Proteomics of barley proteins by combination of in-gel digestion and MALDI-TOF MS

It is shown that combination of gel electrophoresis, matrix assisted laser desorption/ionization time-of-flight mass spectrometry and database searching is a suitable procedure for rapid protein identification. This approach let us to identify some proteins from barley leaves and grains. Identification of barley proteins can be useful for characterization of varieties of malting barley which is important especially in food and brewing industry

Vytváření aplikace v prostředí Microsoft Office

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Ekonomická fakulta. Katedra (155) informatiky v ekonomic

Ekonomicko-ekologické zhodnocení změny topných médií v podmínkách obce Háj ve Slezsku

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Ekonomická fakulta. Katedra (156) makroekonomik

Extrakce miRNA z buněčného lyzátu bez fenol/chloroformu s využitím nosiče na bázi TiO2

While microRNAs are considered as excellent biomarkers of various diseases, there are still several remaining challenges regarding their isolation. In this study, we aimed to design a novel RNA isolation method that would help to overcome those challenges. Therefore, we present a novel phenol/chloroform-free, low-cost method for miRNA extraction. Within this method, RNA is extracted from cell lysate with an isopropanol/water/NaCl system, followed by solid-phase extraction using TiO2 microspheres to effectively separate short RNAs from long RNA molecules. We also demonstrated the pH-dependent selectivity of TiO2 microspheres towards different sizes of RNA. We were able to regulate the size range of extracted RNAs with simple adjustments in binding conditions used during the solid-phase extraction.Ačkoliv jsou microRNA považovány za excelentní biomarkery pro řadu onemocnění, jejich izolace má stále řadu limitací. V této studii jsme se zaměřili na vývoj nové metody izolace RNA, která by některé z těchto limitů pomohla překonat. V této práce je uvedena nová, ekonomicky dostupná metoda izolace RNA, která nevyužívá systému fenol/chloroform. RNA je extrahována z buněčného lyzátu pomocí systému isopropanol/voda/NaCl, který je následován extrakcí na pevnou fázi s využitím TiO2 mikrosfér. Je zde také popsán vliv pH na selektivitu TiO2 mikrosfér k různým velikostem RNA molekul. Pomocí úpravy vazebných podmínek je možné regulovat, jaké rozmezí velikostí RNA molekul bude během extrakce na pevnou fázi ze vzorku izolováno

Frakcionace obohacených fosfopeptidů s využittím separace na mikrokoloně pomocí pH/acetonitril-reverzně fázového gradientu v kombinaci s LC-MS/MS analýzou

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.Hmotnostní spektrometrie (MS) je výkonná a citlivá metoda, která je často využívána k identifikaci fosfoproteinů. Ve fosfoproteomice však byla zjištěna potřeba kompenzovat jejich malou abundanci, nedostatečnou ionizaci a potlačující efekt nefosforylovaných peptidů. To může bránit následné analýze pomocí kapalinové chromatografie a hmotnostní spektrometrie/hmotnostní spektrometrie (LC-MS/MS), což vede k neúplné charakterizaci fosfoproteomu, i když se používají přístroje s vysokým rozlišením. Abychom překonali tyto nevýhody, je zde předkládána efektivní chromatografická technika s mikrogradientem, která poskytuje specifické frakce obohacených fosfopeptidů přímo kompatibilních s LC-MS/MS analýzou. Účelem naší studie bylo zvýšit počet identifikovaných fosfopeptidů, a tím i pokrytí fosfoproteomu v analyzovaném vzorku pomocí reprodukovatelné a přímé metody frakcionace. Tento protokol zahrnuje krok obohacení fosfopeptidů, po kterém následuje optimalizovaná mikrogradientní frakcionace obohacených fosfopeptidů a konečná LC-MS/MS analýza získaných frakcí. Jednoduchý frakcionační systém se skládá z plynotěsné mikrostříkačky sloužící k tlačení optimalizovaného gradientu mobilní fáze do mikrokolony s reverzní fází. Naše data ukazují, že kombinace obohacení fosfopeptidů s mikrogradientní separací je slibnou technikou pro hloubkovou fosfoproteomickou analýzu i díky nenáročným požadavkům na vstupní materiál a navíc poskytuje více než trojnásobně lepší identifikaci proteinů

New Interface for Purification of Proteins: 1D TiO2 Nanotubes Decorated by Fe3O4 nanoparticles

In this work, a high surface area interface, based on anodic 1D TiO2 nanotubes homogenously decorated by Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs) is reported for the first time for an unprecedented purification of His-tagged recombinant proteins. Excellent purification results were achieved from the model protein mixture, as well as from the whole cell lysate (with His-tagged ubiquitin). Compared to a conventional Immobilized-Metal Affinity Chromatography (IMAC) system, specific isolation of selected His-tagged proteins on behalf of other proteins was significantly enhanced on TiO2NTs@Fe3O4NP´s interface under optimized binding and elution conditions. The combination of specific isolation properties, magnetic features, biocompatibility, and ease of preparation of this material consisting of two basic metal oxides, makes it a suitable candidate for future purification of recombinant proteins in biotechnology. The principally new material bears a large potential to open new pathways for discoveries in nanobiotechnology and nanomedicine

TCR triggering induces the formation of Lck-RACK1-actinin-1 multiprotein network affecting Lck redistribution

The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases (SFKs), Lck. Upon TCR triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, Receptor for Activated C Kinase 1 (RACK1), was chosen as a viable option and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1-Lck complexes were detectable in primary CD4+ T-cells with their maximum levels peaking 10 seconds after TCR-CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394Lck which co-purifies with high molecular weight cellular fractions. The formation of RACK1-Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1-pY394Lck-α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with nocodazole, which disrupts the microtubular network, also blocked the formation of RACK1-Lck complexes. Importantly, activation-induced Lck redistribution was diminished in primary CD4+ T-cells by an adenoviral-mediated knock-down of RACK1. These results demonstrate that in T cells, RACK1 as an essential component of the multiprotein complex which upon TCR engagement links the binding of kinase active Lck to elements of the cytoskeletal network and affects the subcellular redistribution of Lck

Influence of metabolic state and body composition on the action of pharmacological treatment of migraine

Migraine is a disabling neurovascular disorder among people of all ages, with the highest prevalence in the fertile years, and in women. Migraine impacts the quality of life of affected individuals tremendously and, in addition, it is associated with highly prevalent metabolic diseases, such as obesity, diabetes mellitus and thyroid dysfunction. Also, the clinical response to drugs might be affected in patients with metabolic disease due to body composition and metabolic change. Therefore, the efficacy of antimigraine drugs could be altered in patients with both migraine and metabolic disease. However, knowledge of the pharmacology and the related clinical effects of antimigraine drugs in patients with metabolic disease are limited. Therefore, and given the clinical relevance, this article provides a comprehensive overview of the current research and hypotheses related to the influence of metabolic state and body composition on the action of antimigraine drugs. In addition, the influence of antimigraine drugs on metabolic functioning and, vice versa, the influence of metabolic diseases and its hormonal modulating medication on migraine activity is outlined. Future exploration on personalizing migraine treatment to individual characteristics is necessary to enhance therapeutic strategies, especially given its increasing significance in recent decades.</p