11th Conference of the Czech Society for Mass Spectrometry (Day 3)
CSMS: 11th Conference of the Czech Society for Mass Spectrometry (Day 3)
Programme of the 11th Czech Mass Spectrometry Conference 2023
ČSHS: Conclusion of the conference, announcement of the best posters
Programme CMSC 2023
Wednesday June 21, 2023
9:00 - 10:20 Section V
9:00 - 9:20 The benefits of trapped ion mobility for single-cell proteomics
- Gary Kruppa
Thought to be an unachievable dream just a few years ago, it is now possible to achieve coverage of the proteome in a single cell from a human cancer cell line to depths of >4000 protein groups, sufficient to begin unraveling biological differences between cells. This talk will present details of a new mass spectrometer with the highest sensitivity to date for single cell proteomics and other proteomics applications requiring ultimate sensitivity such as immunopeptidomics and PTM analysis. The mass spectrometer includes a trapped ion mobility separation (TIMS) device in the front end and the advantages of this configuration for single cell proteomics will be discussed. The best currently achievable results will be presented as well as perspectives for future developments.
9:20 - 9:40 Zapping ions – using powerful lasers for more informative mass spectrometry
- Alan Kádek
Ever since the deciphering of human genome, both structural biologists and mass spectrometrists working in the field of proteomics have realized, that there exists yet another level of complexity involved in the regulation and controlling of protein functions. Through splicing, mutations as well as post-translational modifications and involvement in larger non-covalent assemblies, individual proteins generate a complex landscape of so-called proteoforms with huge implications on their functioning in organisms. Therefore, over the last decade or so proteomics has been aiming increasingly at the analysis of specific proteoforms through the means of top-down mass spectrometry, especially in its native implementation working with whole protein assemblies. However, this approach relies heavily on efficient and informative means of fragmentation for which diverse MS dissociation techniques and possibly their combinations are often sorely needed.
This contribution will report on the successful implementation of both 10.6 μm CO2 and 193 nm ArF lasers for infrared multi-photon dissociation (IRMPD) and ultraviolet photodissociation (UVPD), respectively, for in-cell dissociation inside the 15T SolariX Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer at BIOCEV in Prague. Their benefits as well as potential for collaborations will be showcased on examples of both proteins and smaller molecules. Also, future directions of research aimed at native mass spectrometry of large non-covalent protein assemblies will be discussed.
9:40 - 10:00 Fast photochemical oxidation of nucleic acids coupled to high-resolution MS analysis
- Marek Polák
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 highly 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 a Insulin Response Element (IRE) and DNA binding domain of FOXO4 transcription factor. The 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. 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 bingind 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.
10:00 - 10:20 Internal standard normalization in MALDI mass spectrometry imaging
- Hynek Mácha
Mass spectrometry generally suffers from the strong influence of an analyzed matrix and its impact on the ionization process, affecting a targeted compound’s yield. This matrix effect is highlighted in the matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging, where two key factors must be considered. The first comes from the principle of ionization, in which the matrix has to be cocrystalized with analytes and ensure efficient ionization. The second is the heterogeneity and the integrity of a sample, usually a tissue section, causing enhancement or suppression of the ionization. Thus, MALDI MSI is recognized as a qualitative method where an internal standard, which would compensate for the matrix effects, is absent. We developed an internal standardization approach to extend a possible use of MALDI MSI to quantitative analysis. With the use of ¹³C labeled glucose, ¹³C labeled glutamic acid, ¹⁵N labeled adenosine monophosphate, and ¹³C labeled and species-specific peptides, a broad range of analytes, including saccharides, amino acids, nucleosides, and peptides can be precisely and spatially mapped to decipher region-specific tissue metabolomic processes. The presented approach was used in the Escherichia coli-induced neuroinfection model and opened new opportunities for relative and absolute quantitation.
ČSHS: Internal standard normalization in MALDI mass spectrometry imaging (Hynek Mácha)
10:20 - 10:50 Coffee break
Sponsors and partners
- Město Brno
- Bruker Daltonics
- Waters Corporation
10:50 - 11:50 Plenary lecture III: Introduction to three dimensional cryo-electron microscopy
- Adam Schröfel (Přírodovědecká fakulta UK, Praha)
"This talk provides a concise introduction to three-dimensional cryo electron microscopy (3D cryo-EM) and electron tomography. We will cover the basic principles of sample preparation, imaging, and data collection using cryo-EM techniques, as well as the reconstruction methods involved in deriving high-resolution structures. Additionally, we will explore the application of electron tomography, a powerful extension of cryo-EM, for studying three-dimensional cellular architectures and larger macromolecular complexes. The talk aims to provide participants with a foundational understanding of 3D cryo-EM and electron tomography, showcasing their potential for advancing structural biology and uncovering intricate cellular details.”
ČSHS: Introduction to three dimensional cryo-electron microscopy (Adam Schröfel)
11:50 - 12:00 Conclusion of the conference, announcement of the best posters
12:30 - 14:00 Lunch
14:00 - 15:30 Excursion at Thermo Fisher Scientific
ČSHS: Conclusion of the conference, announcement of the best posters
List of posters
Sulfated phenolic acids are common plant metabolites
- Klára Supíková
Sulfated metabolites are mainly typical for marine organisms, including seagrasses (e.g. zosteric acid), algae (e.g. sulfated phenolic acids and sulfated polysaccharides), invertebrates (e.g. sulfated steroids) and sponges (e.g. psammaplin A sulfate). On the contrary, plant sulfated metabolites, represented by e.g. glucosinolates, sulfated flavonoids and phytosulfokines, are much less common and explored. Here, we report a novel group of plant sulfated compounds, sulfated phenolic acids, that were originally detected only in algae and seagresses.
The untargeted UHPLC-QTOF-MS metabolomic analysis of more than 50 methanolic extracts from plants, fungi and algae was performed. Based on the detection of the neutral loss of sulfur trioxide (monoisotopic mass 79.9568 Da) in MS/MS spectra, we identified ferulic acid 4-sulfate, vanillic acid 4-sulfate, 4-(sulfooxy)benzoic acid and 4-(sulfooxy)phenylacetic acid in plants for the first time. In addition, we detected zosteric acid in land plants. These compounds were quantified in several edible species, including oat, wheat, barley, tomato, carrot, broccoli, celery, cabbage, banana, pineapple, radish and olive oil. While concentrations of sulfated phenolic acids ranged from 0.34 - 22.18 μg/g DW, the corresponding non-sulfated acids were mostly undetected or present at lower concentrations.
Development of sample pretreatment methods for the analysis of selected growth factors in various biological fluids by CZE-MS
- Martina Opetová
Growth factors are a heterogeneous group of proteins that are secreted by various tissues in the human body. Their presence in biological matrices may indicate the development of multiple diseases, thus there is a growing need to develop new high-performance analytical methods and procedures for their reliable identification and quantification. However, these substances are present in biological fluids at very low concentration levels compared to other high-abundant proteins that can mask the presence of growth factors and make their analysis impossible. This work aims to develop a sample pretreatment method for the purification of complex biological samples before their analysis by online hyphenation of capillary zone electrophoresis and mass spectrometry (CZE-MS). Optimization and comparison of different conditions for solid phase extraction and protein precipitation to reach the compatibility with the CZE-MS method and online sample preconcentration was performed on biological fluids (urine, plasma and serum) fortified with a mixture of three selected intact growth factors, namely IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), and TGF-α (transforming growth factor-α).
Optimisation of infliximab middle-up analysis by capillary electrophoresis - mass spectrometry
- Jana Havlikova
Infliximab (IFX) is a chimeric monoclonal antibody (mAb) widely used for treatment of multiple types of diseases. IFX is a 150 kDa protein consisting of heavy and light chain subunits of molecular weight approximately 50 kDa and 25 kDa, respectively. Middle-up mass spectrometry (MS) mAb analysis has gained popularity over the last years as an alternative to a full protein enzymatic digestion. Capillary electrophoresis (CE) is a high-resolution separation electromigration technique that was shown to be advantageous for separation of proteins. In this work, two reducing conditions were investigated and compared while the parameters for CE-MS analysis of IFX reference standard were optimised. A range of concentrations of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) being 50 - 500 mM and 50 μM – 1 mM, respectively, were tested for the IFX reduction. The analysis of reduced IFX was performed on a 6520 qTOF mass spectrometer (Agilent, Santa Clara, US). CE experiments, focused on optimisation of IFX middle-up analysis, were carried out on a CE 7100 instrument coupled to a 6410 triple-quadrupole mass spectrometer (both Agilent, Santa Clara, US). Background electrolyte (BGE) systems (including formic and acetic acids) were tested and the optimal results were achieved by using 1 M acetic acid. Both reducing agents (with appropriate concentration) were shown as suitable and heavy and light chain subunits were observed in the mass spectra. Generally, a difference in charge state distribution of both subunits was observed for the two of the investigated reducing agents. Middle-up CE-MS analysis was capable of detecting mAb light chain in single ion monitoring and scan modes, showing a promise for the future mAb CE-MS analyses.
Development of modern analytical methods for the characterization of keyhole limpet hemocyanin (KLH) as immunogenic carrier protein in biopharmaceuticals
- Ondrej Štefánik
Keyhole limpet hemocyanin (KLH) is a large glycoprotein composed of KLH1 and KLH2 subunits, each of which has a molecular weight in the range of 400 kDa. The aforementioned subunits then assemble into decameric (4 million Da), di-decameric, and multi-decameric structures of enormous sizes.
Its antigenic properties cause strong antibody responses. Therefore, KLH is widely used in the field of immunobiology. The conjugation of nonimmunogenic peptides or other haptens with KLH results in highly immunogenic preparations. KLH has high potential in the treatment of various types of cancer. It can be used alone in the treatment of bladder cancer or as a carrier in vaccines against non-Hodgkin lymphoma, skin melanoma, or breast cancer. Similarly, KLH can be conjugated to HIV or HPV-4 peptides or proteins for potential AIDS therapy and prevention of HPV-related diseases.
The aim of the present study was to develop sufficient and reliable analytical methods for the assessment of KLH structure. Comprehensive characterization was achieved using three approaches: size exclusion chromatography, peptide mapping (LC-MS), and intact protein analysis (MS – Q-TOF). Moreover, specific KLH peptides (characteristic for each KLH subunit) were selected for perspective quantitative approach based on targeted LC-MS analysis.
An application of UPLC-QQQ MS to characterize changes in free cholesterol and cholesterol esters in lung cells and lung tissue after mice exposure to metal oxide nanoparticles
- Markéta Laštovičková
Cholesterol is essential compound for normal cell function. It is a component of cell membranes and a precursor of steroid hormones and other key metabolites. In animal tissues, cholesterol occurs in free form (free cholesterol, FC), esterified to long-chain fatty acids (cholesterol esters, CEs) or bound in lipoproteins. CEs are the preferred form for transport of cholesterol in plasma and as a biologically inert storage form to compensate for the excess. Abnormal cholesterol levels have been observed in a variety of diseases. Thus, quantitative or qualitative changes in various forms of cholesterol can be used to monitor the progress of treatment or as useful biomarkers revealing the molecular mechanism of disease.
The lead oxide nanoparticles (PbO NPs) are emitted into the environment during high-temperature and other technological processes. The inhalation of PbO NPs severely damaged target organs of exposed mice. These nanoparticles crossed the lung barrier and were distributed through the blood to secondary target organs where they caused numerous pathological changes. Our optimized LC-MS method, including reverse-phase UPLC combined with triple quadrupole mass spectrometry (UHPLC-QQQMS), was used to compare the profile of lipids extracted from a control lung cell line (and lung tissue from control mice) compared to lung cells and tissue from mice exposed to elevated concentrations of airborne PbO NPs. Preliminary results showed that the levels of selected CEs (e.g. CE 18:1; CE 18:2, CE 22:6, CE 24:5; CE 24:6) in lung cells were increased after exposure to PbO NPs. Our study revealed a association of alteration of cholesterol metabolism after exposure to metal NPs, which is the subject of our further detailed study.
Monitoring of conformational changes in transmembrane proteins using HDX and FFAP radical labeling
- Lukáš Fojtík
The chloride channel family is divided into two groups: channels and antiporters. Channels are known for their large conformational changes of subunits during ion transport. In contrast, antiporters have only tiny movements near the transport pathway. Transporters are involved in many cellular processes, and their mutation can cause serious diseases. The CLC-ec1 is a Cl-/H+ antiporter of a single proton for two chloride ions from Escherichia coli. Ion transport is supposed to be accompanied by a structural change between inward and outward-facing conformation, but this transition has not been captured so far. During the transport cycle, the protonation of key Glu residues should induce an outward-facing state. This was mimicked through a mutation of three Glu residues for Gln. We used this mutation to reveal the role of protonation in the ion transport mechanism of CLC-ec1. CLC-ec1 and CLC-QQQ mutant were expressed in the E. coli system and purified by IMAC chromatography in ƞdecyl maltoside as a solubilization reagent. Solubilized protein was transferred to the saposin nanodisc by size exclusion chromatography. We then used hydrogen/deuterium exchange and radical labeling with Togni reagent (FFAP) coupled to high-resolution mass spectrometry to capture the structural changes alongside the backbone as well as on the side chains of aromatic residues The optimization of the digest workflow for both methods provided full sequence coverage with reasonable lengths of the peptides. As the best conditions for the HDX we determined online digest on the column of co-immobilized pepsin-nepentesin 2 whereas for FFAP we got better sequence coverage by offline digest via cyanogen bromide and trypsin. HDX and FFAP were subsequently used for the study of the structural differences between CLC-ec1 and the CLC-QQQ mutant at pH 7.4. To prove the role of protonation, a comparison experiment at four different pH levels, from 4.4 to pH 7.4, was performed for both proteins. Results showed a critical connection between protonation and structural changes in the ion transport path and conformational change in the transmembrane helixes.
The ion mobility/mass spectrometry method for analysis of phosphorylated tau peptides in cerebrospinal fluid
- Andrej Kovac
A variety of neurodegenerative disorders, including Alzheimer´s disease (AD), are associated with neurofibrillary tangles composed of tau protein in the form of toxic tau oligomers. Aberrant tau phosphorylation is a hallmark in many tauopathies, and hyperphosphorylation promotes the formation of paired helical filaments which are the main constituents of neurofibrillary tangles in the brain. While cerebrospinal fluid levels of total tau and tau phosphorylated at threonine residue 181 are established core biomarkers for AD, the values of alternative phosphorylation sites, which may have more direct relevance to pathology, for early diagnosis are not yet known. To characterize the specific phosphorylation of tau peptides in CSF, we have applied an innovative mass spectrometry workflow. Using ion mobility separation, we were enabled to provide valuable structural information and separate different isobaric tau peptides. This is the most critical step for the development of ultra-sensitive immunoassays allowing the quantification of novel biomarkers at sub-picogram levels.
LC-MS analysis of internal DNA modification-method development
- Kristína Spustová
Methylation of nucleic acids can play a crucial role in biological processes including regulation of gene expression. The most common DNA modifications are methylations of adenosine and cytosine such as 5-methyldeoxycytosine, 5-hydroxymethyl-deoxycytosine, N4-methyl-deoxycytosine and N6-methyldeoxyadenine (m6dA). These modifications are mostly detected by LC-MS in the form of nucleosides with reverse phase chromatography as a separation method. This protocol first requires an enzymatic digestion by Nuclease P1 (NUP1) followed by dephosphorylation using alkaline phosphatase (CIP).
Our aim was to simplify the sample preparation and to increase the sensitivity of the method. The optimized protocol includes only one enzymatic digestion of DNA by NUP1 followed by hydrophilic interaction chromatography (HILIC). The modifications are analysed in the form of nucleoside monophosphates by UHPLC (Acquity H-class, Waters) equipped with BEH Amide HILIC column coupled to high resolution mass spectrometer (Xevo G2-XS qTOF, Waters). The developed method was used to detect and quantify the most widespread internal modification on DNA, m6dA. Moreover, we also studied the changes of dAMP methylation in Bacillus subitilis under different growth conditions.
Analysis of Insulin-like Growth Factor 1 in Pharmaceutical Preparations by On-line Capillary Electrophoresis-Mass Spectrometry
- Radovan Tomašovský
Insulin-like growth factor-1 (IGF-1) is a 70-amino acid single-chain polypeptide which has found application in diagnostics as a biomarker of growth hormone disorders and as a therapy for growth failure in children and adolescents. Due to its strong anabolic effects, it is often abused by athletes for doping purposes in various pharmaceutical forms, including tablets, capsules, and injectable solutions. Here, we developed on-line hyphenated method based on capillary zone electrophoresis and triple quadrupole mass spectrometry detection with electrospray ionization for the determination of IGF-1 in pharmaceutical matrices. We achieved an efficient separation and well-shaped peaks with favorable migration times (<15 min) by optimizing the background electrolyte (500 mM formic acid + 5% acetonitrile, pH 1.96) and a separation voltage (+30 kV). Highly efficient, accurate, repeatable, sensitive (sub μg/mL levels), and selective analysis of IGF-1 was achieved with further optimization of ESI and MS parameters. Optimized and validated CZE-ESI-MS method was successfully applied for the determination of IGF-1 in injectable solutions (Increlex®) and its presence was also confirmed in various nutritional preparations.
This is the first work that uses validated CZE-ESI-MS method for the determination of IGF-1 in pharmaceutical matrices and reveals the potential of capillary electrophoresis for its use in drug quality control laboratories with benefits such as high separation efficiency, high-speed analysis, low sample consumption, as well as environmental and cost aspects compared to the more established liquid chromatography methods.
The effect of domain order on modulation of fusion two-domain proteins structure and its dynamics studied by hydrogen-deuterium exchange
- Martin Hubálek
Is the function of the multidomain proteins only determined by the existence of domains that are embedded in the sequence or the position and order of the domains matters? In such protein, hypothetically, each domain structure or function could be influenced by close neighbours. To address this hypothesis, we used hydrogen deuterium exchange structural method (HDX-MS) to investigate possible perturbations in structure of PDZ3 domain of Zonula occludens 1 protein (PDZ3). This domain has a natural peptide ligand with known binding site. We took PDZ3 domain and fused with TrpCage, in silico designed artificial domain, in both forward (PDZ3-TrpCage: PsLT) and reverse (TrpCage-PDZ3: TlLP) version of the sequence. Our aim was to determine peptide regions directly influenced by TrpCage position and find the differences between PsLT and TlLP proteins.
We applied HDX analysis in a traditional HDX set-up and also in set-up with low temperature and short labelling times to characterize the deuterium accessibility of PDZ3 peptides and regions. Subsequently, we performed the experiments with peptide ligand (JAMAP6).
The data shows that, in general, TrpCage destabilizes the PDZ3 structure. The peptide localiation also shows that some of the PDZ3 peptides with different behaviour in PsLT compared to TlLP were localized in ligand binding site suggesting that TrpCage affects also function of PDZ3. The results of the ligand binding experiments, surprisingly, counteract the effect of TrpCage more effectively in TlLP compared to PsLT. According to our results, we suggest the methodology to study the domain structural differences in the context of other domains using HDX-MS.
Simple UHPLC-MS amino acid analysis for determination of immunogenic proteins in biopharmaceuticals
- Juraj Piešťanský
We developed a modern and progressive analytical approach for the determination of innovative protein therapeutics present in biopharmaceuticals, based on the highly sensitive and selective amino acid analysis performed by reversed phase ultra high-performance liquid chromatography (UHPLC) and single quadrupole mass spectrometry. Such analytical approach is accompanied with the use of fast and effective targeted amino acids derivatization procedure which enables to enhance the sensitivity and selectivity of the analysis and also to ensure appropriate retention properties of the analytes in reversed phase chromatographic separation mode. Moreover, the on-line connection of ultra high-performance liquid chromatography with single quadrupole mass spectrometer enabled an unambiguous identification of amino acids derivatives and their exact quantification. The proposed method was successfully validated and applied in the field of quality control of modern therapeutic proteins present in biopharmaceuticals. The UHPLC-MS can be advantageously used as routine method in quality control laboratories, and with promising potentialities also for other and/or more complex matrices.
Single particle inductively coupled plasma mass spectrometry imaging of immunochemically labeled spheroid sections
- Vilém Svojanovský
Spheroids are one of the 3D biological models in which the arrangement of proliferating and necrotic cells resembles the tumor microenvironment. One of the markers of proliferation is Ki-67 protein, which is expressed by cells in the active phase of the cell cycle. Immunohistochemical labeling with a primary antibody and a fluorophore-labeled secondary antibody followed by fluorescence microscopy analysis is commonly employed for two- and three-dimensional imaging of the target protein in spheroid sections. We have successfully switched from fluorophore labeling of Ki-67 to nanoparticle labeling combined with LA ICP MS. Recently, gold nanoparticles were used as very sensitive immunochemical tags in conventional UV laser ablation ICP MSI. In our approach, intact 20 nm gold nanoparticles tags were desorbed from the spheroid section with 2940 nm laser ablation system.