The properties of staphylococcal phages from the Siphoviridae, Podoviridae, and Myoviridae families were monitored using capillary electrophoretic methods on fused-silica capillaries with different morphology of surface roughness. Isoelectric points of the examined phages were determined by capillary isoelectric focusing in the original, smooth fused-silica capillary, and they ranged from 3.30 to 3.85. For capillary electrophoresis of phages, fused-silica capillaries with the "pock" and "cone" roughened surface types were prepared by etching a part of the capillary with supercritical water. The best resolution of the individual phages (to range from 3.2 to 4.6) was achieved with the "cone" surface-type fused-silica capillary. Direct application of phage K1/420 at the infection site, represented by human plasma or full blood spiked with Staphylococcus aureus, was on-line monitored by micellar electrokinetic chromatography. The phage particles were dynamically adhered onto the roughened surface of the capillary from 10 μL of the prepared sample at the optimized flow rate of 6.5 μL min-1. The limit of detection was determined to be 104 phage particles. The linearity of the calibration lines was characterized by the regression coefficient, R2 = 0.998. The relative standard deviation (RSD) of the peak area, calculated from ten independent measurements, was (±) 2%. After analysis, viability of the detected phages was verified by the modified "double-layer drop assay" method, and collected phage fractions were simultaneously off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Graphical abstract.

• MeSH
• bakteriofágy patogenita   MeSH
• lidé MeSH
• odběr vzorku krve přístrojové vybavení   MeSH
• oxid křemičitý chemie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Amphotericin B (AmB) is still, despite its severe nephrotoxicity, the first-line agent in the management of serious systemic fungal infections. A sensitive and reliable method is therefore required to control AmB concentration in body fluids of a patient. This study demonstrates the potential of the off-line combination of preparative isoelectric focusing (IEF) with capillary isoelectric focusing (CIEF) or capillary zone electrophoresis (CZE) in the determination of AmB in human blood serum. The required value of the isoelectric point of AmB was determined to be 6.1 using the CIEF technique. Preparative IEF served as a pre-separation and concentration technique. The pH gradient was traced by colored low molecular pI markers. The collected fraction with AmB was easily processed and then analyzed by CIEF and CZE. Tens of picograms of AmB in human blood serum sample can be determined by a combination of preparative IEF with CZE. The method was linear in the AmB concentration range of 0.3-600ngmL-1. The recovery ranged from 93% to 98%. Copyright © 2018 Elsevier B.V. All rights reserved.

• MeSH
• amfotericin B * izolace a purifikace   krev   MeSH
• biochemická analýza krve * metody   MeSH
• elektroforéza kapilární * metody   MeSH
• isoelektrická fokusace MeSH
• lidé MeSH
• limita detekce * MeSH
• Check Tag
• lidé MeSH

This review focuses on the latest development of microseparation electromigration methods in capillaries and microfluidic devices coupled with MS for detection and identification of important analytes. It is a continuation of the review article on the same topic by Kleparnik (Electrophoresis 2015, 36, 159-178). A wide selection of 161 relevant articles covers the literature published from June 2014 till May 2016. New improvements in the instrumentation and methodology of MS interfaced with capillary or microfluidic versions of zone electrophoresis, isotachophoresis, and isoelectric focusing are described in detail. The most frequently implemented MS ionization methods include electrospray ionization, matrix-assisted desorption/ionization and inductively coupled plasma ionization. Although the main attention is paid to the development of instrumentation and methodology, representative examples illustrate also applications in the proteomics, glycomics, metabolomics, biomarker research, forensics, pharmacology, food analysis, and single-cell analysis. The combinations of MS with capillary versions of electrochromatography, and micellar electrokinetic chromatography are not included.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• analýza potravin metody   MeSH
• biologické markery analýza   MeSH
• buněčné linie MeSH
• chromatografie metody   MeSH
• elektroforéza kapilární přístrojové vybavení   metody   MeSH
• glykomika MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací přístrojové vybavení   metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• isoelektrická fokusace přístrojové vybavení   metody   MeSH
• laboratoř na čipu * MeSH
• lidé MeSH
• metabolomika metody   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice přístrojové vybavení   metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND AND AIMS: Proteolytic enzymes contribute to the progression of various cancers. We previously reported increased expression of the proline specific peptidases dipeptidyl peptidase-IV (DPP-IV) and its closest paralogue fibroblast activation protein (FAP) in human glioblastomas. Here we analyze the molecular heterogeneity of DPP-IV and FAP in glioblastomas. METHODS: ELISA, isoelectric focusing, 1D and 2D electrophoresis followed by WB or enzyme overlay assay were utilized to analyze DPP-IV and FAP isoforms. Cell fractionation using a Percoll gradient and deglycosylation with PNGase F were performed to analyze the possible basis of DPP-IV and FAP microheterogeneity. RESULTS: Molecular forms of DPP-IV with an estimated molecular weight of 140-160 kDa and a pI predominantly 5.8 were detected in human glioblastoma; in some tumors additional isoforms with a more acidic (3.5-5.5) as well as alkaline (8.1) pI were revealed. Using 2D electrophoresis, two to three molecular forms of FAP with an alkaline (7.0-8.5) pI and an estimated MW of 120-140 kDa were identified in glioblastoma tissues. In glioma cell lines in vitro, several isoforms of both enzymes were expressed, however the alkalic forms present in glioblastoma tissues were not detected. Removal of N-linked oligosaccharides decreased the estimated molecular weight of both enzymes; the overall pattern of molecular forms nevertheless remained unchanged. CONCLUSION: Several isoforms of DPP-IV and FAP are present in glioblastoma tissue. The absence of alkaline isoforms of both enzymes in glioma cell lines however suggests that isoforms from other, most likely stromal, cell types contribute to the overall pattern seen in glioblastoma tissues.

• MeSH
• dipeptidylpeptidasa 4 fyziologie   MeSH
• elektroforéza MeSH
• ELISA MeSH
• frakcionace buněk MeSH
• genetická heterogenita MeSH
• glioblastom enzymologie   MeSH
• imunohistochemie MeSH
• isoelektrická fokusace MeSH
• lidé MeSH
• membránové proteiny fyziologie   MeSH
• mozek enzymologie   patologie   MeSH
• nádorové buňky kultivované MeSH
• serinové endopeptidasy fyziologie   MeSH
• stabilita enzymů MeSH
• želatinasy fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

A capillary electroseparation technique for focusing and selective pre-concentration of metal chelates with subsequent on-line isotachophoresis (ITP) analysis was developed and verified. The ions of alkali earth metals (Mg, Ca, Sr, and Ba) were pre-concentrated from the mixture and analyzed. The focusing of the metals was carried out in a ligand step gradient, which was created by the addition of a convenient ligand agent to the regular stationary pH step gradient. The analytical procedure consisted of three steps. During the first step, the metal ions were electrokinetically continuously dosed into the column where they were selectively trapped on the stationary ligand step gradient in the form of unmoving zones of chelate complexes with effectively zero charge. After a detectable amount of analyte was accumulated, the dosing was stopped. The accumulated zones were mobilized to the analytical column, where they were analyzed by the ITP method with conductivity or photometric detection. The proper electrolyte systems for dosing, mobilizing, and analyzing in isoelectric focusing (IEF), moving boundary electrophoresis (MBE), and ITP modes were consequently developed and put into practice. The trapping selectivity can be regulated by the choice of pH and convenient complexing agents. A mixture of alkali earth metals were used as model analytes. Using a 3000 s dosing time, the proposed method improved the detection limit by 5-29 times in comparison to analysis by ITP with classical injection.

• MeSH
• chelátory chemie   MeSH
• elektrolyty chemie   MeSH
• izotachoforéza metody   MeSH
• koncentrace vodíkových iontů MeSH
• kovy alkalických zemin analýza   chemie   MeSH
• senzitivita a specificita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Intensive research and development of electrophoresis methodology and instrumentation during past decades has resulted in unique methods widely implemented in bioanalysis. While two-dimensional electrophoresis and denaturing polyacrylamide gel electrophoresis in sodium dodecylsulfate are still the most frequently used electrophoretic methods applied to analyses of proteins, new miniaturized capillary and microfluidic versions of electromigrational methods have been developed. High-throughput electrophoretic instruments with hundreds of capillaries for parallel separations and laser-induced fluorescence detection of labeled DNA strands have been of key importance for the scientific and commercial success of the Human Genome Project. Another powerful method, capillary isoelectric focusing with pressurized and pH-driven mobilization, provides efficient separations and on-line sensitive detection of proteins, bacteria and viruses. Electrophoretic microfluidic devices can integrate single-cell injection, cell lysis, separation of its components and fluorescence or mass spectrometry detection. These miniaturized devices also proved the capability of single-molecule detection.

• MeSH
• bakteriální proteiny analýza   MeSH
• elektroforéza přístrojové vybavení   metody   trendy   MeSH
• imunoanalýza metody   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• proteom analýza   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA metody   MeSH
• stereoizomerie MeSH
• virové proteiny analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18) (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20) detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr(18) was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser(20) and Thr(18) kinase signalling cascades to modulate p53 activity.

• MeSH
• buněčné linie MeSH
• epitopy imunologie   metabolismus   MeSH
• fosfatasy metabolismus   MeSH
• fosforylace MeSH
• fosfoserin imunologie   metabolismus   MeSH
• fosfothreonin imunologie   metabolismus   MeSH
• izoelektrický bod MeSH
• jaderné proteiny * MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• nádorový supresorový protein p53 genetika   imunologie   metabolismus   MeSH
• nádory prsu imunologie   metabolismus   MeSH
• peptidové fragmenty chemická syntéza   imunologie   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• protein - isoformy genetika   imunologie   metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny * metabolismus   MeSH
• rekombinantní proteiny imunologie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• specificita protilátek MeSH
• Spodoptera MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

• Klíčová slova
• elektroforéza kapilární,

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• fyzika, biofyzika
• biomedicínské inženýrství

• Klíčová slova
• Chemie, Imunita,
• MeSH
• imunochemie MeSH
• imunologické techniky MeSH
• Publikační typ
• laboratorní příručky MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• alergologie a imunologie

• MeSH
• biologie buňky MeSH
• buňky MeSH
• molekulární biologie MeSH
• Publikační typ
• příručky MeSH

• Konspekt
• Buněčná biologie. Cytologie
• NLK Obory
• cytologie, klinická cytologie