Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a tool to probe the reactions of trans-Hex-2-enal with proteins.
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Aldehydes↗
Biomedical subjects
M V Novotny
Publications and source records attributed to M V Novotny.
Microcolumn liquid chromatography in biochemical analysts.
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Adrenal Medulla↗
End-label, free-solution capillary electrophoresis of highly charged oligosaccharides.
The effect of fluorescent tags on the separation of negatively charged oligosaccharides, derived from a partially hydrolyzed kappa-carrageenan, was studied. When the charge-to-friction ratio of oligosaccharides is increased by the end-label, the migration order is from smaller to larger oligomers, and the resolution of larger oligomers could be improved by using a sieving medium. The migration order can be entirely reversed when the charge-to-friction ratio of the solute is decreased by the end-label. The experimental electrophoretic mobilities obtained in this work are in excellent agreement with the recently reported theoretical model (Mayer, P.; Slater, G. W.; Drouin, G. Anal. Chem. 1994, 66, 1777-1780). The maximum number of separated oligomers (Mmax) as a function of applied voltage and injection time was also studied, but no strong dependencies were found. Resolution between small oligomers could be significantly improved by following this separation principle.
Carrageenan↗
Stereoselectivity in mammalian chemical communication: male mouse pheromones.
Two male mouse pheromones, 3,4-dehydro-exo-brevicomin (DHB) and 2-sec-butyldihydrothiazole (SBT), are chiral molecules which were previously tested in their respective bioassays as racemic mixtures. The focus of this study has been to determine the absolute configuration of their natural forms and its relation to stereospecific biological action. DHB was established as the R,R-enantiomer possessing biological activity. Due to an extremely easy racemization of SBT under very mild conditions, enantioselectivity of its transmission and its action at the receptor site appear to be of secondary importance.
Animals↗
Hydrolytically stable cellulose-derivative coatings for capillary electrophoresis of peptides, proteins and glycoconjugates.
A method for the preparation of versatile, hydrolytically stable coatings of fused silica capillaries is described. First, the HNO3-pretreated silica surface is reacted with 7-oct-1-enyltrimethoxysilane to form a stable self-assembled alkylsilane monolayer. Secondly, the copolymer of hydroxypropylcellulose and 2-hydroxyethyl methacrylate is statically coated onto a hydrophobized wall. This allows the formation of a highly crosslinked copolymer layer which is tightly bound to the wall surface. Hydrolytical stability in the pH range of 2-10 for several weeks has been demonstrated. The coating generates a low electroosmotic flow at alkaline pH, which can be easily masked by using special buffers. The versatility of this column technology is documented by separations of peptides, glycoproteins and derivatized oligosaccharides.
Capillary Action↗
Maltooligosaccharides as chiral selectors for the separation of pharmaceuticals by capillary electrophoresis.
Complexation between the linear maltodextrin oligosaccharides and certain enantiomeric compounds of pharmaceutical interest in buffered solutions can lead to an analytically desirable chiral recognition. Different maltodextrins were assessed in their capacity to cause enantiomeric separations under various conditions of capillary electrophoresis. The mechanism of chiral recognition has been probed through electrophoretic mobility and selectivity measurements for different buffer solutions and organic solvent additives. A differential interaction of chiral solutes with the maltodextrin helical entities emerges as the basis of such enantioselectivity. This notion is further supported by 1H- and 13C-NMR experiments. Optimized separations of simendan, ibuprofen, warfarin, and ketoprofen enantiomers are demonstrated together with a chiral determination of ibuprofen in a blood serum sample at the therapeutic level.
Blood Chemical Analysis↗
Mixed polymer networks in the direct analysis of pharmaceuticals in urine by capillary electrophoresis.
Two-component polymer mixtures of polyethylene oxide-polydextran have been investigated as unique separation media for capillary electrophoresis. The effects of concentration of the individual polymers and their mixtures on the electroosmotic velocity and electrophoretic mobility of small pharmaceutical compounds were investigated. The molecular masses of polymers, buffer concentrations and percentages of organic solvents and cyclodextrins were varied to explore their effects on the separation process. The plate height against field strength curves were also generated for a better understanding of the kinetic processes involved. The two-component polymer mixtures were found as stable and selective media for the analysis of an anti-ulcer drug famotidine directly in untreated urine.
Buffers↗
Separation of large DNA fragments by capillary electrophoresis under pulsed-field conditions.
Capillary zone electrophoresis (CZE), using an entangled polyacrylamide solution, was applied to large DNA samples under pulsed-field conditions. Highly efficient separations were achieved under biased sinusoidal field and field-inversion pulsing regimes. The separations that were obtained with 8.3-48.5 kb lambda DNA standards and 48.5 kb-1 Mb lambda DNA concatamers (modified with ethidium bromide) clearly demonstrate a dramatic improvement in the separation time (roughly, 10-50 times) over the conventionally used slab-gel techniques. Moreover, the CZE method appears much more sensitive and amenable to component quantification and method automation.
DNA↗
Pulsed-field capillary electrophoresis: optimizing separation parameters with model mixtures of sulfonated polystyrenes.
The electrophoretic transport of high molecular weight charged solutes, both flexible and stiff polymers, has been studied by capillary electrophoresis under constant-field and pulsed-field conditions. Sulfonated polystyrenes were used as model solutes in different entangled polymer solutions. First, changes of the end-to-end distance vectors of flexible polymers were examined through the mobility/potential-gradient curves. Under pulsed-field conditions, the influence of different pulse shapes, frequencies, and amplitudes of forward and backward pulses on the electrophoretic mobilities of model solutes was studied. Resolution of the mixture components was strongly affected by changes in frequency of both sine-wave and square-wave pulses. The experimental results obtained under pulse-field conditions are roughly in agreement with the existing theories of electrophoretic transport.
Electrophoresis↗
Inhibition of glycolytic enzymes by endogenous aldehydes: a possible relation to diabetic neuropathies.
Endogenous saturated and unsaturated aldehydes were found in significant elevations in serum of diabetic humans and rats. These compounds, originating from the lipid peroxidation processes, are shown here to be potent inhibitors of the glycolytic enzymes, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. The inhibition process is non-competitive and progressive. The aldehyde mixture, when supplemented to the standard rat diet at 1/100 ratio, caused nerve damage that is reminiscent of diabetic polyneuropathies.
Aldehydes↗
Separation of amino acid homopolymers by capillary gel electrophoresis.
Gel-filled capillaries utilizing highly concentrated and moderately cross-linked acrylamide-type gels in capillary electrophoresis were successfully applied to the separation of the individual oligomers of various poly(amino acids). Mixtures of both anionic and cationic nature were adequately resolved. While UV detection at 220 nm was mostly utilized, the polyanions with N-terminal groups can also be tagged with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) for a more sensitive detection by a laser-induced fluorescence detector.
Amino Acids↗
Chiral separations of basic drugs and quantitation of bupivacaine enantiomers in serum by capillary electrophoresis with modified cyclodextrin buffers.
Modified cyclodextrin derivatives were evaluated as the buffer additives in capillary electrophoresis of several racemic pharmaceutical bases. Uncoated and polyacrylamide-modified silica capillaries were compared for their effectiveness in the enantiomeric resolution and migration reproducibility of model solutes. Using cationic detergents in the mixed-micellar mode, optimized separations of the racemic drug bupivacaine are demonstrated in a spiked serum sample at the therapeutic level. Precision, linearity and sensitivity of the method appear adequate for reliable quantitation required in pharmacokinetic and clinical studies.
Buffers↗
Peptide mapping of complex proteins at the low-picomole level with capillary electrophoretic separations.
A variety of different peptide-mapping schemes are presented, with emphasis on the development of procedures which can be done with limited quantities (i.e. 5 pmol) of protein. Results are obtained from model proteins which contain disulfide bonds, which must be broken prior to fragmentation of the protein. A reaction involving the simultaneous use of tributylphosphine and 2-methylaziridine to reduce and alkylate the disulfide bonds is employed, due to favorable attributes of these reagents for the scaled-down procedure. The traditional performic acid oxidation reaction to cleave cystine groups is also successfully used with low-picomole quantities of protein. Three different protein digestion reagents are used: trypsin, chymotrypsin, and cyanogen bromide. Each reagent produces a unique mixture of peptides. Capillary electrophoresis is used to separate the peptides, offering high separation efficiencies, short analysis times, and compatibility with small sample sizes. In addition to the conventional use of UV detection for underivatized peptides, laser-induced fluorescence detection is employed in conjunction with an arginine-selective derivatization reaction. This latter procedure for derivatization and detection offers an alternative peptide-mapping mode, in which only the arginine-containing peptides are detected, and is useful in simplifying the peptide maps of large proteins.
Alkylation↗
Sensitive, laser-assisted determination of complex oligosaccharide mixtures separated by capillary gel electrophoresis at high resolution.
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Electrophoresis↗
Selective determination of arginine-containing and tyrosine-containing peptides using capillary electrophoresis and laser-induced fluorescence detection.
The use of two different amino acid-selective fluorogenic reagents for the derivatization of peptides is investigated. One such scheme utilizes a selective reaction of benzoin with the guanidine moiety to derivatize arginine residues occurring in a peptide. The second scheme involves the formylation of tyrosine, followed by reaction with 4-methoxy-1,2-phenylenediamine. The use of capillary electrophoresis and laser-induced fluorescence detection allows enhanced efficiencies and sensitivities to be obtained for the separations of either arginine- or tyrosine-containing peptides. A helium-cadmium laser (325 nm) is ideally suited for the laser-based detection system due to a close match of the excitation maxima of derivatized peptides from both reactions. A detection limit of 270 amol is achieved for model arginine-containing peptides, while the detection limit for model tyrosine-containing peptides is measured at 390 amol. Both derivatization reactions are found to be useful for high-sensitivity peptide mapping applications in which only the peptides containing the derivatized amino acids are detected.
Amino Acid Sequence↗
Electrochromatographic solid-phase extraction for determination of cimetidine in serum by micellar electrokinetic capillary chromatography.
A highly effective electrochromatographic solid-phase extraction and preconcentration method is reported for the determination of cimetidine in serum in the concentration range 0.233-11.4 micrograms/ml. Preconcentrated samples were determined by micellar electrokinetic capillary chromatography while ranitidine was used as an internal standard. Sample preparation included retention of the analyte on a C18 solid-phase cartridge, followed by elution assisted by an applied voltage of 150 V. From 0.5-ml serum samples, 20-50-microliters aliquots were collected for electrophoretic analysis. Within the studied concentration range, the method was linear and provided adequate precision.
Chromatography↗
Recent advances in capillary electrophoresis of proteins, peptides and amino acids.
The current status of high-performance capillary electrophoresis as an analytical separation method for proteins, peptides and amino acids is assessed. Recent advances in suppressing the effects of electroosmotic flow and irreversible adsorption of proteins at the capillary wall are reviewed, together with procedures for optimal separations of peptides and amino acids. The detection aspects emphasize the role of laser-induced fluorescence and capillary electrophoresis/mass spectrometry in high-sensitivity measurements.
Amino Acid Sequence↗
Capillary zone electrophoresis of oligonucleotides. Factors affecting separation.
The influence of various parameters affecting separation of oligonucleotides by capillary zone electrophoresis has been examined. The effects of pH, ionic strength, and various additives, including highly charged cations such as spermine, were studied. Using polycytidines as model compounds, it was demonstrated that pH in the range of 5-8 and ionic strength in the range of 20-200 mmol/l do not influence the separation of oligonucleotides substantially. However, with the addition of spermine to the background electrolyte, migration order was inverted as the effective mobilities of the larger oligonucleotides were greatly decreased. With the addition of spermine and sodium dodecyl sulfate, the separation+ of these model oligonucleotides was also significantly affected. The best separation of a homologous series of polycytidines was obtained with a background electrolyte containing 60 mmol/l histidine, 30 mmol/l glutamic acid, 50 mmol/l sodium dodecyl sulfate and 3 mmol/l spermine.
Electrophoresis↗