Úvod: Nemoc těžkých řetězců gama je vzácné onemocnění dosud popsané přibližně u 150 případů. Cílem práce byla laboratorní diagnostika nemoci těžkých řetězců imunoglobulinu. Materiál a metody: Do FN Ostrava byl referován 60letý pacient pro suspektní lymfom marginální zóny z biopsie žaludku. U pacienta byla doplněna stagingová vyšetření vč. trepanobiopsie kostní dřeně a PET/CT. Ze speciálních vyšetření byla požadována elektroforéza sérových proteinů, imunofixační elektroforéza, stanovení polyklonálních imunoglobulinů, volných lehkých řetězců a párů těžkých/lehkých řetězců imunoglobulinů. Z důvodu nejasného nálezu byla doplněna izoelektrická fokusace v agarózovém gelu s následným afinitním imunoblottingem a SDS elektroforéza. Výsledky: V kostní dřeni bylo nalezeno 0,1 % plazmatických buněk, z toho 87 % klonálních (patologických) plazmocytů, s následujícím imunofenotypem: cyt LAMBDA+ CD38+ CD138+ CD45+ CD19+ CD56– CD27+ CD81– CD117–. V séru pacienta byly nalezeny monoklonální těžké řetězce gama. V moči monoklonální těžké ani lehké řetězce imunoglobulinu detekovány nebyly. PET/CT vyšetření prokázalo generalizovanou lymfadenopatii, splenomegalii a nehomogenní akumulaci fluorodeoxyglukózy v axilárním i apendikulárním skeletu, nicméně bez přítomnosti typických osteolytických ložisek. Závěr: Monoklonální těžké řetězce imunoglobulinů jsou vzácným onemocněním. Pro jejich potvrzení je nutné použít na rozdíl od průkazu kompletní molekuly paraproteinu další pomocné metody. Nález monoklonálního těžkého řetězce gama v séru studovaného pacienta souvisí s přítomností lymfomu marginální zóny, který byl prokázán z biopsie žaludku.
Background: Gamma-heavy chain disease is a rare disease, described so far in approximately 150 cases. The aim of this work was laboratory diagnostics of immunoglobulin heavy chain disease. Materials and methods: A 60-year-old patient was referred to the University Hospital in Ostrava for suspected marginal zone lymphoma from gastric biopsy. Staging examinations including bone marrow trepanobiopsy and PET/CT were added; special examinations required serum protein electrophoresis, immunofixation electrophoresis, determination of polyclonal immunoglobulins, free light chains, and immunoglobulin heavy/light chain pairs. Isoelectric focusing in agarose gel followed by affinity immunoblotting and SDS electrophoresis was added due to unclear findings. Results: 0.1 % of plasma cells were found in the bone marrow, of which 87 % were clonal (pathological) plasma cells, followed by the cyt cytotype LAMBDA + CD38 + CD138 + CD45 + CD19 + CD56- CD27 + CD81- CD117-. Monoclonal heavy chains were found in the patient‘s serum. No monoclonal immunoglobulin heavy or light chains were detected in urine. The PET/CT examination showed generalized lymphadenopathy, splenomegaly and inhomogeneous accumulation of fluorodeoxyglucose in axillary and appendicular skeleton, but without the presence of typical osteolytic lesions. Conclusion: Monoclonal heavy chains of immunoglobulins are a rare disease. In contrast to the detection of a complete paraprotein molecule, additional methods must be used to confirm them. The finding of monoclonal heavy chain gamma in the serum of the study patient is related to the presence of marginal zone lymphoma, which was proven from a gastric biopsy.
- MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- elektroforéza metody MeSH
- isoelektrická fokusace metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- nemoc z těžkých řetězců * diagnóza patologie MeSH
- výsledek terapie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
Reliable determination of protein complex composition or changes to protein levels in whole cells is challenging. Despite the multitude of methods now available for labeling, analysis, and the statistical processing of data, this large variety is of itself an issue: Which approach is most appropriate, where do you set cutoffs, and what is the most cost-effective strategy? One size does not fit all for such work, but some guidelines can help in terms of reducing cost, improving data quality, and ultimately advancing investigations. Here we describe two protocols and algorithms for facile sample preparation for mass spectrometric analysis, robust data processing, and considerations of how to interpret large proteomic datasets in a productive and robust manner.
- MeSH
- datové soubory jako téma MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- hmotnostní spektrometrie metody MeSH
- multiproteinové komplexy izolace a purifikace metabolismus MeSH
- proteomika metody MeSH
- protozoální proteiny izolace a purifikace metabolismus MeSH
- Trypanosoma brucei brucei metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We introduce an optimized periodic acid-Schiff´s staining of glycoproteins on 1D electrophoretic gels. Thanks to heating in a household microwave oven the protocol of standard periodic acid-Schiff´s staining has been accelerated from 6 h to below 10 min employing standard chemistry. At the same time, we show that the microwave-assisted glycoprotein staining is at least as sensitive as the conventional approach. All glycoproteins stained by the microwave-accelerated procedure were successfully identified using MALDI TOF/TOF mass spectrometry. The ensuing reduction in gel staining time and simplification of the staining protocol should significantly increase laboratory throughput when glycoprotein detection on electrophoretic gels is required in large numbers.
- MeSH
- barvení a značení metody MeSH
- barvicí látky chemie MeSH
- časové faktory MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- glykoproteiny analýza chemie MeSH
- kyselina jodistá chemie MeSH
- mikrovlny * MeSH
- reprodukovatelnost výsledků MeSH
- rosanilinová barviva chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The methicillin-resistant Staphylococcus aureus causes difficult-to-treat healthcare-associated infections in humans. For fast and effective selection of an appropriate antibiotic therapy, it is essential to have rapid and reliable methods for differentiation of methicillin-resistant S. aureus from less dangerous methicillin-sensitive S. aureus. There have been many methods for the identification of methicillin-resistant S. aureus described but none has been accepted as an international standard. The most commonly used techniques such as phenotyping and genotyping have a few disadvantages, for instance, these techniques are not reproducible and stable. In addition, they are time-consuming, expensive, and they are not capable to distinguish all S. aureus strains. In this study, the methicillin-resistant and methicillin-sensitive S. aureus isolates obtained from patients were extracted in hot water. The released proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing. These two methods were able to differentiate among tested bacterial strains. The proposed methods are time saving, they are applicable in standard biochemical laboratories, and they do not require any expensive equipment.
- MeSH
- bakteriologické techniky metody MeSH
- časové faktory MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- isoelektrická fokusace metody MeSH
- lidé MeSH
- rezistence na methicilin * MeSH
- Staphylococcus aureus chemie klasifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Metallothioneins (MTs) are involved in heavy metal detoxification in a wide range of living organisms. Currently, it is well known that MTs play substantial role in many pathophysiological processes, including carcinogenesis, and they can serve as diagnostic biomarkers. In order to increase the applicability of MT in cancer diagnostics, an easy-to-use and rapid method for its detection is required. Hence, the aim of this study was to develop a fully automated and high-throughput assay for the estimation of MT levels. Here, we report the optimal conditions for the isolation of MTs from rabbit liver and their characterization using MALDI-TOF MS. In addition, we described a two-step assay, which started with an isolation of the protein using functionalized paramagnetic particles and finished with their electrochemical analysis. The designed easy-to-use, cost-effective, error-free and fully automated procedure for the isolation of MT coupled with a simple analytical detection method can provide a prototype for the construction of a diagnostic instrument, which would be appropriate for the monitoring of carcinogenesis or MT-related chemoresistance of tumors.
- MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- gelová chromatografie metody MeSH
- játra chemie MeSH
- králíci MeSH
- krysa rodu rattus MeSH
- laboratorní automatizace MeSH
- magnetické nanočástice chemie MeSH
- metalothionein analýza izolace a purifikace MeSH
- potkani Wistar MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- elektroforéza kapilární metody přístrojové vybavení využití MeSH
- elektroforéza v polyakrylamidovém gelu metody přístrojové vybavení využití MeSH
- elektroforéza metody využití MeSH
- imunoglobulinové izotypy MeSH
- imunoglobuliny - kappa-řetězce MeSH
- kryoglobuliny MeSH
- lehké řetězce imunoglobulinů * analýza krev moč MeSH
- lidé MeSH
- těžké řetězce imunoglobulinů * analýza krev moč MeSH
- Waldenströmova makroglobulinemie * diagnóza klasifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Fast-staining protocols based on the use of Coomassie blue dye for SDS-PAGE separated proteins, represent a quick and simple solution for protein visualization. It has been shown however, that in some cases a phenomenon of missing spots or spot discoloration may be observed in the proteome pattern when the standard fast-staining protocol is used. In this work, it is demonstrated that this occurrence is affected by the biological variability of samples, and therefore, cannot be observed in all samples. Moreover, it is demonstrated that the phenomenon is manifested exclusively in nonfixed gels, and that including a fixation step into the fast-staining protocol prevented this phenomenon. In conclusion, it has been demonstrated that standard Coomassie blue dye based fast staining for SDS-PAGE resolved proteins is affected by the biological variability of samples in nonfixed gels.
Protein phosphorylation is the most abundant and best studied protein posttranslational modification, dedicated to the regulation of protein function and subcellular localization as well as to protein-protein interactions. Identification and quantitation of the dynamic, conditional protein phosphorylation can be achieved by either metabolic labeling of the protein of interest with (32)P-labeled ATP followed by autoradiographic analysis, the use of specific monoclonal or polyclonal antibodies against the phosphorylated protein species and finally by phosphoproteome delineation using mass spectrometry.Hereby we present a fourth alternative which relies on the enforced-affinity-based-electrophoretic separation of phosphorylated from non-phosphorylated protein species by standard SDS-PAGE systems co-polymerized with Phos-Tag™ and Mn(2+) or Zn(2+) cations. Phosphate groups of phosphorylated Ser, Thr, and Tyr residues form complexes with Mn(2+) and Zn(2+) cations with polyacrylamide immobilized Phos-Tag™. Following appropriate treatment of the gels, separated proteins can be quantitatively transferred to PVDF or nitrocellulose membranes and probed with common-not phosphorylation state specific-antibodies and delineate the occurrence of a certain phosphoprotein species against its non-phosphorylated counterpart.
- MeSH
- akrylamid chemie MeSH
- Arabidopsis enzymologie růst a vývoj MeSH
- bakteriofág lambda enzymologie MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- fenol chemie MeSH
- fosfatasy metabolismus MeSH
- fosfoproteiny izolace a purifikace metabolismus MeSH
- fosforylace MeSH
- kultivační techniky MeSH
- membrány umělé MeSH
- mitogenem aktivované proteinkinasy izolace a purifikace metabolismus MeSH
- polyvinyly chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization.
The major objective of proteomics is to identify and examine the large numbers of proteins extracted from complex biological systems. This is generally achieved by combining various techniques of protein separation with a mass spectrometric analysis of proteins that are digested enzymatically. Recently, several alternatives to this standard protocol have been developed for efficient and fast protein digestion. One option is the use of modified trypsin instead of native trypsin for the in-gel digestion of proteins. Microwave, ultrasonic-assisted protein enzymatic digestion and proteolysis accelerated by infrared radiation are other suitable alternatives. The application of the variable performance of the fast enzymatic digestion of proteins by using different techniques is reported here. The advantage of these methods is to have the ability to detect proteins in a shorter span of time. For example, using alternative protein digestion takes only minutes, in contrast to the several hours required by conventional methods. To demonstrate the suitability of this fast procedure, the digestion of carbonic anhydrase, bovine serum albumin, lysozyme and proteins extracted from plants (Hordeum vulgare, Arabidopsis thaliana) were used. Considering that the required reaction time for the conventional method is much longer, these applied methodic approaches tend to give in-gel digestion a much higher efficiency rating. This study examines the fast, efficient and low-cost proteolytic strategies for the digestion process, and for protein identification based on the use of ultrasound and infrared technology. In addition, comparisons of the applied techniques were studied. Several differences were found, suggesting the potential use of proteolysis accelerated by infrared radiation.
- MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- molekulární sekvence - údaje MeSH
- peptidové fragmenty analýza chemie metabolismus MeSH
- proteiny analýza chemie metabolismus MeSH
- proteomika metody MeSH
- rostlinné proteiny MeSH
- sekvence aminokyselin MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- trypsin metabolismus MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
