2026-02-14
00:01 |
Method development for antibody analysis by CZE: From UV detection towards native CE-MS
Horkelová, Simona ; Koval, Dušan ; Kašička, Václav ; Coufal, P.
Therapeutic antibodies are biologics widely used to treat various diseases. Their thorough characterisation is essential for ensuring quality and efficacy in drug development. Among the key aspects are post–translational modifications, which can significantly affect antibody activity. Native proteomics aims to analyse proteins under conditions that preserve their noncovalent interactions, which are crucial for functional assemblies such as antibody–antigen or protein–protein complexes. This project focuses on developing analytical methods for native protein analysis by coupling capillary electrophoresis with high–resolution mass spectrometry using a nanospray interface. Capillary electrophoresis is employed as an initial separation technique to reduce sample complexity prior to mass spectrometry analysis, with separation conditions optimised to maintain nativelike environments and compatibility with mass spectrometry. In this study, we examined the monoclonal antibodies Trastuzumab (pI = 9.1), Cetuximab (pI = 8.1), and Rituximab (pI = 9.4). Method development was initiated using capillary zone electrophoresis with UV detection. Tested capillary coatings included polyvinyl alcohol, hydroxypropyl cellulose, polyacrylamide, polyethylene oxide, a cationic polyacrylamide derivative (APTAC, 5% and 30% of the cationic component), and five layer successive multiple ionic polymer layer coatings based on diethylaminoethyl-dextran and poly(diallyldimethylammonium chloride) both in combination with poly(sodium styrene sulfonate). Background electrolytes included formic acid (0.12M, 0.5M, 1.0M, pH = 1.87–2.35) and acetic acid (0.83 M, 1.69 M, 4.0 M, pH = 2.07–2.41). Based on performance and repeatibility, polyethylene oxide, successive multiple ionic polymer layer, and APTAC (30%) were selected for further use. For background electrolyte, we decided to proceed with 0.12 M formicacid, along with 0.83 M, and 1.69 M acetic acid. Currently, separations at varying pH values are being explored. This research lays the foundation for a robust capillary zone electrophoresis–mass spectrometry method for native protein analysis, with ongoing efforts to refine conditions and transfer the system to mass spectrometry detection.
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2025-12-15
16:33 |
Základy hmotnostní spektrometrie: MS skeny a vysoké rozlišení
Cvačka, Josef
Přednáška představuje různé typy skenů používané v hmotnostní spektrometrii a v technikách s iontovou mobilitou. U jednotlivých typů analyzátorů je stručně vysvětleno technické pozadí realizace těchto skenů. V závěrečné části se přednáška zaměřuje na problematiku hmotnostně-spektrometrického rozlišení.
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2025-04-20
01:11 |
Separation of cyclic diadenosine diphosphorothioate and the diastereomers of its difluorinated derivative and estimation of binding constants of their complexes with 2-hydroxypropyl-β-cyclodextrin by affinity capillary electrophoresis
Štěpánová, Sille ; Břehová, Petra ; Kašička, Václav
Cyclic dinucleotides (CDNs) are known to activate the stimulator of interferon genes (STING), a protein that is a member of the cyclic-guanosine-adenosine synthase-STING signaling pathway and that is important for the innate immune response in eukaryotic cells. CDNs contain two ribonucleoside monophosphates linked via 3′–5′ or 2′–5′ phosphodiester bonds. Many therapeutic oligonucleotides contain phosphorothioate modification. The aim of this work was to develop a new affinity capillary electrophoresis (ACE) method for the separation of a potential anticancer drug, 2′,3′-cyclic diadenosine diphosphorothioate (Rp, Rp) (ADU-S100), and three recently synthesized diastereomers of its difluorinated derivative, 3′,3′-cyclic di(2′-fluoro,2′-deoxyadenosine phosphorothioate) [1] (see Fig. 1) using native (α-, β-, and γ-cyclodextrins (CDs)) or modified CDs (2-hydroxypropyl-β-CD (HP-β-CD) and 2-hydroxypropyl-γ-CD (HP-γ-CD)) as chiral selectors. In addition, the background electrolyte (BGE) composition and the concentration of the suitable CD should be optimized. Moreover, ACE should be applied for the estimation of the average apparent binding constants of the complexes of the analyzed CDNs with the chiral selector providing best separation.
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2025-04-20
01:11 |
Analysis and physico-chemical characterization of peptides and lipopeptides regulating food intake by capillary electrophoresis and isotachophoresis
Šolínová, Veronika ; Křížková, Adéla ; Maletínská, Lenka ; Kašička, Václav
New lipidized analogs of peptides (ghrelin, prolactin-releasing peptide (PrRP) and cocaine- and amphetamine-regulated transkript peptide (CARTP)) are studied for regulation of food intake. Ghrelin (28 amino acids) is native peptide hormone, which is secreted in the stomach and acts both peripherally and centrally [1]. PrRP (31 amino acids) was discovered as a ligand for an orphan G-protein coupled receptor GPR 10 and as a regulator of prolactin release [2]. The CARTP (42 amino acids) is a brain-born and brain-acting neuropeptide with anorexigenic properties regulating hormone leptin [3]. The inability of these peptides to cross blood-brain barrier to their target receptors may be overcome by their lipidization at biologically inactive sites of their molecules. The set of ten (lipo)peptides was synthesized to be tested as potential drugs. Prior their application, they have to be analyzed and characterized. For that reason, the aim of this work was to check their purity and to characterize their acid-base properties by capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP).
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2025-04-20
01:11 |
Separation of selected catecholamines and determination of binding constants of their complexes with HS-β-CD by capillary electrophoresis
Sázelová, Petra ; Jiráček, Jiří ; Kašička, Václav
Catecholamines play an important role in carbohydrate and fat metabolism, cardiovascular system regulation, unstriped musculature function, blood coagulation, and the regulation of the acute adaptive reactions of the body. Analyses of the most important neurotransmitters such as dopamine, adrenaline and noradrenaline are of great significance for diagnostics and treatment of various brain diseases. The main path of formation of catecholamines in the body is: tyrosine - dihydroxyphenylalanine (DOPA) - dopamine - noradrenaline - adrenaline. L-DOPA (levodopa) is a chiral drug used in the treatment of Parkinson's disease, which is related to the depletion of the dopamine in the brain. Only this enantiomer is converted to dopamine while D-DOPA may cause side effects. Hence, control of enantiomeric purity of L-DOPA is necessary. Capillary electrophoresis (CE) using chiral selectors, among them especially cyclodextrins, is powerful tool for separation of enantiomers [1].
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2025-04-20
01:11 |
Chiral separation of ketamine and its metabolites from rat plasma samples using partial filling CE-ESI/MS
Konášová, Renáta ; Koval, Dušan ; Tůma, P. ; Kašička, Václav
Ketamine is a chiral drug used for a nearly 60 years to induce and maintain anaesthesia. In addition, ketamine and especially its metabolites act as antidepressants. For that reason, the mechanisms of action of these species have been studied. Different effects of enantiomers of ketamine and its metabolites were observed. S-ketamine has about twice the analgesic potency than the clinically used racemic mixture [1] and is supposed to be responsible for high abuse liability of ketamine [2]. The metabolism of R-ketamine to 2R,6R-hydroxynorketamine is essential for its antidepressant effect [3]. Ketamine and its main metabolites were successfully separated by capillary zone electrophoresis (CZE) [4] but a complete separation of enantiomers of ketamine (Ket) and its main metabolites, norketamine (NK), dehydronorketamine (DHNK) and hydroxynorketamine (HNK), has not yet been achieved. The recently developed LC-MS method requires one achiral and two chiral columns for separation of six enantiomers [5]. Therefore, the aim of this work was to develop a new CZE method that could separate the enantiomers of all above species in a single run.
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2025-04-20
01:11 |
Determination of the effective charge and ionic mobilities of highly sulfated cyclodextrins by capillary isotachophoresis and zone electrophoresis
Kašička, Václav ; Šolínová, Veronika ; Sázelová, Petra ; Koval, Dušan ; Vigh, G.
Sulfated cyclodextrins (SCDs) belong to the most popular chiral selectors in capillary electrophoresis [1, 2]. SCDs are available as mixtures of randomly highly sulfated species (HSCDs) or as well defined single isomer compounds (SI-CDs). For the randomly HS-CDs, only an average degree of substitution (DS) is estimated but the isomeric heterogeneity and the charge distribution are not known. In the SI-CDs, the sulfatation positions and the DS are well specified. However, due to close vicinity of the sulfate groups in both types of SCDs, counterion condensation can occur in their molecules and their actual effective charge can be lower than number of sulfate groups. For that reason, the aim of this work was to estimate the degree of charge reduction in both types of SCDs using the procedure based on separation of SCDs and reference compounds by capillary isotachophoresis (CITP) and zone electrophoresis (CZE). \n
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2025-03-15
00:00 |
ELIXIR CZ Annual Conference 2024: Data management
Vondrášek, Jiří ; Strachotová, Anna ; Votrubová, Tereza
Data management is becoming an increasingly vital part of modern research, and this conference is here to help you navigate it with ease. Over three days, more than 20 experts will guide us through the world of data management, from its role in EU projects to its impact on everyday scientific work. You’ll discover the tools that simplify data management and explore best practices tailored to the needs of the scientific community. Plus, we’ll dive into how data management is being handled on a national level. Get ready to enhance your research with practical insights and solutions!
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2025-01-19
00:08 |
Physical stabilization of water-soluble PVA nanofibrous materials functionalized with biologically active substances
Lisnenko, M. ; Holeček, M. ; Kuželová Košťáková, E. ; Valtera, J. ; Rejman, Dominik ; Müllerová, J. ; Běhálek, L. ; Jenčová, V.
Tissue engineering aims to develop materials that enhance biological activity and promote tissue healing and regeneration. One promising approach is to functionalize nanofibrous materials with antimicrobial substances, such as lipophosphonoxin (LPPO), and use water-soluble polymers like polyvinyl alcohol (PVA) to incorporate bioactive molecules into fibers. However, water-soluble materials often face the issue of burst release, releasing over 90% of the active substances within the initial 24 hours. This research focuses on preparing functionalized nanofibrous materials based on PVA containing the experimental antimicrobial compound LPPO and subsequent physical stabilization of the materials using the Heat treatment method. The applied stabilization successfully reduced the incorporated substance's release rate by up to 50%. The resulting materials have the potential to provide functional cross-linked PVA nanofiber scaffolds for regenerative medicine applications in large and chronic skin injuries.
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2025-01-12
00:00 |
Hmotnostní spektrometrie ve strukturní analýze lipidů
Cvačka, Josef
Přednáška na konkrétních příkladech ukazuje využití hmotnostní spektrometrie pro určování struktury lipidů v různých typech biologických vzorků. Diskutovány jsou nové analytické postupy, které zahrnují chemickou derivatizaci a fragmentaci iontů v plynné fázi s využitím různých aktivačních technik.\nVýstup byl podpořen projektem Národní institut pro výzkum metabolických a kardiovaskulárních onemocnění (Program EXCELES, ID LX22NPO5104) – Financováno Evropskou unií – Next Generation EU.
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