Biomedical subjects

C G Huber

Publications and source records attributed to C G Huber.

33 records · Page 2Linked to original sources

PubMed1999

Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography-electrospray ionization mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry of proteins. I. Liquid chromatography.

Proteins ranging in molecular mass from 14,000 to 80,000 were analyzed by reversed-phase high-performance liquid chromatography-electrospray mass spectrometry (RP-HPLC-ESI-MS) using 60 x 1.0 mm I.D. microbore-columns packed with 2.3 microns highly crosslinked, octadecylated poly(styrene-divinylbenzene) particles. Proteins were eluted at temperatures of 80-90 degrees C with gradients of acetonitrile in 0.10-0.50% aqueous solutions of trifluoroacetic acid, formic acid or acetic acid. Substitution of trifluoroacetic acid, the most commonly used mobile phase additive for RP-HPLC, by formic acid resulted in a 35-160-fold improvement in analyte detectability at the cost of an only 32-104% increase in peak width at half height of eluting chromatographic peaks. The lower limits of detection for carbonic anhydrase (M(r) 29,022.7) in full scan and selected ion monitoring mode were 37 and 2.3 fmol, respectively. Measurement of protein masses by RP-HPLC-ESI-MS was accurate and highly reproducible with maximum mass deviations of 0.025% and relative standard deviations of less than 0.011%. Calibration plots of peak area versus concentration allowed the reliable quantitation of proteins in a concentration range of 0.010-1.0 mg/ml. Finally, the optimized method was applied to the separation, identification and quantification of proteins in real samples such as commercial protein preparations, monoclonal antibody fragments, allergen extracts and whey drinks.

Chromatography, High Pressure Liquid↗

PubMed1999

Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography-electrospray ionization mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry of proteins. II. Capillary electrophoresis.

Peptides and proteins were separated by capillary electrophoresis (CE) in fused-silica capillaries coated with an irreversibly adsorbed monolayer of derivatized polystyrene nanoparticles. Whereas phosphate buffer, pH 3.10, enabled the highly efficient separation of basic proteins with plate counts up to 1,400,000 m-1, volatile buffer components such as formic acid or acetic acid titrated with ammonia to the desired pH had to be used for the direct coupling of CE with electrospray ionization mass spectrometry (ESI-MS). Compared to 40 mM phosphoric acid-sodium hydroxide, pH 3.10, a background electrolyte containing 125 mM formic acid-ammonia, pH 4.00, was shown to yield equivalent separation efficiency. Investigation of the influence of buffered electrolytes on the ESI-MS signal of lysozyme at pH 2.70-4.00 showed that the charge state distribution shifted to lower charge states at higher pH with a concomitant five-fold decrease in signal intensity of the most abundant signal. The presence of trifluoroacetic acid in the background electrolyte greatly increased the level of baseline noise and completely inhibited the observation of any mass signals related to proteins. Full scan spectra could be acquired from 50-500 fmol amounts of proteins during coupled CE-ESI-MS utilizing 100-125 mM formic acid-ammonia, pH 3.10. However, compared to UV detection, considerable band broadening is observed with ESI-MS detection which is mainly attributed to column overloading, band spreading in the interface, and scanning data acquisition. Finally, the major whey proteins beta-lactoglobulin A, beta-lactoglobulin B, and alpha-lactalbumin were identified in a whey drink by comparison of molecular masses determined by CE-ESI-MS to molecular masses calculated from the amino acid sequence.

Chromatography, High Pressure Liquid↗

PubMed1999

Identification of illudins in Omphalotus nidiformis and Omphalotus olivascens var. indigo by column liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry.

Reversed-phase liquid chromatography was used to separate toxins in mushrooms of the genus Omphalotus. Crude ethyl acetate extracts of cultures were injected directly onto a 150 x 2 mm I.D. column packed with 3 microns octadecylsilica and eluted with a gradient of acetonitrile in 0.1% aqueous acetic acid at a flow-rate of 200 microliters/min. Monitoring of the column effluate by atmospheric pressure ionization tandem mass spectrometry allowed the identification of the toxins. The fungal toxins illudin M and illudin S were detected and identified for the first time in cultures of the Australian Omphalotus nidiformis and the North American Omphalotus olivascens var. indigo (Boletales, Basidiomycetes) and confirmed the valuable taxonomic character of illudins for the genus Omphalotus.

Agaricales↗

PubMed1999

Unexpected BII conformer substate population in unoriented hydrated films of the d(CGCGAATTCGCG)2 dodecamer and of native B-DNA from salmon testes.

Conformational substates of B-DNA had been observed so far in synthetic oligonucleotides but not in naturally occurring highly polymeric B-DNA. Our low-temperature experiments show that native B-DNA from salmon testes and the d(CGCGAATTCGCG)2 dodecamer have the same BI and BII substates. Nonequilibrium distribution of conformer population was generated by quenching hydrated unoriented films to 200 K, and isothermal structural relaxation toward equilibrium by interconversion of substates was followed by Fourier transform infrared spectroscopy. BI interconverts into BII on isothermal relaxation at 200 K, whereas on slow cooling from ambient temperature, BII interconverts into BI. Our estimation of the dodecamer's BI-to-BII conformer substate population by curve resolution of the symmetrical stretching vibration of the ionic phosphate is 2.4 +/- 0.5 to 1 at 200 K, and it is 1.3 +/- 0.5 to 1 between 270 and 290 K. Pronounced spectral changes upon BI-to-BII interconversion are consistent with base destacking coupled with migration of water from ionic phosphate toward the phosphodiester and sugar moieties. Nonspecific interaction of proteins with the DNA backbone could become specific by induced-fit-type interactions with either BI or BII backbone conformations. This suggests that the BI-to-BII substate interconversion could be a major contributor to the protein recognition process.

Animals↗

PubMed1998

On-line cation exchange for suppression of adduct formation in negative-ion electrospray mass spectrometry of nucleic acids.

One major difficulty in the analysis of nucleic acids by electrospray mass spectrometry is represented by the affinity of the polyanionic sugar-phosphate backbone for nonvolatile cations, especially ubiquitous sodium and potassium ions. A simple on-line sample preparation system comprising a microflow pumping system and 45 x 0.8-mm-i.d. microcolumns packed with weak or strong cation-exchange resins is described for the efficient removal of cations from nucleic acid samples. Samples were analyzed by flow injection analysis at a 3-5 microL/min flow of 10 mM triethylamine in 50% water-50% acetonitrile. After on-line desalting, mass spectra of oligonucleotides revealed no significant sodium adduct peaks. Moreover, signal-to-noise ratios were greatly enhanced compared to direct injection of the samples. Electrospray mass spectrometry with on-line sample preparation allowed accurate molecular mass determinations of picomole amounts of crude oligonucleotide preparations ranging in size from 8 to 80 nucleotides within a few minutes. The good linearity of the calibration plot (R2 = 0.9988) over at least 2 orders of magnitude and a relative standard deviation in peak areas of less than 9% permitted the sensitive quantitative measurement of oligonucleotides in a concentration range of 0.2-20 microM with selected-ion monitoring. Finally, the on-line sample preparation system was evaluated for the mass spectrometric analysis of complex oligonucleotide mixtures.

Cations↗

PubMed1998

Micropellicular stationary phases for high-performance liquid chromatography of double-stranded DNA.

The central role of nucleic acids in biosciences has effectuated the rapid development of numerous techniques for their isolation, separation, characterization and quantitation. Advances in high-performance liquid chromatography, particularly the introduction of novel microparticulate sorbents, have greatly promoted the separation and quantitation of nucleic acids. Because of their favorable mass transfer properties, micropellicular packing materials are advantageous for fast and high-resolution separations of double-stranded (ds) DNA molecules. With micropellicular packings, anion-exchange and ion-pair reversed-phase chromatography are the most popular chromatographic separation modes for dsDNA. The effective separation mechanisms in both chromatographic techniques are preferably described by nonstoichiometric models, that are founded on a better physicochemical background than traditional stoichiometric models. Column efficiency, retention characteristics, and size or sequence dependency of retention of dsDNA are greatly influenced by the chosen operational variables in both chromatographic modes. The applicability of HPLC with micropellicular stationary phases nucleic acids research includes preparative DNA fractionation, DNA restriction mapping, analysis of polymerase chain reaction products and purification of plasmid DNA.

Chemical Phenomena↗

PubMed1998

Capillary electrophoresis of peptides and proteins in fused-silica capillaries coated with derivatized polystyrene nanoparticles.

High-resolution capillary electrophoretic separation of proteins and peptides was achieved by coating the inner wall of 75 microm ID fused-silica capillaries with 40-140 nm polystyrene particles which have been derivatized with alpha-omega-diamines such as ethylenediamine or 1,10-diaminodecane. A stable and irreversibly adsorbed coating was obtained upon deprotonation of the capillary surface with aqueous sodium hydroxide and subsequent flushing with a suspension of the positively charged particles. At pH 3.1, the detrimental adsorption of proteins to the capillary inner wall was suppressed efficiently because of electrostatic repulsion of the positively charged proteins from the positively charged coating which enabled protein separations with maximum efficiencies of 400000 plates per meter. A substantial improvement of separation efficiency in particle-coated capillaries was observed after in-column derivatization of amino functionalities with 2,3-epoxy-l-propanol, resulting in a more hydrophilic coating. Five basic and four acidic proteins could be separated in less than 7 min with efficiencies up to 1900000 theoretical plates per meter. Finally, coated capillaries were applied to the high-resolution analysis of protein glycoforms and bioactive peptides.

Animals↗

PubMed1997

Capillary electrochromatography with gradient elution.

A capillary electrochromatograph incorporating a gradient-forming system generally employed in HPLC is described, and the use of gradient elution in reversed phase electrochromatography is demonstrated by the separation of PTH-amino acids and steroid hormones. The gradient former employs two reciprocating displacement pumps to control the composition of the eluent in the reservoir at the column inlet with time in a controlled manner. Thus, the composition of the mobile phase flowing through the column and driven by electrosmotic forces can be changed with time in a controlled fashion as customary in HPLC with gradient elution. The design of the system allows also for isocratic elution by pumping the eluent of constant composition through the cavity at the column inlet and thus continuously supplying fresh buffer. The eluent gradient is generated by the two pumps and a 10 microL mixer. From there the liquid passes at a flow rate of 0.1-0.2 mL/min through the 17 microL cavity housing the column inlet and an electrode. The flow of the mobile phase was electrosmotic at an effective overall electric field strength of 500-1500 V/cm through a 50 microns x 20/12 cm capillary column packed with 3.5 microns octadecylated silica particles. Gradient profiles generated in this manner were highly reproducible. The same-day and day-today reproducibilities of the electrosmotic flow were found to be better than 3%. The use of the capillary electrochromatographic system was demonstrated with isocratic and gradient elution for the separation of complex mixtures of biologically interesting substances. The influence of the column temperature on the electrosmotic flow velocity and retention of PTH-amino acids was also investigated.

Chromatography, High Pressure Liquid↗

PubMed1994

High-resolution liquid chromatography of fluorescent dye-labeled nucleic acids.

Using 100 mM of triethylammonium acetate as ion-pairing reagent, phosphodiester oligonucleotides labeled fluorescently at their 5' terminus could be separated successfully on alkylated nonporous 2.3-microns poly(styrene-divinylbenzene) particles by means of high-resolution liquid chromatography. Applying excitation wavelengths of 490, 520, 550, and 575 nm, respectively, optimum sensitivity was achieved for the fluorophores 5-carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein, N,N,N',N'-tetramethyl-6-carboxyrhodamine, and 6-carboxy-X-rhodamine (FAM, JOE, TAMRA, and ROX, respectively) at emission wavelengths of 520, 550, 580, and 605 nm, respectively. With calibration curves being linear over at least three orders of magnitude, the lower detection limits were 0.5, 2, 2, and 3 fmol, respectively. Depending on the type of fluorescent dye attached, retention times increased in the order JOE < FAM < TAMRA < ROX. Subsequently, fluorescent oligonucleotides were employed to prime polymerase chain reactions (PCR). Again the fluorophores were found to increase the retention times of double-stranded nucleic acids, but to a lesser degree than those of single-stranded oligonucleotides. Using a single FAM label attached to one of the two PCR primers, the sensitivity of fluorescence detection was found to be approximately 1 fmol or 30-70 times higher than that of uv absorbance detection depending on the length of the PCR product. Since the technique allows the separation of PCR products differing only 4 to 8 base pairs in length within a size range of 50 to 200 base pairs, it may be employed for the quantitative assessment of competitive PCR.

Base Sequence↗

PubMed1994

High-performance liquid chromatography for routine analysis of hepatitis C virus cDNA/PCR products.

Ion-pair reversed-phase high-performance liquid chromatography on alkylated nonporous polystyrene-divinylbenzene particles with a mean diameter of 2.1 microns was used to analyze PCR products according to their chain length within a few minutes. The simple and reliable procedure allows the simultaneous separation and isolation of DNA fragments differing in chain length by 1%-5% up to a size of 500 base pairs with recovery rates exceeding 97%. A greater than 70-fold increase in sensitivity could be achieved through the use of a fluorescein-labeled primer, which allowed the determination of a 127-bp hepatitis C virus cDNA/PCR product with a lower mass detection limit of 2 fmol. Calibration curves showed excellent linearity over a range of at least 4 magnitudes. Finally, the stationary phase allowed the routine analysis of hundreds of PCR products with high reproducibility of both retention times and peak areas.

Base Sequence↗

PubMed1993

High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene-divinylbenzene copolymers.

Phosphodiester-oligonucleotides were separated by ion-pair reversed-phase HPLC on nonporous polystyrene-based particles having a diameter of 2.1 +/- 0.12 microns. With unmodified poly(styrene-divinylbenzene) beads it was not possible to resolve oligonucleotides efficiently. However, upon addition of polyvinyl alcohol during polymerization baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases was obtained, with triethylammonium acetate serving as ion-pairing reagent. An even higher separation efficiency was achieved by Friedel-Crafts alkylation of poly(styrene-divinylbenzene) particles. At a column temperature of 50 degrees C, the number of theoretical plates exceeded 6 x 10(5) per meter. The maximum loading capacity for a 50 x 4.6-mm i.d. column still resulting in the highest resolution was 0.1 microgram. Calibration curves showed excellent linearity over a range of at least 4 magnitudes, with a lower mass detection limit of 0.16 ng. Recoveries ranged from 96.7 to 100.8%. The same stationary phase also allowed the separation of phosphorylated from dephosphorylated oligonucleotides, the former ones being eluted earlier.

Alkylation↗

PubMed1993

High-resolution liquid chromatography of DNA fragments on non-porous poly(styrene-divinylbenzene) particles.

DNA restriction fragments and PCR products were separated by means of ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles with a mean diameter of 2.1 microns. Optimum resolution was obtained by using an acetonitrile gradient in 100 mM of triethylammonium acetate and a column temperature of 50 degrees C. This allowed the separation of DNA fragments differing in chain length by 1-5% up to a size of 500 base pairs. PCR products could be analyzed directly in less than two minutes with a concentration sensitivity of at least 300 ng/ml. Compared with anion-exchange chromatography or gel electrophoresis no desaltation of the purified DNA molecules is required because the volatile buffer system can be readily evaporated. Subsequently, the method was used for the semiquantitative evaluation of the expression of multidrug resistance genes in mononuclear white blood cells.

Base Sequence↗

PubMed1992

Comparative study of capillary zone electrophoresis and high-performance liquid chromatography in the analysis of oligonucleotides and DNA.

Capillary zone electrophoresis and high-performance liquid chromatography were compared with regard to the separation of oligonucleotides and double-stranded DNA. Both anion-exchange and reversed-phase high-performance liquid chromatography on non-porous particles are considered to be superior to capillary electrophoresis in terms of speed and selectivity in the analysis of oligonucleotides up to 30 bases in length. Moreover, reversed-phase chromatography allows the simultaneous purification of detritylated oligonucleotides with recoveries > 90%. Compared with anion-exchange chromatography, there is no need for a subsequent desaltation step because the volatile buffer system can be readily evaporated. With regard to dsDNA, however, the resolving power of capillary electrophoresis cannot be matched by anion-exchange chromatography at present. Moreover, the combined use of hydroxyethylcellulose and ethidium bromide not only yielded a separation efficiency equal to that achieved by means of gel-filled capillaries but also avoids some of their limitations such as the destruction of the gel matrix at high current densities and the bias involved in electrokinetic injection.

Base Sequence↗

PubMed1992

High-performance liquid chromatographic separation of detritylated oligonucleotides on highly cross-linked poly-(styrene-divinylbenzene) particles.

Detritylated oligonucleotides were separated by reversed-phase high-performance liquid chromatography on highly cross-linked polystyrene-based particles having a mean particle diameter of 2.3 microns. The addition of poly(vinyl alcohol) during polymerization, which resulted in the presence of hydroxyl groups on the surface of the poly(styrene-divinylbenzene) beads, was necessary to obtain baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases. The impact of temperature was investigated and optimum resolution was achieved at 40 degrees C. At pH 7.0, the retention times of oligonucleotides were found to depend on the ratio of bases and to increase in the order of C less than G less than A less than T. Under the same conditions, it was possible to separate phosphorylated from dephosphorylated oligonucleotides, the former being eluted earlier. Recoveries ranged from 92 to 100%.

Base Sequence↗

PubMedDate not supplied

Analysis of nucleic acids by on-line liquid chromatography-mass spectrometry.

The numerous problems posed by modern biochemistry, biology, and medicine, as well as the growing significance of genetic engineering require the application of fast and reliable methods of utmost sensitivity and selectivity for the analysis of nucleic acids. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) represent established analytical techniques for the characterization and structural elucidation of single- and double-stranded nucleic acids, ranging in size from a few nucleotides to several thousand base pairs. Although both techniques are independently applicable for nucleic acid analysis, the on-line hyphenation significantly enhances their potential for the robust and fully automable routine analysis of minute amounts of biological samples. Among the various chromatographic and mass spectrometric modes available in principle, ion-pair reversed-phase HPLC and electrospray ionization mass spectrometry (ESI-MS) have been shown to be the most suitable for the direct interfacing of liquid chromatography (LC) and MS. Instrumental setup, as well as chromatographic and mass spectrometric experimental conditions, need to be carefully selected in order to maximize the performance of the hyphenated analytical system. Applications of HPLC-ESI-MS include the characterization of oligodeoxynucleotides synthesized by solid-phase synthesis, the analysis of antisense oligodeoxynucleotides, oligonucleotide metabolites, and DNA adducts, the analysis of genomic segments specifically amplified by the polymerase chain reaction (PCR), the characterization of ribonucleic acids, the sizing of double-stranded DNA restriction fragments, the genotyping of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), the detection of mutations in nucleic acid sequences, and the sequencing of nucleic acids.

Animals↗