Capillary electrophoresis-mass spectrometry Dotaz Zobrazit nápovědu
Coupling of capillary electrophoresis (CE) with mass spectrometry (MS) represents a powerful combination for performing rapid, efficient, and sensitive analysis of a variety of compounds. Here we describe a construction, operation, and application of a microfabricated liquid junction CE-MS interface. The interface is designed as a microfabricated unit with an integrated liquid junction and electrospray tip made from polyimide, which is positioned in a plastic connection block securing the separation CE capillary and attachable to the CE instrument. The application was demonstrated by CE-MS analysis of dextran oligomers labeled by (2-aminoethyl)trimethylammonium (AETMA) salt.
A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism--AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry.
- MeSH
- aminoimidazolkarboxamid analogy a deriváty farmakologie chemie MeSH
- elektroforéza kapilární metody přístrojové vybavení MeSH
- erytrocyty * cytologie metabolismus MeSH
- hmotnostní spektrometrie * metody MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- nukleotidy analýza chemie izolace a purifikace MeSH
- puriny chemie MeSH
- ribonukleosidy farmakologie chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Capillary electrophoresis hyphenated with electrospray mass spectrometry (CE-ESI-MS) has emerged in the past decade as one of the most powerful bioanalytical techniques. As the sensitivity and efficiency of new CE-ESI-MS interface designs are continuously improving, numerical modeling can play important role during their development. In this review, different aspects of computer modeling and simulation of CE-ESI-MS interfaces are comprehensively discussed. Relevant essentials of hydrodynamics as well as state-of-the-art modeling techniques are critically evaluated. Sheath liquid-, sheathless-, and liquid-junction interfaces are reviewed from the viewpoint of multidisciplinary numerical modeling along with details of single and multiphase models together with electric field mediated flows, electrohydrodynamics, and free fluid-surface methods. Practical examples are given to help non-specialists to understand the basic principles and applications. Finally, alternative approaches like air amplifiers are also included. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 34: 558-569, 2015.
- MeSH
- algoritmy MeSH
- chemické modely MeSH
- elektroforéza kapilární přístrojové vybavení metody MeSH
- elektromagnetická pole MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací přístrojové vybavení metody MeSH
- hydrodynamika MeSH
- lidé MeSH
- počítačová simulace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
One of the challenging instrumental aspects in coupling an automated CE instrument with ESI mass spectrometry (CE-MS) is finding the balance between the stability, reproducibility and sensitivity of the analysis and compatibility with the standard CE instrumentation. Here, we present a development of a new liquid junction based electrospray interface for automated CE-MS, with a focus on the technical design followed by computer modeling of transport conditions as well as characterization of basic performance of the interface. This hybrid arrangement designed as a microfabricated unit attachable to the automated CE instrument allows using of a wide range of separation capillaries with respect to their diameter, length or internal coating (e.g., for suppressed electroosmotic flow). Different compositions of the ESI liquid and background electrolyte solutions can be used if needed. The microfabricated part, prepared by laser machining from polyimide, includes a self-aligning liquid junction, a short transport channel, and a pointed sprayer for the electrospray ionization. This microfabricated part is positioned in a plastic connection block securing the separation capillary and flushing ports. Transport conditions were modelled using computer simulation and the real life performance of the interface was compared to that of a commercial sheath liquid interface. The basic performance of the interface was demonstrated by separations of peptides, proteins, and oligosaccharides.
- MeSH
- chemické modely MeSH
- elektroforéza kapilární přístrojové vybavení MeSH
- hmotnostní spektrometrie přístrojové vybavení MeSH
- laboratorní automatizace MeSH
- mikrofluidní analytické techniky přístrojové vybavení metody MeSH
- proteiny analýza izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Alzheimer's disease is the most common cause of dementia, currently afflicting almost 40 million patients worldwide. According to the amyloid cascade hypothesis, the pathogenesis of the disease could be slowed down or even stopped by the inhibition of beta-secretase, making this aspartic acid protease a potentially important drug target site. Capillary electrophoresis is a promising technique for screening putative enzyme inhibitors due to highly effective separations, minuscule sample and other chemicals consumption, compatibility with a variety of detection techniques, and high throughput via automation. This chapter presents a method based on capillary electrophoresis coupled to mass spectrometry detection for kinetic and inhibition assays of the beta-secretase reaction with a decapeptide derived from an amyloid precursor protein.
- MeSH
- Alzheimerova nemoc farmakoterapie metabolismus MeSH
- amyloidový prekurzorový protein beta metabolismus MeSH
- aspartátové endopeptidasy metabolismus MeSH
- elektroforéza kapilární metody MeSH
- hmotnostní spektrometrie metody MeSH
- inhibitory enzymů farmakologie MeSH
- inhibitory proteas farmakologie MeSH
- kinetika MeSH
- lidé MeSH
- plošný screening metody MeSH
- sekretasy antagonisté a inhibitory MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The optimization of working parameters controlling the transfer of an analyte from the separation into the spray capillary at the liquid junction interface is a complex problem. The numerical models of hydrodynamic flow, electric field strength and the consequential mass transfer provide a valuable insight into the function of the miniaturized device. The results revealed that the most important parameter is the electric field strength inside the gap between the separation and spray capillaries. In a strong electric field, the analyte leaving the separation capillary is immediately transferred into the spray capillary at its maximum concentration. Although the losses of analyte outside the interface are dominant in this case, the sprayed concentration determines the detection sensitivity. Since only a small amount of the sprayed material enters mass spectrometer, the losses at the interface do not influence the sensitivity. At low electric field strength the total amount of analytes is transferred into the spray capillary. In this case, however, the analytes enter the capillary slowly and, as a result, are significantly diluted. The electric field strength, pressure and dimensions determine the mass transfer in the interface and must be considered when the optimum conditions of an analysis are chosen. Several fold improvement in sensitivity and efficiency of the method can be expected when working under the optimum conditions.
In this work, interactions of carboxylated core shell magnetic nanoparticles with polymyxin B sulfate were studied by connecting capillary electrophoresis with inductively coupled plasma mass spectrometry. The interaction was probed by affinity mode of capillary electrophoresis with 25 mM phosphate buffer at physiological pH. 54Fe, 56Fe, 57Fe, 34S, and 12C isotopes were used to monitor the migration of an electroosmotic flow marker and the interaction of the nanoparticles with polymyxin B. The analysis of interaction data showed two distinct interaction regions, one with low polymyxin B concentration, the second with high polymyxin B concentration. These regions differed in the strength of the interaction, 1.49 × 107 M-1 and 1.60 × 104 M-1, and in the stoichiometry of 0.7 and 3.5, respectively. These differences can be explained by the decrease of electrostatic repulsion between nanoparticles caused by polymyxin B. This is also in agreement with the nanoparticles peak shapes: sharp for low polymyxin B concentrations and broad for high polymyxin B concentrations.
Open tubular capillary enzyme reactors were studied for rapid protein digestion and possible on-line integration into a CE/ESI/MS system. The need to minimize the time of the analyte molecules to diffuse towards the surface immobilized enzyme and to maximize the surface-to-volume (S/V) ratio of the open tubular reactors dictated the use of very narrow bore capillaries. Extremely small protein amounts (atto-femtomoles loaded) could be digested with enzymes immobilized directly on the inside wall of a 10 microm I.D. capillary. Covalently immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin and pepsin A were tested for the surface immobilization. The enzymatic activity was characterized in the flow-through mode with on-line coupling to electrospray ionization-time of flight-mass spectrometer (ESI/TOF-MS) under a range of protein concentrations, buffer pH's, temperatures and reaction times. The optimized reactors were tested as the nanospray needles for fast identification of proteins using CE-ESI/TOF-MS.
- MeSH
- biokompatibilní potahované materiály MeSH
- elektroforéza kapilární metody MeSH
- elektroforéza mikročipová MeSH
- elektrolyty MeSH
- enzymy imobilizované chemická syntéza klasifikace MeSH
- financování organizované MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- mikrochemie MeSH
- on-line systémy MeSH
- pepsin A metabolismus MeSH
- proteiny analýza chemie MeSH
- sekvenční analýza proteinů metody MeSH
- senzitivita a specificita MeSH
- studie proveditelnosti MeSH
- trypsin metabolismus MeSH
