Vestibular schwannoma is the most common benign neoplasm of the cerebellopontine angle. Its first symptoms include hearing loss, tinnitus, and vestibular symptoms, followed by cerebellar and brainstem symptoms, along with palsy of the adjacent cranial nerves. However, the clinical picture has unpredictable dynamics and currently, there are no reliable predictors of tumor behavior. Hence, it is desirable to have a fast routine method for analysis of vestibular schwannoma tissues at the molecular level. The major objective of this study was to verify whether a technique using in-sample specific protein digestion with trypsin would have the potential to provide a proteomic characterization of these pathological tissues. The achieved results showed that the use of this approach with subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of released peptides allowed a fast identification of a considerable number of proteins in two differential parts of vestibular schwannoma tissue as well as in tissues of control healthy samples. Furthermore, mathematical analysis of MS data was able to discriminate between pathological vestibular schwannoma tissues and healthy tissues. Thus, in-sample protein digestion combined with LC-MS/MS separation and identification of released specific peptides followed by mathematical analysis appears to have the potential for routine characterization of vestibular schwannomas at the molecular level. Data are available via ProteomeXchange with identifier PXD045261.
- MeSH
- chromatografie kapalinová metody MeSH
- lidé MeSH
- peptidové fragmenty * analýza chemie metabolismus MeSH
- peptidy metabolismus MeSH
- proteolýza MeSH
- proteomika metody MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- trypsin chemie MeSH
- vestibulární schwannom * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Nonaqueous capillary electrophoresis (NACE) using methanol (MeOH) as a solvent of the BGEs and quantum mechanical density functional theory (DFT) have been applied to determine the thermodynamic acidity (ionization) constants (pKa ) of mono- and diaza[5]helicenes, mono- and diaza[6]helicenes, and their dibenzo derivatives in MeOH and water. First, the mixed acidity constants, pKa,MeOHmix${\rm{p}}K_{{\rm{a,MeOH}}}^{{\rm{mix}}}$ , of ionogenic pyridinium groups of azahelicenes and their derivatives in MeOH were obtained by nonlinear regression analysis of pH dependence of their effective electrophoretic mobilities. The effective mobilities were measured by NACE in a large series of methanolic BGEs within a wide conventional pH range (pHMeOH 1.6-12.0) and at ambient temperature (21-26°C) in a home-made CE device. Prior to mixed acidity constant calculation, the effective mobilities were corrected to reference temperature (25°C) and constant ionic strength (25 mM). Then, the mixed acidity constants were recalculated to the thermodynamic acidity constants pKa,MeOH by the Debye-Hückel theory of nonideality of electrolyte solutions. Finally, from the methanolic thermodynamic pKa,MeOH values, the aqueous thermodynamic pKa,H2O${\rm{p}}{K_{{\rm{a,}}{{\rm{H}}_{\rm{2}}}{\rm{O}}}}$ constants were estimated using the empirical relations between methanolic and aqueous acidity constants derived for structurally related pyridine derivatives. Depending on the number and position of the nitrogen atoms in their molecules, the analyzed azahelicenes were found to be weak to moderate bases with methanolic pKa,MeOH in the range 2.01-8.75 and with aqueous pKa,H2O${\rm{p}}{K_{{\rm{a,}}{{\rm{H}}_{\rm{2}}}{\rm{O}}}}$ in the range 1.67-8.28. The thermodynamic pKa,MeOH obtained by the DFT calculations were in a good agreement with those determined experimentally by NACE.
For the understanding of pathological states of bone tissues in oral surgery, it would be desirable to have the possibility to simulate these processes on bone cell models in vitro. These cultures, similarly to bone tissues, contain numerous proteins entrapped in the insoluble matrix. The major goal of this study was to verify whether a method based on direct in-matrix protein digestion could be suitable for the discrimination between different induced pathological states of bone cell models cultivated in vitro. Using in-sample specific protein digestion with trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released peptides, 446 proteins (in average per sample) were identified in a bone cell in vitro model with induced cancer, 440 proteins were found in a model with induced inflammation, 451 proteins were detected in control in vitro culture, and 491 proteins were distinguished in samples of vestibular laminas of maxillary bone tissues originating from six different patients. Subsequent partial least squares - discrimination analysis of obtained liquid chromatography-tandem mass spectrometry data was able to discriminate among in vitro cultures with induced cancer, with induced inflammation, and control cultivation. Thus, the direct in-sample protein digestion by trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released specific peptide fragments from the insoluble matrix and mathematical analysis of the mass spectrometry data seems to be a promising tool for the routine proteomic characterization of in vitro human bone models with induced different pathological states.
- MeSH
- chromatografie kapalinová metody MeSH
- lidé MeSH
- peptidy analýza MeSH
- proteiny chemie MeSH
- proteolýza MeSH
- proteomika metody MeSH
- stomatochirurgické výkony * MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- trypsin chemie MeSH
- zánět MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
We present a method for finely adjustable electroosmotic flow (EOF) velocity in cathodic direction for the optimization of separations in capillary electrophoresis. To this end, we use surface modification of the separation fused silica capillary by the covalently attached copolymer of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), that is, poly(AM-co-AMPS) or PAMAMPS. Coatings were formed by the in-capillary polymerization of a mixture of the neutral AM and anionic AMPS monomers premixed in various ratios in order to control the charge density of the copolymer. EOF mobility varies in the 0 to ∼40 × 10-9 m2 V-1 s-1 interval for PAMAMPS coatings ranging from 0 to 60 mol.% of charged AMPS monomer. For EOF in PAMAMPS-treated capillaries, we observed (i) a negligible dependence on pH in the 2-10 interval, (ii) a minor variance among background electrolytes (BGEs) in function of their components and (iii) its standard decrease with increasing ionic strength of the BGE. Interest in variable cathodic EOF was demonstrated by the amelioration of separation of two kinds of isomeric anionic analytes, that is, monosaccharides phosphates and helquat enantiomers, in counter-EOF mode.
In this work, open-tubular capillary electrochromatography (OT-CEC) method with bare gold nanoparticles (GNPs)-based stationary phase has been developed and applied for separation of tryptic peptide fragments of native and glycated proteins, bovine serum albumin (BSA), and human transferrin (HTF). The GNPs-based stationary phase was prepared by immobilization of bare GNPs, freshly reduced from tetrachloroaurate(III) ions by citrate reduction, on the sol-gel pretreated inner wall of the fused silica capillary. The separation efficiency, peak capacity, and peptide recovery of this open-tubular capillary column were investigated by varying the experimental parameters such as type and concentration of the buffering constituent and pH of the background electrolyte (BGE), temperature, and separation voltage. The best separations of the above tryptic peptides were achieved in the BGE composed of aqueous 100 mmol/L sodium phosphate buffer, pH 2.5, at separation voltage 10 kV per 47-cm long, 50 μm inside diameter capillary thermostated at 25°C. OT-CEC with bare GNPs stationary phase is shown to be a suitable technique for separation of complex peptide mixtures arising from tryptic digestion of native and glycated BSA and HTF, and for investigation of glycation (nonenzymatic glycosylation) of these proteins.
- MeSH
- adsorpce MeSH
- glykosylace MeSH
- kapilární elektrochromatografie přístrojové vybavení metody MeSH
- lidé MeSH
- nanočástice chemie MeSH
- peptidy analýza MeSH
- proteiny chemie MeSH
- trypsin chemie MeSH
- zlato chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The review brings a comprehensive survey of the recent developments of high-performance electroseparation methods in capillary and microchip formats: zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography and electrochromatography. Applications of these techniques to analysis, isolation, purification and physicochemical and biochemical characterization of peptides are described. Advances in the investigation of electromigration properties of peptides, and in the methodology of their analysis, such as sample preparation, adsorption suppression, EOF control and detection, are presented. New developments, in particular, CE and CEC modes are reported and several types of their applications to peptide analysis are described: conventional qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC techniques to provide relevant physicochemical characteristics of peptides are demonstrated.
- MeSH
- elektroforéza kapilární metody MeSH
- kapilární elektrochromatografie metody MeSH
- lidé MeSH
- peptidy analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Thermodynamic acid-base dissociation (ionization) constants (pK(a)) of seven zwitterionic heterocyclic bases, first representatives of new heterocyclic family (2,3,5,7,8,9-hexahydro-1H-diimidazo[1,2-c:2',1'-f][1,3,2]diazaphosphinin-4-ium-5-olate 5-oxides), originally designed as chiral Lewis base catalysts for enantioselective reactions, were determined by capillary zone electrophoresis (CZE). The pK(a) values of the above very weak zwitterionic bases were determined from the dependence of their effective electrophoretic mobility on pH in strongly acidic background electrolytes (pH 0.85-2.80). Prior to pK(a) calculation by non-linear regression analysis, the CZE measured effective mobilities were corrected to reference temperature, 25°C, and constant ionic strength, 25 mM. Thermodynamic pK(a) values of the analyzed zwitterionic heterocyclic bases were found to be particularly low, in the range 0.04-0.32. Moreover, from the pH dependence of effective mobility of the bases, some other relevant characteristics, such as actual and absolute ionic mobilities and hydrodynamic radii of the acidic cationic forms of the bases were determined.
ACE was applied to the quantitative evaluation of noncovalent binding interactions between benzo-18-crown-6-ether (B18C6) and several alkali metal ions, Li(+), Na(+), K(+), Rb(+) and Cs(+), in a mixed binary solvent system, methanol-water (50/50 v/v). The apparent binding (stability) constants (K(b)) of B18C6-alkali metal ion complexes in the hydro-organic medium above were determined from the dependence of the effective electrophoretic mobility of B18C6 on the concentration of alkali metal ions in the BGE using a nonlinear regression analysis. Before regression analysis, the mobilities measured by ACE at ambient temperature and variable ionic strength of the BGE were corrected by a new procedure to the reference temperature, 25 degrees C, and the constant ionic strength, 10 mM. In the 50% v/v methanol-water solvent system, like in pure methanol, B18C6 formed the strongest complex with potassium ion (log K(b)=2.89+/-0.17), the weakest complex with cesium ion (log K(b)=2.04+/-0.20), and no complexation was observed between B18C6 and the lithium ion. In the mixed methanol-water solvent system, the binding constants of the complexes above were found to be about two orders lower than in methanol and about one order higher than in water.
- MeSH
- algoritmy MeSH
- alkalické kovy chemie MeSH
- benzenové deriváty chemie MeSH
- crown ethery chemie MeSH
- elektroforéza kapilární metody MeSH
- kinetika MeSH
- methanol chemie MeSH
- osmolární koncentrace MeSH
- regresní analýza MeSH
- teplota MeSH
- voda chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The review article is devoted mainly to the description of applications of gold nanoparticles (GNPs) in separation sciences, especially in electromigration and chromatographic techniques. The applications of GNPs in particular separation methods, CE, microchip CE, MEKC, CEC, HPLC and GC, are classified according to the molecular size of the analytes from low-molecular-mass compounds via medium sized substances to biopolymers (proteins and nucleic acids). A very recent and promising utilization of GNPs for sample preparation, preconcentration and preseparation of selected analytes from complex matrices is presented as well. Moreover, in two introductory sections, typical preparation procedures of the GNPs and their modifications are presented and physico-chemical and analytical methods employed for characterization of the native and modified GNPs are briefly introduced.
- MeSH
- chemická frakcionace přístrojové vybavení metody MeSH
- chemické techniky analytické přístrojové vybavení metody MeSH
- chromatografie micelární elektrokinetická kapilární MeSH
- chromatografie plynová MeSH
- elektroforéza kapilární MeSH
- kovové nanočástice chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zlato MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
