Fast Photochemical Oxidation of Proteins (FPOP) is a promising technique for studying protein structure and dynamics. The quality of insight provided by FPOP depends on the reliability of the determination of the modification site. This study investigates the performance of two search engines, Mascot and PEAKS, for the data processing of FPOP analyses. Comparison of Mascot and PEAKS of the hemoglobin--haptoglobin Bruker timsTOF data set (PXD021621) revealed greater consistency in the Mascot identification of modified peptides, with around 26% of the IDs being mutual for all three replicates, compared to approximately 22% for PEAKS. The intersection between Mascot and PEAKS results revealed a limited number (31%) of shared modified peptides. Principal Component Analysis (PCA) using the peptide-spectrum match (PSM) score, site probability, and peptide intensity was applied to evaluate the results, and the analyses revealed distinct clusters of modified peptides. Mascot showed the ability to assess confident site determination, even with lower PSM scores. However, high PSM scores from PEAKS did not guarantee a reliable determination of the modification site. Fragmentation coverage of the modification position played a crucial role in Mascot assignments, while the AScore localizations from PEAKS often become ambiguous because the software employs MS/MS merging.
Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.
Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.
- MeSH
- glykopeptidasa MeSH
- glykoproteiny * analýza MeSH
- glykosylace MeSH
- polysacharidy * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Botulismus je v České republice raritní život ohrožující onemocnění. Od roku 1960 bylo hlášeno celkem 155 případů, přičemž od roku 2013, s výjimkou výskytu familiárního botulismu v roce 2013, se jednalo dle údajů ISIN (dříve EPIDAT) pouze o 3 izolované případy. V naší práci uvádíme výskyt botulismu po požití paštiky nedohledatelného původu u manželského páru, jenž byl hospitalizován v červenci 2022 pro otravu botulotoxinem. Z neurologických příznaků dominovala dysartrie, po aplikaci antibotulinního séra došlo k výraznému zlepšení klinického obrazu. Vzorky pacientů byly analyzovány pomocí afinitních nosičů a MALDI hmotnostní spektrometrie. Jedná se o moderní vysoce citlivou techniku k zjištění přítomnosti botulinních neurotoxinů. Na rozdíl od klasického průkazu obtížným a finančně nákladným biologickým pokusem na myších nedochází při výše uvedené analýze k usmrcování laboratorních zvířat.
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Intermediate filaments (IFs) are essential constituents of the metazoan cytoskeleton. A vast family of cytoplasmic IF proteins are capable of self-assembly from soluble tetrameric species into typical 10-12 nm wide filaments. The primary structure of these proteins includes the signature central 'rod' domain of ~ 300 residues which forms a dimeric α-helical coiled coil composed of three segments (coil1A, coil1B and coil2) interconnected by non-helical, flexible linkers (L1 and L12). The rod is flanked by flexible terminal head and tail domains. At present, the molecular architecture of mature IFs is only poorly known, limiting our capacity to rationalize the effect of numerous disease-related mutations found in IF proteins. Here we addressed the molecular structure of soluble vimentin tetramers which are formed by two antiparallel, staggered dimers with coil1B domains aligned (A11 tetramers). By examining a series of progressive truncations, we show that the presence of the coil1A domain is essential for the tetramer formation. In addition, we employed a novel chemical cross-linking pipeline including isotope labelling to identify intra- and interdimeric cross-links within the tetramer. We conclude that the tetramer is synergistically stabilized by the interactions of the aligned coil1B domains, the interactions between coil1A and the N-terminal portion of coil2, and the electrostatic attraction between the oppositely charged head and rod domains. Our cross-linking data indicate that, starting with a straight A11 tetramer, flexibility of linkers L1 and L12 enables 'backfolding' of both the coil1A and coil2 domains onto the tetrameric core formed by the coil1B domains. Through additional small-angle X-ray scattering experiments we show that the elongated A11 tetramers dominate in low ionic strength solutions, while there is also a significant structural flexibility especially in the terminal domains.
Transcription factors exert their regulatory potential on RNA polymerase II machinery through a multiprotein complex called Mediator complex or Mediator. The Mediator complex integrates regulatory signals from cell regulatory cascades with the regulation by transcription factors. The Mediator complex consists of 25 subunits in Saccharomyces cerevisiae and 30 or more subunits in multicellular eukaryotes. Mediator subunit 28 (MED28), along with MED30, MED23, MED25 and MED26, belong to presumably evolutionarily new subunits that seem to be absent in unicellular eukaryotes and are likely to have evolved together with multicellularity and cell differentiation. Previously, we have shown that an originally uncharacterized predicted gene, F28F8.5, is the true MED28 orthologue in Caenorhabditis elegans (mdt-28) and showed that it is involved in a spectrum of developmental processes. Here, we studied the proteomic interactome of MDT-28 edited as GFP::MDT-28 using Crispr/Cas9 technology or MDT-28::GFP expressed from extrachromosomal arrays in transgenic C. elegans exploiting the GFPTRAP system and mass spectrometry. The results show that MDT-28 associates with the Head module subunits MDT-6, MDT-8, MDT-11, MDT-17, MDT- 20, MDT-22, and MDT-30 and the Middle module subunit MDT-14. The analyses also identified additional proteins as preferential MDT-28 interactants, including chromatin-organizing proteins, structural proteins and enzymes. The results provide evidence for MDT-28 engagement in the Mediator Head module and support the possibility of physical (direct or indirect) interaction of MDT-28 with additional proteins, reflecting the transcription-regulating potential of primarily structural and enzymatic proteins at the level of the Mediator complex.
- MeSH
- alely MeSH
- Caenorhabditis elegans metabolismus MeSH
- jaderné proteiny metabolismus MeSH
- mediátorový komplex metabolismus MeSH
- podjednotky proteinů metabolismus MeSH
- proteiny Caenorhabditis elegans metabolismus MeSH
- proteomika * MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
As a part of the innate immunity, NK (Natural Killer) cells provide an early immune response to different stimuli, e.g. viral infections and tumor growths. However, their functions are more complex; they play an important role in reproduction, alloimmunity, autoimmunity and allergic diseases. NK cell activities require an intricate system of regulation that is ensured by many different receptors on a cell surface which integrate signals from interacting cells and soluble factors. One way to understand NK cell biology is through the structure of NK receptors, which can reveal ligand binding conditions. We present a modified protocol for recombinant expression in Escherichia coli and in vitro refolding of the ligand-binding domain of the inhibitory Nkrp1b (SJL/J) protein. Nkrp1b identity and folding was confirmed using mass spectrometry (accurate mass of the intact protein and evaluation of disulfide bonds) and one-dimensional nuclear magnetic resonance spectroscopy. The intention is to provide the basis for conducting structural studies of the inhibitory Nkrp1b protein, since only the activating Nkrp1a receptor structure is known.
- MeSH
- buněčná inkluze MeSH
- disulfidy chemie MeSH
- Escherichia coli metabolismus MeSH
- hmotnostní spektrometrie MeSH
- lektinové receptory NK-buněk - podrodina B biosyntéza chemie genetika MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- refolding proteinů MeSH
- rekombinantní proteiny biosyntéza MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rychlý rozvoj badatelského výzkumu v moderní glykobiochemii a glykobiologii vedl k širokému zpřístupnění nových experimentálních postupů přejímaných vzápětí biotechnologickým průmyslem pro produkci glykoproteinových terapeutik a diagnostik. Pokrok posledního období je založen na důkladných a systematických znalostech detailů enzymové mašinérie vedoucí ke specifické glykosylaci proteinů, která zůstává jednou z nejčastějších a nejdramatičtějších ko- a posttranslačních modifikací proteinů. Klonování genů kódujících jednotlivé glykosylační enzymy umožnilo jejich použití v buněčných technologiích a buněčném inženýrství vedoucím k široké dostupnosti linií produkujících spolehlivě humánní typ glykosylace. Pokrok v těchto technikách probíhal paralelně s rozvojem metod umožňujících provést komplexní analýzu proteinové glykosylace, a byl nyní rozsáhle využit biotechnologickými firmami k produkci více než dvou stovek schválených terapeutických proteinů, z nichž má více než polovina charakter glykoproteinů.
Rapid progress in contemporary glycobiology led to widespread availability of new technologies employed by biotechnology industry for production of glycoprotein therapeutics and diagnostics. This progress has been based on our detailed knowledge of enzyme machinery enabling protein glycosylation, one of the most often and most extensive posttranslational protein modification. Cloning of individual genes coding for glycosylation enzymes enabled their use in cellular technologies and cellular engineering resulting in wide availability of cell lines able to produce human glycosylation efficiently and precisely. Progress in this direction went in parallel with that in the techniques suitable for analytical evaluation of glycosylation, and has been now widely adopted at biotechnological companies producing more then two hundred therapeutical proteins, more then half of them being glycosylated.
