Adam Pruska

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Pruska

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Adam

ORCID

0000-0002-5304-2541

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Publications 1 - 10 of 15

Mass Spectrometric Methods for Studying Thermodynamic Properties of Biomolecular Systems
Pruska, Adam (2023)
To fully understand how biomolecules interact in living organisms, we need to look at their energy levels and physical properties in their unbound and bound states. By combining this information, researchers can elucidate how biomolecules form, work, and affect cellular processes. Understanding the relationship between energy, shape, and function of biomolecules is essential in a wide range of studies, from fundamental research to clinical and industrial applications. Over the years, technological advancements have led to the development of numerous techniques and sophisticated approaches that allow us to deepen our understanding of the thermodynamics and kinetics of biomolecular systems. With a variety of types, such as photometric, calorimetric, and other methods, these techniques have transformed the field of biophysical and biochemical research. This thesis discusses the latest progress of temperature-controlled electrospray ionization (TC-ESI-MS) as one of the newest methods for studying thermodynamics in biomolecular systems. The first section of the thesis provides a comprehensive overview of the current knowledge about the structures of nucleic acids and their diverse biological roles, ranging from the packaging of DNA into chromatin to the regulation of translation processes. It also includes an overview of protein structures and their tendency to undergo aggregation. The ensuing chapter discusses the current position of temperature-controlled electrospray ionization mass spectrometry (TC-ESI-MS) within the context of various analytical techniques and other MS-based methods and presents the particular technological advancements in TC source designs. The discussion in the second part of this thesis focuses on the potential applications of TC-ESI-MS in studying of various biomolecular assemblies. Firstly, the impact of DNA G-quadruplexes (GQs) on different secondary structures, utilizing TC-nESI-MS to obtain unprecedently detailed thermodynamic information, were investigated. The results indicated that the order and primary structure of domains are critical for their stability and mutual destabilization. The findings further support the fact that this technique is useful for characterizing oligonucleotide interactions, with potential applications in drug design, aptamer characterization, and biosensing. Follow-up work revealed that inosine can incorporate into incomplete RNA GQs, thereby changing the unfolding mechanism and stability. In both studies, TC-ESI-MS benefited from the possibility to identify and quantify intermediates occurring during the melting experiments. x Furthermore, this thesis presents a study on the complicated unfolding mechanism of three-way junctions (TWJs) and the effect of TWJ-binding ligands on the stability of TWJ complex. We utilized both solution and gasphase techniques, including surface-induced dissociation, to interpret the results obtained from TC-ESI-MS. The last chapter presents development and optimization of a novel methodology for analyzing protein aggregation using a high-resolution cyclic ion mobility mass spectrometry device in combination with the TC-nESI source. The study demonstrates many advantages of TC-nESI in the detection of protein aggregation, highlighting its effectiveness in investigating the hexameric MDa urease protein pre-aggregates with implemented ion mobility separation increasing a number of detected temperature-induced intermediate species. Overall, the dissertation highlights the potential of using TC-ESI-MS as a powerful analytical tool for studying the thermodynamics of biological systems. The studies provide a comprehensive overview of both the applications and technological developments of this method, along with the challenges and future scope associated with its implementation. By advancing TC-ESI-MS, researchers can continue to expand their knowledge of not only thermodynamics but also the kinetics of biological systems, and contribute to advancements in the field of biochemistry, biophysics and the pharmaceutical industry.
Stoichiometry and architecture of the human pyruvate dehydrogenase complex
Zdanowicz, Rafal; Afanasyev, Pavel; Pruska, Adam; et al. (2024)
Science Advances
The pyruvate dehydrogenase complex (PDHc) is a key megaenzyme linking glycolysis with the citric acid cycle. In mammalian PDHc, dihydrolipoamide acetyltransferase (E2) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) form a 60-subunit core that associates with the peripheral subunits pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3). The structure and stoichiometry of the fully assembled, mammalian PDHc or its core remained elusive. Here, we demonstrate that the human PDHc core is formed by 48 E2 copies that bind 48 E1 heterotetramers and 12 E3BP copies that bind 12 E3 homodimers. Cryo-electron microscopy, together with native and cross-linking mass spectrometry, confirmed a core model in which 8 E2 homotrimers and 12 E2-E2-E3BP heterotrimers assemble into a pseudoicosahedral particle such that the 12 E3BP molecules form six E3BP-E3BP intertrimer interfaces distributed tetrahedrally within the 60-subunit core. The even distribution of E3 subunits in the peripheral shell of PDHc guarantees maximum enzymatic activity of the megaenzyme.
Additive manufacturing of Zn with submicron resolution and its conversion into Zn/ZnO core-shell structures
Nydegger, Mirco; Pruska, Adam; Galinski, Henning; et al. (2022)
Nanoscale
Electrohydrodynamic redox 3D printing (EHD-RP) is an additive manufacturing (AM) technique with submicron resolution and multi-metal capabilities, offering the possibility to switch chemistry during deposition “on-the-fly”. Despite the potential for synthesizing a large range of metals by electrochemical small-scale AM techniques, to date, only Cu and Ag have been reproducibly deposited by EHD-RP. Here, we extend the materials palette available to EHD-RP by using aqueous solvents instead of organic solvents, as used previously. We demonstrate deposition of Cu and Zn from sacrificial anodes immersed in acidic aqueous solvents. Mass spectrometry indicates that the choice of the solvent is important to the deposition of pure Zn. Additionally, we show that the deposited Zn structures, 250 nm in width, can be partially converted into semiconducting ZnO structures by oxidation at 325 °C in air.
SNAPpa: A Photoactivatable SNAP-tag for the Spatiotemporal Control of Protein Labeling
Mandl, Sabrina; Maiwald, Barbara; Adlmanninger, Elena; et al. (2025)
JACS Au
SNAP-tag is one of the most commonly used self-labeling protein tags for cell imaging studies. To achieve selective spatiotemporal imaging of cells, we set out to engineer a photoactivatable SNAP-tag. For this, we incorporated the well-established and readily available photocaged unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) into all three tyrosine positions of SNAP. In-gel imaging analysis and fluorescence polarization measurements revealed that placing ONBY in position Y114 of the SNAP-tag facilitates the most effective and most efficient photoactivation of the irreversible self-labeling reaction with (sulfonated) benzyl guanine substrates, which is why we dubbed this photoactivatable SNPA-tag variant "SNAPpa". To demonstrated its potential for live-cell imaging, we further tested SNAPpa in HEK293 cells, either fused to a nuclear localization domain for intracellular imaging or fused to either a transmembrane region or the glucagon-like peptide 1 receptor for extracellular imaging. Each SNAPpa construct produced no fluorescence signal when ONBY remained in its photocaged state by keeping the cells in the dark. However, a clear fluorescence signal appeared after light-induced decaging of ONBY. Applying a localized light beam thereby highlighted the precise spatiotemporal control of cell imaging. In conclusion, SNAPpa can be used for the efficient light-induced activation of fluorescence labeling and can be easily established, readily implemented and effectively combined with the broad repertoire of substrates that is already available for SNAP.
Lactic Acidosis Interferes With Toxicity of Perifosine to Colorectal Cancer Spheroids: Multimodal Imaging Analysis
Pavlatovská, Barbora; Machálková, Markéta; Brisudová, Petra; et al. (2020)
Frontiers in Oncology
Colorectal cancer (CRC) is a disease with constantly increasing incidence and high mortality. The treatment efficacy could be curtailed by drug resistance resulting from poor drug penetration into tumor tissue and the tumor-specific microenvironment, such as hypoxia and acidosis. Furthermore, CRC tumors can be exposed to different pH depending on the position in the intestinal tract. CRC tumors often share upregulation of the Akt signaling pathway. In this study, we investigated the role of external pH in control of cytotoxicity of perifosine, the Akt signaling pathway inhibitor, to CRC cells using 2D and 3D tumor models. In 3D settings, we employed an innovative strategy for simultaneous detection of spatial drug distribution and biological markers of proliferation/apoptosis using a combination of mass spectrometry imaging and immunohistochemistry. In 3D conditions, low and heterogeneous penetration of perifosine into the inner parts of the spheroids was observed. The depth of penetration depended on the treatment duration but not on the external pH. However, pH alteration in the tumor microenvironment affected the distribution of proliferation- and apoptosis-specific markers in the perifosine-treated spheroid. Accurate co-registration of perifosine distribution and biological response in the same spheroid section revealed dynamic changes in apoptotic and proliferative markers occurring not only in the perifosine-exposed cells, but also in the perifosine-free regions. Cytotoxicity of perifosine to both 2D and 3D cultures decreased in an acidic environment below pH 6.7. External pH affects cytotoxicity of the other Akt inhibitor, MK-2206, in a similar way. Our innovative approach for accurate determination of drug efficiency in 3D tumor tissue revealed that cytotoxicity of Akt inhibitors to CRC cells is strongly dependent on pH of the tumor microenvironment. Therefore, the effect of pH should be considered during the design and pre-clinical/clinical testing of the Akt-targeted cancer therapy.
Aptapaper ─ An Aptamer-Functionalized Glass Fiber Paper Platform for Rapid Upconcentration and Detection of Small Molecules
Martinez Jarquin, Sandra; Begley, Alina; Lai, Yin-Hung; et al. (2022)
Analytical Chemistry
We tested a paper-based platform (“Aptapaper”) for the upconcentration and analysis of small molecules from complex matrices for two well-characterized aptamers, quinine and serotonin binding aptamers (QBA and SBA, respectively). After incubating the aptapaper in conditions that ensure correct aptamer folding, the aptapaper was used to upconcentrate target analytes from complex matrices. Aptapaper was rinsed, dried, and the target analyte was detected immediately or up to 4 days later by paper spray ionization coupled to high resolution mass spectrometry (PS-MS). The minimum concentrations detectable were 81 pg/mL and 1.8 ng/mL for quinine and serotonin, respectively, from 100 mM AmAc or water. Complementary characterization of the QBA aptapaper system was performed using an orthogonal fluorescence microscopy method. Random adsorption was analyte-specific and observed for quinine, but not serotonin. This aptapaper approach is a semi-quantitative (10-20% RSD) platform for upconcentration of small metabolites by mass spectrometry.
Novel Insight into Proximal DNA Domain Interactions from Temperature‐Controlled Electrospray Ionization Mass Spectrometry
Pruska, Adam; Marchand, Adrien Henri; Zenobi, Renato (2021)
Angewandte Chemie. International Edition
Quadruplexes are non‐canonical nucleic acid structures essential for many cellular processes. Hybrid quadruplex‐duplex oligonucleotide assemblies comprised of multiple domains are challenging to study with conventional biophysical methods due to their structural complexity. Here, we introduce a novel method based on native mass spectrometry (MS) coupled with a custom‐built temperature‐controlled nanoelectrospray ionization (TCnESI) source designed to investigate interactions between proximal DNA domains. Thermal denaturation experiments were planned to observe unfolding of multi‐stranded oligonucleotide constructs derived from biologically relevant structures and to identify unfolding intermediates. Using the TCnESI MS, we observed changes in T m and thermodynamic characteristics of proximal DNA domains depending on the number of domains, their position, and order in a single experiment.
Conformational Dynamics of Hemoglobin in Solution and the Gas Phase Elucidated by Mass Spectrometry
Harrison, Julian A.; Gabriel, Janic; Pruska, Adam; et al. (2024)
Analytical Chemistry
Solution and gas-phase measurements can provide valuable insights into biomolecular conformational dynamics. By comparing the data from such experiments, it is possible to elucidate the nature of the interactions governing a biomolecule's stability. Here, we measured human, bovine, and porcine hemoglobin stability in solution and the gas phase using collision-induced dissociation, collision-induced unfolding, surface-induced dissociation, and temperature-controlled nanoelectrospray mass spectrometry. Hemoglobin dimer and tetramer stability in solution and gas phases did not correlate, likely due to differences in the composition of positive and negative amino acids on the surface of these molecules. Specifically, the absence of Lys-116 on the beta-subunit makes it easier for the human hemoglobin dimer to dissociate in the gas phase. However, the presence of Lys-60 makes the subunit more rigid thus it cannot unfold to the same extent as the other hemoglobin. Hemoglobin tetramers of different origins had similar stability in the gas phase, as there was no difference in the composition of charged amino acids at the tetramer interface. These results highlight how temperature-controlled mass spectrometry and collision-induced unfolding can elucidate the structural reasons behind differences in the gas-phase and solution stability of protein complexes.
Temperature-Controlled Electrospray Ionization: Recent Progress and Applications
Harrison, Julian Alexander; Pruska, Adam; Oganesyan, Irina; et al. (2021)
Chemistry - A European Journal
Native electrospray ionization (ESI) and nanoelectrospray ionization (nESI) allow researchers to analyze intact biomolecules and their complexes by mass spectrometry (MS). The data acquired using these soft ionization techniques provide a snapshot of a given biomolecules structure in solution. Over the last thirty years, several nESI and ESI sources capable of controlling spray solution temperature have been developed. These sources can be used to elucidate the thermodynamics of a given analyte, as well as provide structural information that cannot be readily obtained by other, more commonly used techniques. This review highlights how the field of temperature-controlled mass spectrometry has developed.
Inosine Substitutions in RNA Activate Latent G-Quadruplexes
Hagen, Timo; Laski, Artur; Brümmer, Anneke; et al. (2021)
Journal of the American Chemical Society
It is well-accepted that gene expression is heavily influenced by RNA structure. For instance, stem-loops and G-quadruplexes (rG4s) are dynamic motifs in mRNAs that influence gene expression. Adenosine-to-inosine (A-to-I) editing is a common chemical modification of RNA which introduces a nucleobase that is iso-structural with guanine, thereby changing RNA base-pairing properties. Here, we provide biophysical, chemical, and biological evidence that A-to-I exchange can activate latent rG4s by filling incomplete G-quartets with inosine. We demonstrate the formation of inosine-containing rG4s (GI-quadruplexes) in vitro and verify their activity in cells. GI-quadruplexes adopt parallel topologies, stabilized by potassium ions. They exhibit moderately reduced thermal stability compared to conventional G-quadruplexes. To study inosine-induced structural changes in a naturally occurring RNA, we use a synthetic approach that enables site-specific inosine incorporation in long RNAs. In summary, RNA GI-quadruplexes are a previously unrecognized structural motif that may contribute to the regulation of gene expression in vivo.

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Adam Pruska

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