Capillary electrophoresis coupled to mass spectrometry (CE/MS) is gaining its space among the most powerful tools in modern (bio)analytical laboratory. The most challenging instrumental aspect in CE/MS is striking the balance between the stability and reproducibility of the signal and sensitivity of the analysis. Several interface designs have been published in the past decade addressing the variety of instrumental aspects and ease of operation. Most of the interfaces can be categorized either into the sheath flow arrangement (considered to be a de facto standard), or sheathless interface, often expected to provide the ultimate sensitivity. In this work we have explored an "interface-free" approach, where the CE/MS analysis was performed in narrow bore (<20 μm ID) electrospray capillary. The separation capillary and electrospray tip formed one entity and the high voltage, applied at the injection end of the capillary served for both the separation and electrospray ionization. Thus the separation voltage was defined as the product of the electrospray current and resistivity of the separation electrolyte. Optimum conditions for the separation and electrospray ionization were achieved with voltage programming. The performance of this simplest possible CE/MS system was tested on peptide separations from the cytochrome c tryptic digest. The subnanoliter sample consumption and sensitivity in the attomole range predetermines such a system for analysis of limited samples.
- MeSH
- Cytochromes c analysis MeSH
- Electrophoresis, Capillary methods MeSH
- Spectrometry, Mass, Electrospray Ionization methods MeSH
- Humans MeSH
- Peptide Fragments analysis MeSH
- Reproducibility of Results MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Keywords
- fotodynamická terapie,
- MeSH
- Apoptosis * MeSH
- Cell Death * MeSH
- Cytochromes c analysis MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Photochemotherapy * adverse effects MeSH
- Caspases analysis MeSH
- Rabbits MeSH
- Humans MeSH
- Melanoma * therapy MeSH
- Mice MeSH
- Check Tag
- Rabbits MeSH
- Humans MeSH
- Mice MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Combination of viscometry and CD spectrometry made it possible to suggest the existence of a new conformational state of cytochrome c at high temperatures and ionic strengths. The molten globule state of cytochrome c was found at acidic pH in the presence of high concentrations of a salt at 20 °C. A low-cooperative conformational change of the cytochrome c to the unfolded state was observed with increasing temperature. Viscometry is a suitable tool for studies of similar processes in biomacromolecules.
AIMS: In this study we have tested the effect of unsaturated fatty acids on the proapoptotic effects of saturated fatty acids in the human pancreatic β-cells NES2Y. RESULTS: We found that unsaturated palmitoleic and oleic acid at a concentration of 0.2 mM and higher are able to completely inhibit the proapoptotic effect of their counterpart saturated palmitic and stearic acid at a concentration of 1 mM. Apoptosis induced by stearic acid was associated with significant activation of caspase-6, -7, -9, -2 and -8, but not with significant activation of caspase-3. The activation of caspases was blocked by coincubation with oleic acid. Stearic acid treatment was not associated with a significant change in mitochondrial membrane potential, reactive oxygen species level and with cytochrome c release from mitochondria. Furthermore, stearic acid treatment was not associated with changes in p21(WAF1/CIP1), PIDD, Fas receptor and Fas ligand expression. However, we detected endoplasmic reticulum (ER) stress markers, i. e. a significant upregulation of BiP and CHOP expression as well as XBP1 mRNA splicing. These changes were inhibited by coincubation with oleic acid. CONCLUSION: Presented data indicate that oleic acid inhibits apoptosis induction by stearic acid in NES2Y cells upstream of caspase activation and ER stress induction. It does not involve an interference with the mitochondrial pathway of apoptosis induction, with p53 activation and PIDD expression as well as with Fas receptor and Fas ligand expression.
- MeSH
- Enzyme Activation MeSH
- Apoptosis drug effects MeSH
- Insulin-Secreting Cells cytology drug effects metabolism MeSH
- Cytochromes c analysis MeSH
- DNA-Binding Proteins genetics metabolism MeSH
- Endoplasmic Reticulum drug effects metabolism MeSH
- Gene Expression drug effects MeSH
- Stress, Physiological MeSH
- Caspases metabolism MeSH
- Oleic Acid pharmacology MeSH
- Fatty Acids, Monounsaturated pharmacology MeSH
- Stearic Acids pharmacology MeSH
- Humans MeSH
- Membrane Potential, Mitochondrial drug effects MeSH
- RNA, Messenger MeSH
- Mitochondria drug effects metabolism MeSH
- Heat-Shock Proteins genetics metabolism MeSH
- Flow Cytometry MeSH
- RNA Splicing MeSH
- Cell Line, Transformed MeSH
- Transcription Factor CHOP genetics metabolism MeSH
- Transcription Factors genetics metabolism MeSH
- Up-Regulation drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
