Új kapilláris izoelektromos fókuszálás tömegspektrometriával

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Új homogén folyadékkromatográfiás és elektrokromatográfiás töltetek kifejlesztése = Development of New Homogeneous Media for Liquid Chromatography and Electrochromatography

A szelektív kémiai rendszerek egyik nagy kihívása, hogy makromolekulákat és akár sejteket analitikai és preparatív módon felismerjen és elválasszon a befoglaló mátrixból. Az ún. imprinting technika csak kismolekulákra adott eddig lehetőséget. Munkánk során a szelektív gélek kifejlesztésével, tesztelésével, és kromatográfiás, valamint elektroforetikus elválasztások alapját biztosító új, homogén (monolit) töltetek kialakításával foglalkoztunk. A kifejlesztett gélek makromolekulák, különböző fehérjék és ennél nagyon biológiai rendszerek, pl. vírusok vagy baktériumok szelektív felismerésére, és megkötésére is alkalmasak. Az újonnan kifejlesztett töltetek alkalmasak kapilláris HPLC és akár miniatürizált elektrokromatográfiás készülékekben is szelektív elválasztásokra. A projekt során kimutattuk, hogy a szelektív gélek nagy érzékenységgel képesek felismerni ugyanazon fehérje különböző konformációs állapotait (vasat tartalmazó és nem tartalmazó transzferrinek), különböző fajtáktól származó, szerkezetileg hasonló fehérjéket (hemoglobinok). A munka eredményeként bebizonyítottuk, hogy a homogén gélek alkalmasak vírusok szelektív megkötésére olyan mértékben, hogy két virus (Semliki Forest Virus vad és mutáns formája) közötti, mindösszesen három aminosav-maradék különbséget is képesek felismerni. S végül bebizonyítottuk, hogy a szelektív gélek sejteket (különböző Gram-negatív baktériumokat) is képesek szelektíven megkötni. | The selective chemical systems have the task to recognize and/or separate macromolecules or even cells from the matrix in analytical or preparative way. The imprinting technique, however, has not provided this opportunity, yet, but the selective recognition of small molecules only. This project included the testing, development and application of selective gels (artficial antibodies) for chromatographic and electrphoretic separations in the form of new, homogen (monolitic) beds. The selective gels are capable of recognizing and binding macromolecules, or larger macro-assemblies, such as viruses of bacteria. The new homogen gels can be used in capillary chromatography or in miniaturized electrochromatographic systems. The results show that with the use of these gels one can differentiate the different conformations of the same protein (e.g. iron-free or iron-contaning transferrin) and proteins with very similar structures (like hemoglobins from different species). We approved that the homogen gels are able to recognize viruses with very small differences (e.g. Semliki Forest Virus wild and mutant forms) differing only by three amino acid residues from each other. Finally we showed that the selective gel-technique is able to recognize cells with high specificity (e.g. differentiation of several Gram-negative bacteria)

Bioanalitikai kérdések: Bioanalytical solutions

Fatty acids everywhere, ionic liquids and proteins, endotoxin structure and function. Kivonat Zsírsavak mindenhol, ionfolyadékok és fehérjék, endotoxin szerkezet és funkció. &nbsp

Sequential injection setup for capillary isoelectric focusing combined with MS detection

Abstract Capillary isoelectric focusing in the presence of electroosmosis with sequential injection of carrier ampholytes and sample was found to be suitable for MS detection. The separate injection of the sample and the ampholytes provides good condition to suppress and overcome the undesirable effect of the presence of ampholytes in MS. By the appropriate selection of ampholyte solutions, whose pH range not necessarily covers the pI values of the analytes, the migration of the components can be controlled, and the impact of the ampholytes on MS detection is decreased. The unique applicability of this setup is shown by testing several parameters, such as the application of volatile electrolyte solutions, the type of the ampholytes, the order and the number of the ampholyte and sample zones. Broad and narrow pH range ampholytes were applied in experiments using uncoated capillaries with different lengths for the analyses of substituted nitrophenol dyes to achieve optimal conditions for the MS detection. Although the sample components are not leaving the pH gradient, due to the decrease in the ampholyte concentration at the position of the components, and because the sample components migrate in charged state, the ionisation is more effective for MS detection

Microfluidic Analysis for the Determination of Protein Content in Different Types of Plant-Based Drinks

The widespread consumption of plant-based drinks, driven by health and dietary reasons (including cow’s milk allergy, lactose intolerance, milk protein intolerance, following a vegetarian or vegan diet) necessitates automated and accurate test methods. Our study demonstrates the simultaneous determination of protein components and total protein concentrations in plant-based milk alternatives using a rapid and reproducible microchip gel electrophoretic method. As expected, the electrophoretic profiles of each plant-based drink differed. Based on our analyses and statistical evaluation, it can be determined that the protein profiles of different plant-based beverages do not differ significantly between different manufacturers or different expiry dates. The measured total protein content was compared with the nominal values, i.e., the values stated on the beverage labels. As the number of consumers of functional and specialized plant-based milk alternatives continues to rise, it is important to prioritize methods that provide qualitative and quantitative information on protein composition and other nutrients

Diagnostic relevance of urinary steroid profiles on ovarian granulosa cell tumors: two case reports

Abstract Background Granulosa cell tumor of the ovary is the most frequent sex cord stromal tumor and represents 2 to 5% of all primary ovarian cancers. Ovarian granulosa cell tumor is a malignant tumor with slow progression and in some cases this tumor is hormonally active. The recurrence of granulosa cell tumor often happens after 5 years. Case presentation We describe two cases of postmenopausal women with adult-type granulosa cell tumors of the ovary. Patient 1 is a 49-year-old European woman with a recurrent tumor; patient 2 is a 55-year-old European woman without recurrence of tumor. Urinary steroid profiles of patient 1 were monitored during a 5-year period starting from before an operation (13 samples). In patient 2, the urinary steroid profiles were monitored during a 3-year period starting from after an operation (six samples). The 24-hour urinary samples were examined and the urinary concentration of 20 androgen, progesterone, and corticoid metabolites was quantitatively determined by gas chromatography-mass spectrometry with selected ion-monitoring mode. Conclusions Based on these cases a correlation could be observed between increased levels of the urinary steroids and the recurrence of ovarian granulosa cell tumor; therefore, we concluded that a urinary steroid profile could be a more effective method to follow-up such patients compared to the traditional serum hormones determinations supplemented with conventional tumor markers