lausanne, Switzerland
4. ETH Zürich, Department of Environmental Systems Science, 8092 Zürich, Switzerland

Biodiversity loss tends to be an essential driver of the deterioration of freshwater ecosystem functions. However, the effect of heavy metals (HMs) is poorly understood as opposed to other factors, such as nutrients or climate change. Therefore, we aimed at exploring how metal distribution contributes to biodiversity changes in microbial communities associated to freshwater sediments. In this study, we collected sediments from 34 streams and lakes in Switzerland and categorized them into three groups based on their HMs concentration and sampling location: Near-Lake Sediment (NLS), Near-River Sediment (NRS), and Other Sediment (OS). Microbial diversity and community composition was determined using 16S rRNA gene amplicon sequencing. Most of the sediments were not significantly contaminated with HMs and only some HMs contents in few samples exceeded the limits, such as Cr, Zn, Cd, Cu, and Pb. A positive correlation between Fe, Cu, Zn, Co, Cd, and Pb concentrations indicated a possible common source. Based on the diversity analyses of microbial communities, our results show that NLS and NRS had a higher microbial richness than OS, but there were no significant differences among them. It was particularly remarkable that Al significantly (p < 0.05) affected the microbial richness and diversity of NRS. Planctomycetota, Cyanobacteria, and Proteobacteria were found to be the major phylum within the three sampling groups. Furthermore, bacteria at the genus level included uncultured bacteria, Pirellula, OM190, Gemmata, and WD2101_soil group. There was a predominantly negative correlation between HMs and microbial abundance. The envfit test further demonstrated the effects of Pb and Al on the microbial communities. FAPROTAX predictions revealed that Mn exhibited a significant positive effect on nitrogen fixation function, whereas Cu, Pb, and Zn displayed a significant negative effect on nitrogen fixation. Overall, our findings advance the association between HMs and their impacts on microbial diversity in freshwater sediments. At the same time, we should understand the relative contribution of heavy metals to biodiversity loss in comparison to other major drivers such as climate change or pollution by synthetic chemical.

From individual MS devices to a core facility. Or is there another way?

Basile Wettstein, Laurent Bigler

University of Zurich

In medium and large sized institutions, managing diverse analytical equipment is a daily challenge. This presentation unveils a server-based MS system, using a University of Zurich case study to vividly elucidate implementation, benefits, and challenges. Server-based MS centralizes instrument control, data processing, reporting and also data storage. This leads to a more cost-efficient framework by easier handling, troubleshooting and maintenance. The University’s transition from a decentralized setup to server-based MS is showcased, emphasizing processes, technologies, and resolutions. This practical example underscores advantages like time-effectiveness and security, while recognizing potential technical and organizational obstacles. The insights into the Chromeleon based solution offer valuable guidance to researchers and analysts engaged in analytical methods.

Solution-phase reactive oxygen species from low-temperature plasma jet treatment leads to peptide oxidation and protein aggregation

Alina Begley, Irina Oganesyan, Dušan Mrđenović, Renato Zenobi

Departement Chemie und Angewandte Biowissenschaften, ETH Zürich, 8093 Zürich, Switzerland

Low-temperature plasmas (LTPs) are used for medical applications from wound healing to selective apoptosis of cancer cells. LTPs form complex mixtures of reactive species and their mechanism of action is still under debate. Here we show that LTP treatment of solutions results in short-lived radicals that oxidize peptides, and if the peptides are sufficiently long in sequence and high in concentration, they form aggregates.

Our atmospheric pressure He-DBD jet is a homogenous LTP that generates He*, which reacts with the atmosphere and forms a cascade of gas-phase species. Ultimately, bombarding the solution with O(3P) and O2- generates short-lived solution-phase reactive oxygen species (ROS) OH• and O2•- without significant changes in pH. The longer the treatment time of the solution, the more solution-phase ROS form. These ROS oxidize peptides without significant fragmentation. 

The same oxidative modifications were found for peptides with increasing chain length (9, 18, 37, 76 amino acids) using high-resolution mass spectrometry. However, the ratio of unmodified to oxidized peptide was higher for longer peptides, because oxidized long peptides formed insoluble aggregates. Ion mobility showed that single oxidation (+O) results in peptide compaction, and multiple oxidation (+xO) results in peptide elongation. The subtle change in peptide and protein conformation leads to aggregation for long peptides, which increases with peptide concentration. On the other hand, short peptides oxidized but remained in solution independent of peptide concentration. These findings highlight the importance of peptide chain length and concentration in the fate of biomolecules after treatment with plasma medicine.

Reactive oxygen species - a friend or a foe?

Irina Oganesyan, Alina Begley, Dušan Mrđenović, Julian A. Harrison, Renato Zenobi

Departement Chemie und Angewandte Biowissenschaften, ETH Zürich, 8093 Zürich, Switzerland

Plasma medicine is an emerging biomedical field that utilizes cold, non-equilibrium plasmas for various applications, including cancer therapy, sterilization, wound treatment, and cosmetics. Cold atmospheric plasma (CAP) generates reactive oxygen and nitrogen species (ROS and RONS) along with other energetic species. While ROS and RONS have been known to negatively impact biomolecules through oxidation, recent studies have shown that controlled supply of these reactive species can aid wound healing and act as potent antiseptics against multidrug-resistant bacteria. CAP treatment has also been found to stimulate hemostasis for fresh wounds through platelet aggregation and local hemolysis of red blood cells (RBCs). 

Safety concerns regarding plasma-generated ROS and RNS in healthcare have led to investigations on their impact on proteins involved in wound blood coagulation. Previous studies have indicated significant structural changes and oxidation of proteins following plasma exposure. However, limited research exists on the specific chemical modifications induced by ROS during plasma treatment and their link to global effects like coagulation. This study aims to explore the molecular mechanisms by which plasma-induced chemical modulation leads to coagulation and aggregation of hemoglobin, the most abundant protein in RBCs. 

In this study, we employed sensitive techniques such as ion mobility and mass spectrometry to monitor hemoglobin species and detect chemical modifications induced by the plasma jet. Microscopy was used to observe changes in RBCs after treatment, and size exclusion chromatography and atomic force microscopy were used to characterize protein aggregation. The study anticipated the production of oxygenation products, detectable through mass spectrometry analysis. Understanding the impact of oxidation-induced structural changes in hemoglobin is crucial in the context of hemoglobin-related diseases and protein complex formation, denaturation, and aggregation.

In conclusion, this study provides insights into the interactions between plasma-generated ROS and RNS and hemoglobin, shedding light on their role in blood coagulation and potential biomedical applications in wound treatment.

How confident are our assignments in our mass spectrometry spectra?

Bruna Falgueras Vallbona, Dr David Kilgour, Dr Quentin Hanley

Nottingham Trent University

Mass spectrometry (MS) is widely used to identify molecules and where the confidence of that assignment is related to how closely the measured mass matches the theoretical or exact mass. So, normally an analyst might only accept assignments within ±5ppm or within ±100ppb depending on their instrument and applications. But, is a global mass error limit such as this sufficient for confident assignments? It is possible to get more information by considering the mass accuracy of assigned peaks in combination with their signal-to-noise: we would expect high signal-to-noise peaks to exhibit a lower mass error distribution than lower signal-to-noise peaks. So, a high signal-to-noise peak could have a mass error that is inside the global limit, but still indicate that the assignment may be incorrect. If we could predict how the mass error distribution would vary as a function of the signal-to-noise ratio, in a spectrum, then it would be possible to identify assignments where the mass error was anomalous, even if it fell within the global limits. Hitherto, finding the most suitable function for this role is done by trial and error. Here, I present my investigation into the underlying maths, peak distributions and background noise of different mass analysers and how we will use this information to aid in higher confidence for automated assignments. I will show how the mass accuracy of different mass spectrometric peaks shapes (Gaussian, Lorentzian and Sinc) are affected by noise, with different statistical distributions, as a function of the signal-to-noise ratio, and how it varies across the mass range.

Structural studies on cyclic neuropeptides in the presence of copper ions by means of gas phase FRET and ion mobility spectrometry

Lukas Raphael Benzenberg, Ri Wu, Despoina Svingou, Renato Zenobi

Department Chemistry and Applied Biosciences, Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland

Introduction: The function of somatostatin (SST), a cyclic neuropeptide, was recently found to be altered in the presence of Cu(II) ions, which leads to self-aggregation and loss of biological function as a neurotransmitter. However, the impact of Cu(II) ions on the structure and function of SST is not fully understood. In this work, transition metal ion Förster resonance energy transfer (tmFRET) and native ion mobility-mass spectrometry (IM-MS) were utilized to study the structures of mass-selected gas-phase ions of SST and of a smaller analogue, octreotide (OCT), and their copper adducts. 

Methods: Primary amines in somatostatin (A1, K4, K9) and octreotide (F1, K4) were used for labeling with carboxyrhodamine 6G NHS ester acting as the donor (D) fluorophore. Labeling isomers were separated by LC-UV/VIS and labeling sites were verified by b- and y-fragments generated in CID experiments. Fluorescence lifetime measurements of labeled peptides and their copper adducts were carried out for every major charge state (z = 2+, 3+) in MS/MS mode. For this, a customized quadrupole ion trap was used that allows for irradiation of the ion cloud by a pulsed fs laser. Additional structural information of the species probed was obtained by ion mobility spectrometry experiments. 

Results: Fluorescence lifetimes of all peptide labeling isomers for every measured charge state did not differ significantly. However, when Cu(II) adducts were probed, a significant decrease in the lifetimes was observed. Gas-phase emission spectra were acquired to prove that the reduction of the donor lifetime in the presence of copper results from tmFRET and not from other photophysical or structural effects. Overall, fluorescence quenching continuously decreased for higher charge states, which is likely a result of stronger charge repulsion, resulting in higher dye-copper distances. Interestingly, lifetime decay curves of copper species in the 2+ charge state for both somatostatin and octreotide exhibit a superposition of two different lifetimes that differ significantly, as determined by a double exponential fit. While this finding may suggest a co-existence of an unfolded and folded state in linear peptides, this is unlikely for the more rigid, cyclic systems studied here and rather suggests the existence of two Cu(II) binding sites. Furthermore, the ratio of the corresponding lifetimes indicates that both binding sites coexist. Native IM-MS measurements were conducted to verify whether the significant difference in r(D-Cu) values can be explained by a conformational change or originates from different copper ion binding sites. The small CCS differences observed in IM-MS experiments disagree with the co-existence of unfolded and folded conformations as could be surmised from the r(D-Cu) values, but is better explained by different copper binding sites. Utilizing multiple dye labeling isomers improves the location accuracy of Cu(II) ions, which were found to either bind to the disulfide bond region or to be complexed by aromatic residues, Phe3-D-Trp4 (OCT) and Phe6-Phe7 (SST). These binding sites are consistent with results obtained from collisional-induced dissociation (CID). While the first may lead to self-aggregation as reported in literature, binding of Cu(II) ions to the latter, novel binding site may impair receptor binding, as the motif directly represents part of the receptor binding sequence.

A mass spectrometry method for N-linked glycans released from a monoclonal antibody

Leonie Widmer¹, Laurent Bigler¹, Ettore Gilardoni²

1. Department of Chemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland
2. Philochem AG, Libernstrasse 3, 8112 Otelfingen 

Since 1980s the number of approved monoclonal antibody (mAbs) drugs has increased rapidly and today they make up the biggest share of biopharmaceuticals. They are used for many different indications such as cancer, inflammatory diseases, infections, and respiratory diseases. Glycosylation is one of the most common posttranslational modifications of proteins in nature and especially of mAbs which usually contain at least two N-glycans. The glycosylation can have a huge effect on the efficacy and safety of the drug hence it is considered a critical quality attribute (CQA). One of the techniques that is commonly used for the analysis of N-glycans is derivatization followed by (ultra)high-performance liquid chromatography (UHPLC) combined with fluorescent detection or mass spectrometry (MS). The derivatization step requires work time, is material consuming and a potential source of error. To overcome these drawbacks, we decided to develop an LC-MS workflow to analyse unlabelled N-glycans released from a mAb.

In-cell LiP-MS: Detecting protein structural states in their native environment

Franziska Elsässer¹, Roberta Florea¹, Felix Räsch², Karsten Weis², Paola Picotti¹

1. Institute of Molecular Systems Biology, Department of Biology, ETH Zürich
2. Institute of Biochemistry, Department of Biology, ETH Zürich 

Protein structures are highly dynamic and adapt to their environment and interaction partners. Limited proteolysis-coupled mass spectrometry (LiP-MS) has been successful in capturing structural alterations on a proteome-wide scale. In LiP-MS, proteins are cleaved by an unspecific protease for a short period of time, which results in structure-specific peptide patterns. Mass spectrometry is used to identify these structural fingerprints. The standard LiP-MS workflow captures protein structural states in native cell lysates. However, cell lysis may expose proteins to non-native conditions and dilution may disrupt labile interactions. 
This is particularly detrimental for structures such as phase separated protein assemblies, that are held together by weak interactions. An example of this are stress granules that are labile macromolecular assemblies of proteins and RNA that form under stress conditions and are poorly characterized at the structural level. Because of their transient nature, established methods are insufficient in capturing stress granule formation.

To circumvent disruption of protein structures that may occur upon cell lysis and study protein structures in their cellular context, we developed in-cell LiP-MS. We are using electroporation to introduce a low-specificity protease, proteinase K into mammalian cells. We observed a significant increase in peptide fragments after electroporation in the presence of proteinase K, while electroporation itself did not fragment proteins. We show that in-cell LiP-MS captures not only known specific structural changes like rapamycin-binding of FKBP1A, but also downstream effects of pathway activation.

We applied in-cell LiP-MS to study stress granule formation in mammalian cells. We observed structural changes of known stress granule components like G3BP1. Moreover, we provide the first in situ dataset on the global structural alterations of proteins during stress granule formation that we used as basis for functional studies. In future applications, in-cell LiP-MS may enable structural studies of other labile protein assemblies in their native environment.

Investigating ligand binding and structural dynamics in amyloid-β complexes by native ion mobility-mass spectrometry and gas-phase transition metal FRET

Despoina Svingou, Ri Wu, Lukas Raphael Benzenberg, Renato Zenobi

Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland 

The oligomerization and aggregation of amyloid-β peptides, as well as the dysregulation of metal homeostasis are the main pathological features of Alzheimer’s disease. Recent therapeutic strategies suggest the use of metal chelators or other small molecules that lead to reduced toxicity or impede oligomerization either by dislodging metals or by inducing structural changes, respectively. This study focuses on the structural characterization of amyloid-β 1-40 (Αβ1-40) and on its binding event with leucine enkephalin-nitrilotriacetic acid (LE-NTA), a chelating neuropeptide, in the presence of Cu(II) ions by gas-phase transition metal FRET (tmFRET) and native ion mobility-mass spectrometry (IM-MS).

Αβ1-40 was initially subjected to labeling with carboxy-rhodamine 6G (cR6G) NHS ester (fluorophore donor), targeting three possible labeling sites (D1, K16 and K28). Fluorescence lifetime experiments of Αβ1-40-cR6G and of its complex with LE-NTA in the presence of Cu(II), a localized transition metal acceptor, were conducted in a modified quadrupole ion trap mass spectrometer that enables gas-phase fluorescence spectroscopy of mass selected ions. Energy transfer efficiencies, calculated directly from lifetime values, revealed inter-chromophore distances, while IM-MS gave further insights into the structural changes of labeled Αβ1-40 copper species upon complexation with LE-NTA.

Fluorescence decay curves obtained for the monomer and its copper adducts revealed an uncoiled N-terminus and a close interaction of the C-terminus with the Cu-bound and hydrophobic regions of the 3+ Αβ species. However, in the more unfolded 4+ state, this interaction tends to be diminished, as reduced quenching for all isomers was observed. Upon formation of the non-covalent complex with the neuropeptide by addition of LE-NTA to the labeled Αβ1-40 Cu(II) adducts, a sole 4+ charge state hinted at the amyloid species retaining its most abundant 3+ charge state. Native IM-MS measurements of the 4+ Cu-free and Cu-bound species of D1, K16 and K28 provided a range of calculated CCS values similar to the non-covalent complexes, suggesting compaction after the addition of the ligand. Additionally, comparing the Αβ1-40-cR6G copper species with their respective LE-NTA complexes revealed noticeable variations in lifetime values which translates to successful dislodging of the metal ion from Αβ1-40.

In conclusion, utilizing native IM-MS and gas-phase tmFRET as orthogonal methodologies allowed us to structurally characterize unambiguously selected monomeric Aβ species and their non-covalent complexes while predicting the ligand’s orientation, as well as deciphering ligand induced conformational changes and metal displacement. This highlights it as a complementary structural probe for locating binding events and structural changes in the rapidly growing field of MS-based gas-phase structural biology.

Poster presentations, session 2

Introduction to IonBench analytical furniture solutions

Franck Subrenat

IONBENCH 

IonBench, the experts in LC GC MS ICP OES laboratory furniture - Introduction to the various solutions ionBench can offer.

Fast industrial method for screening wine cork stoppers for 2,4,6-trichloroanisole

Manuel Hutterli, Luca Cappellin, Luigi Ciotti

TOFWERK, Thun, Switzerland

41 years ago, Buser et al. [J. Agric. Food. Chem., 1982] from the Swiss Federal Research Station in Wädenswil identified 2,4,6-trichloroanisole (TCA) as the source for the musty cork taint contamination of wine, which causes economic losses for the wine industry estimated at several billion dollars yearly. The human threshold for TCA is extremely low: wine contaminated by 1-2 ng/L TCA can be perceived as tainted. Contaminations with <0.5 ng/L TCA are commonly considered negligible and are not perceivable. The possibility of reliably and non-destructively screening and sorting individual cork stoppers for TCA contamination below the perception threshold on an industrial scale prior to their use can efficiently prevent TCA taint in wine. Previously used, typically GC-based, analytical methods have so far struggled to provide a fast and reliable solution, whereas sensory analysis by trained panelists is expensive and time consuming. Here, we present a novel approach based on real-time chemical ionization – time-of-flight (CI-TOF) mass spectrometry employing the Vocus ion source and ion-molecule reactor (TOFWERK, Switzerland). TCA ions are produced with chemical ionization (CI) via a charge transfer reaction. The ‘Vocus Cork Analyzer’ (VCA) cork stopper autosampler consists of heated, individual cork stopper chambers allowing real-time sampling of the headspace air for the quantification of TCA concentrations by the time-of-flight MS. The technique proved capable of non-destructively quantifying TCA contamination in a single cork stopper in 2 s with a limit of quantification of 0.1 ng/L, well below the perception threshold. The comparison with the destructive and time-consuming standard methods (ISO 20752:2014 and Method OIV-MA-AS315-16) for releasable TCA quantification reveals a significantly better analytical performance of the VCA. A demonstration on industrial scale, quantifying TCA contamination in about 1 million cork stoppers per month is presented, proving the capability of the technique in an industrial environment. Finally, the possibility to simultaneously measure other off flavors besides TCA is also reported.

Insights into the phosphorylation dynamics of the mTOR pathway in response to growth-impacting conditions: A targeted phosphoproteomics approach

Nikolai Huwa¹, Adele C. Blatter^1,2, René Schönenberger¹, Ksenia Groh¹

1. Swiss Federal Institute of Aquatic Science and Technology, Eawag, 8600 Dübendorf, Switzerland
2. Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich, 8092 Zurich, Switzerland 

Reversible protein phosphorylation plays a central role in many cellular processes, controlling the regulation of protein function and transmission of information within signaling cascades. Among these, the mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator of several cellular processes associated with growth and proliferation. Understanding how this pathway responds to different environmental cues, including not only nutrient availability and energy levels but also chemical exposures, could help uncover new targets for toxicity prediction. Traditionally, protein phosphorylation has been studied by antibody-based methods. However, this approach can be laborious and costly, particularly when evaluating multiple protein targets. In addition, suitable antibodies may not be available for non-mammalian model organisms. Therefore, we developed an alternative method using mass spectrometry-based targeted (phospho)proteomics and applied it to investigate the phosphorylation dynamics within the mTOR pathway in zebrafish (Danio rerio) PAC2 cell line. Our workflow starts with an optimised sample preparation, including fast cell lysis (<1 min) with 5% SDS, in-column (S-Trap™) protein trapping and digestion, followed by phosphopeptide enrichment with NTA-Fe3+-beads. The recovery rate in the complex lysate matrix, monitored by spiking heavy stable isotope labelled peptides of the endogenous peptide targets, was found to be overall high at 70% (±17%) for all 42 targeted monophosphopeptides. Our developed multiple reaction monitoring (MRM) method provides rapid and sensitive detection of multiple peptide targets simultaneously, which allowed us to effectively track changes in both the protein abundance and phosphorylation status of 15 protein targets within the mTOR pathway. We investigated the response dynamics of the mTOR pathway under different conditions, including nutrient deprivation and exposure to an mTOR inhibitor. Our results demonstrate the applicability of this method for monitoring time-dependent responses in protein phosphorylation along the mTOR pathway in zebrafish and provide insights into several key checkpoints that regulate cell growth and proliferation.

Ultra-high sensitivity for single cell proteome analysis with the timsTOF SCP and timsTOF Ultra

Pascal Looser¹, Torsten Müller¹, Christoph Krisp¹, Anjali Seth², David Hartlmayr², Guilhem Tourniaire², Markus Lubeck¹ and Gary Kruppa³ 

1. Bruker Daltonics GmbH & Co. KG, Bremen, Germany
2. Cellenion, Lyon, France
3. Bruker s.r.o., Brno, Czech Republic 

For single cell proteome analysis, ultra-high sensitivity mass spectrometry is a key to reach proteome coverages necessary for understanding the cellular heterogeneity on a cell-by-cell level. Latest enhancements in ion transfer with a larger transfer capillary, an additional higher-pressure segment for more effective ion collection and two orthogonal deflections, to maintain robustness, and high-capacity trapped ion mobility spectrometry (TIMS) pushes the limits of detection to single cell level.
Here, we assessed the sensitivity of a timsTOF Ultra mass spectrometer using a dilution series of K562 cell digest showing excellent identification rates, reproducibility, and quantification accuracy per concentration replicates. Processing of the dia-PASEF data identified >1,000 protein groups out of 15 pg, and >7,000 protein groups out of 16 ng K562 peptides loaded on column. The quantitative accuracy improved inversely with loaded peptide amounts with 19% at 15 pg to 4% at loads of 4, 8 and 16 ng. Analysis of the isolated HeLa cells resulted in good identification rates and good reproducibility per individual cell count group with expected increase in protein abundance from the single cells to 20 cells. 
The timsTOF Ultra combined with automated single cell isolation and sample preparation using the cellenONE® platform for protein-loss reduced preparation and transfer with the proteoCHIP format leads to deep proteome coverage and high reproducibility.

Determination of chemically induced deamidation sites of gluten peptides using ion mobility or ultra-high resolution mass spectrometry

Julien Bourquin¹, Matthew E. Daly¹, Qianying Xu², Lisa Reid¹, Lee A. Gethings¹, Emma Marsden-Edwards¹ and E. N. Clare Mills²

1. Waters Corporation, Wilmslow, UK
2. Manchester Institute of Biotechnology, Division of Immunology, Immunity to Infection and Respiratory Medicine, University of Manchester, Manchester, UK

Introduction: Immune-mediated adverse reactions to food include IgE-mediated allergy and coeliac disease, both of which can be elicited by ingestion of gluten containing products. In coeliac disease, epitopes within these proteins known as coeliac toxic motifs can reach the gut intact and specific glutamine residues are deamidated by tissue transglutaminase, increasing their immunotoxicity. Deamidated gluten is used as a food ingredient as it is more dispersible in water than the non-deamidated form. However, the pattern of deamidation caused by treatment with food-safe acids such as tartaric acid compared to tissue transglutaminase is unclear. Peptides with multiple deamidation sites may co-elute under the same chromatographic peak and therefore other methods of site determination are required. 

Methods: Peptides derived from a chymotryptic digest of wheat flour were synthesised encompassing coeliac toxic motifs and chemically deamidated with 0.8M tartaric acid for 18 hours at 70°C. Peptides were either directly infused or chromatographically separated using an ACQUITY™ Premier System with the eluate directed to either a SELECT SERIES™ Cyclic™ IMS or a SELECT SERIES MRT mass spectrometer (Waters Corporation, Wilmslow, UK) in positive ion mode. The SELECT SERIES Cyclic IMS was run in either single-pass or multi-pass mode where ions traverse the Cyclic IMS device more than once. Resulting data were compared and the ability to identify sites of deamidation was considered. 

Preliminary data: Mass spectrometry profiling of peptides subject to chemical deamidation revealed a complex pattern and number of deamidation sites within the peptide sequence due to the large number of glutamine residues present. Analysis with the SELECT SERIES Cyclic IMS using multi-pass allowed the separation of peptides with similar degrees of deamidations, however with different sites due to the unique conformational changes. Parallel analysis using the SELECT SERIES MRT demonstrated the additional benefit of increased mass resolution of unequivocal identification of fragments produced in the collision cell. The degree of deamidation of the peptides was never observed to reach 100% of susceptible residues after 18 hours of treatment with tartaric acid, and the terminal deamidation pattern was different within the same peptide. Acid deamidation can therefore mimic the action of tissue transglutaminase in deamidation of specific glutamine residues, and possibly increase the immunotoxicity of the deamidated gluten food ingredient and convey an increased risk to the those with coeliac disease. 
Treatment of peptides with food-grade tartaric acid mimicked the action of tissue transglutaminase in deamidation of glutamine residues.

From sample to result in seconds: DART ionization with trapped ion mobility QTOF MS for fast identification of seized drugs

Nicolas Fraysse¹, Leon Gröschel^1,2, Birgit Schneider¹, Ilona Nordhorn¹, Carsten Baessmann¹

1. Bruker Daltonics GmbH & Co. KG, Bremen, Germany
2. University of Münster, Münster, Germany 

Background and aim: Forensic analytics are constantly facing growing demands including an increasing number of targets due to emerging new psychoactive substances and the need to shorten response time and increase throughput. The use of Direct Analysis in Real Time with High Resolution Mass Spectrometry (DART-HRMS) as a forensic tool is praised for its ability to quickly and easily generate valuable information. Herein, we present a workflow based on the combination of DART-HRMS with trapped ion mobility spectrometry (TIMS) for the identification of seized drugs including isomeric substances. 

Methods: A DART JumpShot source (Bruker Daltonics) and a timsTOF Pro 2 mass spectrometer (Bruker Daltonics) were used for the analysis. 3 µL aliquots of sample solutions containing mixtures of different drugs including opioids, benzodiazepines, amphetamines etc. and isomeric substance pairs were deposited onto QuickStrip wire mesh grid cards. Alternatively, a small amount of drug powder could be deposited on a glass stick and ionized from there without any additional sample preparation. The DART source was operated with helium in pulsed gas flow ionization mode at a temperature of 350°C. Full scan and parallel accumulation serial fragmentation (PASEF)MS/MS spectra were acquired in positive ionization mode and searched against home-built libraries containing about 250 drugs and toxins as well as 3rd party libraries, e.g., from NIST. 

Results: Up to 20 different drugs could be separated and identified in one sample. Using TIMS, pairs of isomeric substances including morphine and norcodeine as well as hydromorphone and norhydrocodone could be separated, yielding clean MS/MS spectra for library searches. Collision cross-sections (CCS) were used as an additional identification criterion to assist in the identification of the drugs. Together with Bruker’s automated library search for DART-MS/MS data, sample reports were received in less than 15 s after sample introduction. 

Conclusion: The proposed workflow provides a comprehensive solution for the characterization of seized drugs. Compared to analysis times of several minutes with chromatographic methods, analyses with DART-MS are completed within 15 seconds. Ion mobility can be used to clean up spectra and improve identification certainty. Additionally, little to no sample preparation is required.

One extraction two methods - combination of LC-MS/MS and GC-MS/MS for the analysis of cannabinoids in hair 

Christian Berchtold, Simon Kramer, Tina Binz

Center for Forensic Hair Analysis, Zurich Institute of Forensic Medicine

Background and aim: At the Center for Forensic Hair Analysis of the Zürich Institute for Forensic Medicine approximately 400 hair samples are analyzed for cannabinoids annually. A robust and reliable LC-MS/MS routine method is used to quantify ∆9-THC, CBD and CBN in hair. This method is based on a APCI LC-MS/MS approach [1]. To reliably detect THC consumption in hair, the active metabolite THC-COOH should be detected [2]. To support the current methodology and workflow, a new GC-MS/MS method was developed for the analysis of ∆9-THC and metabolites. The method enables the reinjection of existing extracts and thus provides a parallel analysis with two methodologies. In addition, the GC-MS/MS as an orthogonal method, would potentially support the identification of other cannabinoids of interest. 

Methods: An Agilent (Santa Clara, CA, USA) 7890B GC-System equipped with a split/splitless injector, a CTC Analytics (Zwingen, CH) PAL LSI 85 Autosampler, and an Agilent 7000C MS Triple Quad equipped with electron impact ionization (EI) were used. An Agilent HP-5ms Ultra Inert column (30m, 0.25mm 0.25 µm) and helium as carrier gas were used for chromatographic separation. Hair samples were extracted according to the method by Scholz et al. [1]. The methanol hair extract is used for LC-MS/MS analysis (as a mix of 100 µL methanol extract and 100 µL buffer) [1]. For GC-MS/MS analysis 750 µL of the methanol extract (Approach A) or the already measured LC-MS/MS injection solution are used (Approach B). For both approaches the samples are dried under nitrogen and derivatized with 50 µL BSTFA 1% TMCS.
 
Results: Approach A (analyzing the methanolic extract) showed sufficient GC-MS/MS sensitivity: LOD (limit of detection) 0.2 pg/mg for THC-OH, THC-COOH, 1 pg/mg for ∆9-THC and CBN, and 5 pg/mg for CBD. Directly compared with LC –MS/MS the median CV for the values of ∆9-THC was 4.2 %. Approach B (analyzing the already injected LC-MS/MS solution) showed LODs at 1 pg/mg for THC-OH, 5 pg/mg for THC-COOH, CBN, ∆9-THC, and 10 pg/mL for CBD. Directly compared with the results of a LC-MS/MS experiment, the median CV for ∆9-THC was found at 4.8 %. Both approaches showed in all TC-positive samples signals for the THC-COOH metabolites (exceptions are spiked QC-Samples), which proves THC consumption. 

Conclusion: The new GC-MS/MS method enables the analysis of the same hair extract from the routine LC-MS/MS method without an additional workup and provides comparable results. Using the methanolic extract (approach A) provides higher sensitivity due to the higher volume analyzed. Alternatively, it is also possible to analyze the samples after the LC-MS/MS measurement (same injection solution, approach B) with slightly reduced sensitivity. In addition, the detection of the THC-metabolite THC-COOH by GC-MS/MS allows the differentiation between THC contamination and consumption. It is also possible to integrate additional cannabinoids such as Hexahydrocannabinol (HHC) or ∆8-THC into the method. Nevertheless, the derivatization step for GC-MS is laborious and therefore it will remain a confirmation method for the fast routine LC-MS/MS method. 

[1] Scholz, C., Madry, M.M., Kraemer, T., Baumgartner, M.R., 2022. LC–MS-MS Analysis of Δ9-THC, CBN and CBD in Hair: Investigation of Artifacts. Journal of Analytical Toxicology 46, 504–511. https://doi.org/10.1093/jat/bkab056.
[2] Franz, T., Skopp, G., Schwarz, G., Musshoff, F., 2018. Proof of active cannabis use comparing 11-hydroxy-∆9-tetrahydrocannabinol with 11-nor-9-carboxy-tetrahydrocannabinol concentrations. Drug Testing and Analysis 10, 1573–1578. https://doi.org/10.1002/dta.2415.

Biomonitoring of bisphenols in children and adolescents

Baptiste Clerc¹, Alexandra Jaus¹, Aline Troxler², Julia Vincentini³, Sandrine Estoppey³ and Gisela Umbricht¹

1. Federal Institute of Metrology METAS, Bern, Switzerland
2. Federal Food Safety and Veterinary Office FSVO, Bern, Switzerland
3. Centre universitaire de médecine générale et santé publique Unisanté, Lausanne, Switzerland
 
Bisphenols (BPs) are used as monomer for the production of polycarbonates, polyvinyl chloride, epoxy resins and thermal paper. Due to their presence in common consumer products as well as their chemical structure, they can migrate from these products into food, beverages, water, air, dust and soil, leading to human exposure by a variety of routes. BPs are suspected to have disrupting effects on the endocrine system of humans and animals. Endocrine-disrupting chemicals may mimic, block, or interfere with the body's hormones and are associated with a wide array of health issues. Human biomonitoring for these compounds is therefore a crucial method to assess the possible presence of these chemicals and their corresponding metabolites in various body fluids to determine the extent of exposure in both a qualitative and quantitative manner.

Glucuronidation of BPs in the intestine and liver is considered the main metabolic pathway for most BPs. These metabolites are mainly excreted through urine, and other metabolites only result when the glucuronidation pathway is saturated. For the sample preparation, urine samples from Swiss kids will first be treated with β-glucuronidase to hydrolyze the glucuronide conjugates and set free the BPs. Internal standards will be added to the samples. Further sample purification will be performed by using a trap column, which means that the substances are focused on the trap column via a first pump and further, after the switch of the columns, the substances are transferred to the analytical column via a second pump and chromatographically separated. The substances are quantified using a UHPLC-MS/MS system with negative polarity electrospray ionization applying scheduled multiple reaction monitoring.

Recently, the Swiss Federal Food Safety and Veterinary Office mandated the first nutrition survey in Swiss kids (menuCH-Kids), which contains, among others, the analysis of various chemicals and elements in different human matrices. The study includes a representative set of urine samples of 1800 Swiss kids (6-17y.), which will constitute the analysed samples. The collection will be spread over one year and begins in fall 2023. The tests will include bisphenol A, S, F, AF and TMC.

Optimized lab-based and field environmental analysis using direct mass spectrometry

David Müller¹, Kara Merkle-Gams¹, Vaughan Langford², Mark Perkins³

1. Syft Technologies, Darmstadt (DE)
2. Syft Technologies, Christchurch (NZ)
3. Element Materials Technology, Cambridge (UK)

Conventional methods for the analysis of trace volatile organic compounds (VOC) in air, soil, and water usually involve significant sample preparation followed by timely chromatographic analysis. Direct mass spectrometry (DMS) provides opportunities for simplification – or even elimination – of sample preparation, plus real-time onsite analysis or high-throughput lab analysis. SIFT-MS (Selected Ion Flow Tube - Mass Spectrometry) is a DMS technique that provides highly sensitive and selective analysis of a wide range of compounds by applying various switchable soft chemical ionization (CI) reagents. Diverse VOC such as nitrosamines, ethylene oxide, benzene, toluene and formaldehyde as well as inorganic gases such as sulfur dioxide and hydrogen sulfide are detected in a direct, single analysis. 
Various environmental applications of SIFT-MS will be discussed, ranging from real-time ambient air monitoring to high-throughput water and soil analysis. 

SIFT-MS utilizes precisely controlled chemical ionization reactions to detect and quantify trace amounts of VOC. Analysis occurs in real-time and with typical LOD in the sub-ppbv range. Positive or negative reagent ions are generated when a microwave is discharged through moist air. Selection of the individual reagent ion is performed with an upstream quadrupole, following controlled ion-molecule reactions with the sample gas in the flow tube chamber. The mass analysis of the product and remaining reagent ions is done in a downstream quadrupole. Software processes the ion counts, together with instrumental parameters, to calculate absolute concentrations of the target compounds.
Field studies will be presented where SIFT-MS was applied for the mobile analysis of VOC in air directly on the site of plants, public traffic or areas like gas stations or convenient stores. Those were performed in public areas in Germany and in the United States of America. 

Additionally, the technology can be used in high-throughput lab analysis. A study will be discussed that was applied to quantify possible odor-causing chemical species at a large New Zealand wastewater treatment plant (WWTP). Results are discussed and correlated in terms of chemical compositions determined by SIFT-MS and by conventional olfactometry.

Fate of the double helix DNA in-vacuo. Ion mobility spectrometry, IR and Circular Dichroism ion spectroscopy

Frédéric Rosu¹, Steven Daly², FELIX peoples³, Valérie Gabelica^1,2

1. University of Bordeaux, CNRS UAR 3033, Institut Européen de Chimie et Biologie (IECB), Pessac, France
2. University of Bordeaux, INSERM U1212, ARNA Laboratory, IECB site, 2 rue Robert Escarpit, 33600 Pessac, France
3. FELIX, The Neederlands

We report the fate of the B-DNA double helix in the gas phase using ion mobility spectrometry, infrared action spectroscopy and electronic circular dichroism ion spectroscopy. Based on theoretical interpretations of our ion mobility results on 12-mer duplex DNA, we have observed that regular B-DNA double helix in solution undergoes conformational changes and become more compact. We have performed molecular dynamics at the semi-empirical level or DFT level to generate ensembles of structures matching the experimental collisional cross sections. The models show that the helicity increased and new hydrogen bonds between the phosphates groups appeared. We performed IRMPD action spectroscopy in a particular wavelength range (1900-2600 cm-1) to demonstrate that existence of these phosphate-phosphate H-bonds. B-DNA shows a band near 2500 cm-1 characteristic of POH-OP bond. Next, we used a recently developed a new spectroscopic methods that is sensitive to the stacking orientation of the nucleic acids bases. Mass resolved circular dichroism action spectroscopy has been used in combination of TD-DFT calculations to obtain information of the new stacking configuration in the compacted helix.

Mixed brominated chlorinated paraffins (BCPs) – the ultimate endeavour when the mass spectrometric analysis of CPs is already a challenge

Jules Hutter^1,2, A. Tell^1,2, M. Knobloch^1,3, U. Stalder³, L. Bigler³, S. Kern², D. Bleiner^1,3, N. V. Heeb¹

1. Swiss Federal Laboratories for Materials Science and Technology Empa, Überlandstrasse 129, 8600 Dübendorf, Switzerland
2. Zürich University of Applied Sciences ZHAW, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland
3. University of Zürich, Department of Chemistry, Winterthurerstrasse 190, 8057 Zürich, Switzerland 

Additives are often added to plastic materials to increase their performance. Such additives can be flame retardants, plasticisers, or surfactants, just to name a few. Depending on the application, these additives must be long-lasting inert chemi-cals that can withstand harsh conditions. Some of these substances, such as hexabromocyclododecanes (HBCDs), have already been declared as persistent organic pollutants (POPs) and their use is now regulated by the Stockholm Convention [1]. As a consequence, novel brominated flame retardants (NBFRs) entered the market [2]. Other examples of additives found in plastics materials are chlorinated paraffins (CPs) [3]. CPs are high production volume chemicals (>1 Mt/y), used as technical mixtures including a wide range of C- (nC ≈ 9–34) and Cl-homologues (nCl ≈ 3–14). Short-chain chlorinated paraffins (SCCP, C10–C13) are listed as POPs under the Stockholm Convention since 2017, and their use has been restricted as well. After the ban, SCCPs were substituted by medium- (MCCP, C14–C17) and long-chain chlorinated paraffins (LCCP, C18–C21), which have similar chemical and physical properties. It is being discussed whether MCCPs should be regulated as the SCCPs. We hypothesize that NBFRs and CPs are present in recycled plastic materials, and when processed may form mixed brominated chlorinated paraffins (BCPs). The analysis of BCPs is considered an ultimate challenge. BCPs can be expressed by the general formula CnH2n+2-x-yBrxCly. We will present a new analytical method to detect and evaluate BCP-spectra.

Mixtures of seven single-chain CPs (C10, C12, C14, C18, C21, C22 and C30) were exposed to heat and bromine. A method of liquid chromatography coupled to an atmospheric pressure chemical ionization mass spectrometer with an Orbitrap analyser (LC-APCI-Orbitrap-MS) was applied. The APCI source produced singly charged chloride-adduct ions [M+Cl]- of CPs and BCPs. The high mass resolution in combination with an adapted version of R-based automated spectra evaluation routine (bromo-RASER) enabled the simultaneous recognition of CP- and BCP-homologues in mass spectra containing about 36000 ions [5]. Besides non-brominated CP-homologues, dozens of mono-, di-, tri- and even some tetra-brominated CPs were detected. Relative abundances of BCP-homologues were 10% of the abundances corresponding to CP-homologues. Mono-BCPs were the most abundant BCP-species followed by di-, tri-, and tetra-BCPs with proportions of 5.2, 4.7, 0.1 and <0.1 %, respectively. We will present measured mass spectra and isotopic clusters of selected BCPs and show their mass accuracies. The combination of the chosen analytical and data evaluation methods is a powerful tool for a selective and fast evaluation of complex spectra containing ten thousands of CP- and BCP-ions. With the ability to determine those new chemicals, we get one step closer to evaluate risks and benefits of recycled plastic materials, which are increasingly used in a circular economy. 

[1] N. Heeb, et al., Chemosphere 80 (2010) 701–708.
[2] R. Zhang, et al., Journal of Hazardous Materials 447 (2023) 130789. 
[3] M. Knobloch, et al., Chemosphere 291 (2022) 132938.
[4] L. Schinkel, et al., Chemosphere 194 (2018) 803-811.
[5] M. Knobloch, et al., Anal. Chem., 94 (2022) 40.

Targeted deconvolution benefits ligand screening and binding analysis in Orbitrap-based protein-ligand interaction applications

Konstantin Nagornov, Anton Kozhinov, Yury Tsybin

Spectroswiss, 1015 Lausanne, Switzerland 

Taking into account the Orbitrap data's specific nature, namely the time-domain transients and isotopic envelopes' profiles, should enhance the feature extraction performance in protein-ligand interaction data analysis. Previously, we reported on developing an Orbitrap data-specific software tool (FTMS Simulator) for accurate simulation of Orbitrap FTMS (profile) mass spectra for any elemental composition. Here, we use these accurately simulated Orbitrap mass spectra to support sensitive, rapid, and targeted deconvolution-based feature extraction and compound identification in protein-ligand interaction workflows. Features are confirmed by similarity scoring between the isotopic envelopes of the experimental and simulated mass spectra. The isotopic envelopes can be matched individually or grouped. First, affinity LC-MS based ligand screening was performed using a Q Exactive Orbitrap. More than 10'000 ligands were sampled and analysed using two (standard vs sample) 96-deep-well plates (about 100 ligands per single well). Compounds identification and similarity filtering in the samples were performed by extracting selecting ion current (SIC) chromatograms using the user-defined number of highest isotopologues. The binding index was calculated as a ratio of under the curve areas between standard and sample SIC features. Secondly, the ligand binding assays experiment was performed using a Q Exactive Orbitrap. Several target ligands were sampled and analyzed using two (non-specific, control vs specific, target) 96-deep-well plates. Each ligand was present in several different concentrations (separate wells). The specific and non-specific dependencies of the under the curve area of SIC features on concentrations were used for calculation of ligand binding affinity. Overall, we demonstrate that small molecules analysis in protein-ligand interactions, where large number (>20k) of reference compounds is searched in the large (up to 1 k) number of files, may be automated, accelerated and improved using the targeted deconvolution method, pre-calculated profile library and automatic detection parameters optimization.

Assessment of various strategies for distinguishing enantiomers by cold ion spectroscopy

Vladimir Kopysov, Oleg V. Boyarkin

SCI SB RB Group, Ecole Polytechnique Fédérale de Lausanne, Station 6, CH-1005, Lausanne, Switzerland

Identification of isomers, and especially enantiomers, remains among the challenging tasks in analytical chemistry. While isomeric species often differ in their physical properties and biological activities, there is no single universal method capable of distinguishing them. Cold ion spectroscopy (CIS) is a technique based on the measurement of ultraviolet (UV) or infrared (IR) photofragmentation spectra of cold gas-phase ions. Cooling ions to cryogenic temperatures drastically suppresses the inhomogeneous spectral broadening, often allowing for vibrational resolution in UV and IR spectra, which makes them highly specific to the 3D structure of the ions. A library of such spectra recorded for a set of isomers can be used to identify them in their solution mixtures. This approach has been successfully employed for identification of isomeric peptides, oligosaccharides, lipids, and drug molecules. However, enantiomers have the same UV and IR spectra. To distinguish them, one can nonetheless record the spectra of their noncovalent complexes with a chiral molecule. Here, we assess the capability of CIS to identify enantiomers of a lipid, sphingosine, using various chiral UV chromophores and a chiral solvent molecule, sec-butanol. We also demonstrate how CIS can be applied in the case when only one sterically pure isomer is available or when even none of the standards are available.

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