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Biomedical subjects

Christian G Huber

Publications and source records attributed to Christian G Huber.

At least 37 records · Page 2Linked to original sources
PubMed2004

Separate analysis of DYS385a and b versus conventional DYS385 typing: is there forensic relevance?

In order to determine to what extent the separate analysis of both copies of DYS385 improves Y-chromosomal short tandem repeat (Y-STR) haplotyping, we followed a recently published protocol for the separate amplification of DYS385a and DYS385b with modifications and compared the results with those obtained by conventional analysis in a population sample comprising 133 unrelated Caucasian males from Austria. Additionally, we typed all markers of the minimal haplotype (minHT) and a set of Y-chromosomal single nucleotide polymorphisms (Y-SNPs) in order to interpret the STR data depending on the Y-SNP haplogroup structure. The separate amplification of DYS385a and b improved the power of discrimination of this marker when compared to the results obtained with the conventional non-locus-discriminating amplification strategy. However, the degree of this improvement varied greatly between different haplogroups and was found to be highest in clade K. In the forensically relevant context of the minHT, the separate analysis of the DYS385 alleles had no effect on the differentiation of paternal lineages in our study. Furthermore, the amplicon lengths of 700-780 base pairs obtained in the course of the locus-discriminating approach restrict the applicability of this amplification strategy to high quality DNA samples.

Austria↗
PubMed2004

Characterization of some physical and chromatographic properties of monolithic poly(styrene-co-divinylbenzene) columns.

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200 microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. Important chromatographic features of the synthesized columns were characterized and critically compared to the properties of columns packed with micropellicular, octadecylated poly(styrene-co-divinylbenzene) (PS-DVB-C18) particles. The permeability of a 60 mm long monolithic column was slightly higher than that of an equally dimensioned column packed with PS-DVB-C18 beads and was invariant up to at least 250 bar column inlet pressure, indicating the high-pressure stability of the monolithic columns. Interestingly, monolithic columns showed a 3.6 times better separation efficiency for oligonucleotides than granular columns. To study differences of the molecular diffusion processes between granular and monolithic columns, Van Deemter plots were measured. Due to the favorable pore structure of monolithic columns all kind of diffusional band broadening was reduced two to five times. Using inverse size-exclusion chromatography a total porosity of 70% was determined, which consisted of internodule porosity (20%) and internal porosity (50%). The observed fast mass transfer and the resulting high separation efficiency suggested that the surface of the monolithic stationary phase is rather rough and does not feature real pores accessible to macromolecular analytes such as polypeptides or oligonucleotides. The maximum analytical loading capacity of monolithic columns for oligonucleotides was found to be in the region of 500 fmol, which compared well to the loading capacity of the granular columns. Batch-to-batch reproducibility proved to be better with granular stationary phases compared to monolithic stationary phase, in which each column bed is the result of a unique column preparation process.

Chromatography, High Pressure Liquid↗
PubMed2004

Intact mass measurements for unequivocal identification of hydrophobic photosynthetic photosystems I and II antenna proteins.

The purpose of this study was to determine the optimal experimental conditions necessary to allow rapid and accurate identification of highly hydrophobic proteins, such as the antenna proteins from photosystems I and II. The antenna proteins were derived from two different species, tomato and Arabidopsis, whose photosynthetic genome is well known. The separation techniques included sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting, microamino acid sequencing, reversed-phase high-performance liquid chromatography (RP-HPLC), intact mass measurements, and peptide mass fingerprinting by mass spectrometry. Immunoblotting was time-consuming, and success was limited due to cross-reactivity between these highly conserved sequences. The best results by far were achieved by separating intact proteins through hyphenation of reversed-phase liquid chromatography on-line to an electrospray ionization mass spectrometer (ESI-MS) and consequently identifying individual proteins from their intact mass measurements (IMMs), whereas peptide mass fingerprinting was hampered by the highly hydrophobic nature of these proteins. RP-HPLC-ESI-MS was superior in the quality of separation. Moreover, the high quality of mass spectra recorded during the RP-HPLC-ESI-MS analysis meant that the relative deviations of the molecular masses determined with a quadrupole ion trap mass analyzer ranged between 100 and 300 ppm. Thus, the correspondence between the intact mass values measured with those deduced from the DNA sequences allowed the different types of antenna proteins to be identified and assigned to their corresponding gene families. By utilizing this correlation, it was possible to spot gene products of previously cloned genes.

Arabidopsis↗
PubMed2004

Multidimensional proteomic analysis of photosynthetic membrane proteins by liquid extraction-ultracentrifugation-liquid chromatography-mass spectrometry.

The membrane protein components of photosystem I (PSI) and II (PSII) from different species were prefractionated by liquid extraction and sucrose gradient ultracentrifugation and subsequently analyzed by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) using poly-(styrene-divinylbenzene)-based monolithic capillary columns. The analytical method was shown to be very flexible and enabled the identification of antenna proteins as well as most of the proteins of the reaction center from PSI and PSII in various plant species with few RP-HPLC-ESI-MS analyses necessitating only minor adaptations in the gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid. The membrane proteins, ranging in molecular mass (Mr) from 4196 (I protein) to more than 80,000 (PSI A/B) as well as isoforms were identified on the basis of their intact Mr and comparison with Mr deduced from known DNA or protein sequences. High quality mass spectra enabled the identification and quantitation of the nonphosphorylated and phosphorylated reaction center subunits D1, D2, and CP43 of PSII, containing five to seven membrane-spanning alpha-helices. Because of its high flexibility and suitability for proteins having a very wide range of Mr and hydrophobicities, the method is generally applicable to the analysis of complex mixtures of membrane proteins.

Arabidopsis↗
PubMed2004

Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry.

We present the first global computer-aided sequencing algorithm for the de novo determination of short nucleic acid sequences. The method compares the fragment ion spectra generated by collision-induced dissociation of multiply charged oligodeoxynucleotide-ions to the m/z values predicted employing established fragmentation pathways from a known reference sequence. The closeness of matching between the measured spectrum and the predicted set of fragment ions is characterized by the fitness, which takes into account the difference between measured and predicted m/z values, the intensity of the fragment ions, the number of fragments assigned, and the number of nucleotide positions not covered by fragment ions in the experimental spectrum. Smaller values for the fitness indicate a closer match between the measured spectrum and predicted m/z values. In order to find the sequence most closely matching the experimental spectrum, starting from a given nucleotide composition all possible oligonucleotide sequences are assembled followed by identification of the correct sequence by the lowest fitness value. Using this concept, sequences of 5- to 12-mer oligodeoxynucleotides were successfully de novo determined. High sequence coverage with fragment ions was essential for obtaining unequivocal sequencing results. Moreover, the collision energy was shown to have an impact on the interpretability of tandem mass spectra by the de novo sequencing algorithm. Experiments revealed that the optimal collision energy should be set to a value just sufficient for complete fragmentation of the precursor ion.

Algorithms↗
PubMed2004

Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides.

An algorithm for the comparative sequencing (COMPAS) of oligonucleotides is shown to be suitable for the sequence verification of nucleic acids ranging in length from a few to 80 nucleotides. The algorithm is based on the matching of a fragment ion spectrum generated by collision-induced dissociation to m/z values predicted from a known reference sequence employing established fragmentation pathways. Prior to mass spectrometric investigation, the oligonucleotides were on-line purified by ion-pair reversed-phase high-performance liquid chromatography using monolithic separation columns. This study evaluated the potential and the limits of COMPAS regarding the length and the charge state of oligonucleotides, the selected collision energy, and the analyzed amount of sample using a quadrupole ion trap mass spectrometer. The results revealed that oligonucleotides could be very reliably re-sequenced up to 60-mers, although the algorithm was successfully used to even verify sequences up to 80-mers. The relative collision energy was typically in the range between 13 and 20%, which allowed in most cases a verification of the reference sequence in a window of at least three consecutive collision energies. To select a proper charge state for fragmentation, a compromise had to be found between high signal intensity and low charge state. Furthermore, by reducing the eluent flow rate during elution of the oligonucleotide, the sequence of a 50-mer was successfully verified from the analysis of 295 fmol of the raw product. COMPAS was proven to be reproducible and was applied to the genotyping of the polymorphic, Y-chromosomal locus M9 contained in a 62-base pair polymerase chain reaction product.

Algorithms↗
PubMed2004

Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry.

While electrospray ionization mass spectrometry has shown great potential for the identification of genotypes in DNA sequences amplified by polymerase chain reaction (PCR), the quantitative determination of allele frequencies remains challenging because of the presence of cationic adducts in the mass spectra which severely impairs accuracy of quantitation. We report here on the elaboration of an optimized desalting protocol for ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) of PCR amplicons which facilitates the genotyping of single nucleotide polymorphisms (SNPs). Chromatographic purification at temperatures between 50 and 70 degrees C using monolithic reversed-phase columns and acetonitrile gradients in aqueous, 20-30 mmol/l butyldimethylammonium bicarbonate enabled the mass spectrometric analysis of nucleic acid solutions containing up to 1.7 mol/l sodium chloride. A further improvement in removal of metal cations was achieved upon the addition of 5-10 mmol/l ethylenediaminetetraacetic acid (EDTA) to the sample solution prior to liquid chromatography. ICEMS was used for the semi-quantitative genotyping of SNPs amplified from the tetraploid genome of potato cultivars. Using a quadrupole ion trap mass spectrometer, allele frequencies were determined with an accuracy of 2-9% by measuring the relative intensities of the signals corresponding to the molecular mass of each of the alleles in the deconvoluted mass spectra. ICEMS results correlated well with those obtained by pyrosequencing, single nucleotide primer extension, and conventional dideoxy sequencing.

Chromatography, High Pressure Liquid↗
PubMed2003

Characterization of a variant of the spinach PSII type I light-harvesting protein using kinetically controlled digestion and RP-HPLC-ESI-MS.

A previously unknown isoform of the type I major antenna protein of photosystem II of spinach was identified, and its amino-terminal sequence was characterized by a novel kinetic digestion approach, in which sequential tryptic digestion was followed by analysis of both released peptides and truncated proteins by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry. Using nonpolar, monolithic, 200-microm-i.d. separation columns based on poly(styrene/divinylbenzene) copolymer and applying gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid, released peptides and truncated proteins could be separated and mass analyzed in a single chromatographic run. This enabled a straightforward identification of the fragments removed from the amino-terminal ends of the protein, which was essential for the characterization of the antenna isomers showing the most significant sequence variation in the amino-terminal region. The sequences of the amino termini were derived from the differences in molecular mass between intact and truncated proteins and were corroborated by sequencing using tandem mass spectrometry and database searching. The sequence of the 23 amino-terminal residues of the previously unknown isoform differed from that of the other two known isoforms only in one and three amino acids, respectively. Such subtle changes in amino acid sequence are supposed to play an important role in the supramolecular organization of photosynthetic antenna proteins.

Chromatography, High Pressure Liquid↗
PubMed2003

Mutation scanning by ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS).

Partially denaturing high-performance liquid chromatography has emerged as the most sensitive physical mutation scanning method. However, there are a few reports of mutations missed or only detectable at unique temperatures. The combined use of ion-pair reversed-phase high-performance liquid chromatography under completely denaturing conditions and electrospray ionization quadrupole ion trap mass spectrometry (ICEMS) obviates the need for selecting appropriate temperatures for resolving heteroduplices and allows the discrimination of different alleles even when they co-elute due to distinct mass differences between nucleobases. This was demonstrated for the detection of four mutations (259G>A, 286A>G, 300T>G, and 331+1G>A) in exon 5 and intron 5 of BRCA1, respectively. Current mass resolution of quadrupole ion trap mass spectrometers limits the identification of single A>T or T>A transversions with a mass difference of 9 Da to fragments <80 base pairs (bp). The presence of all other mutations can be detected in fragments up to approximately 105 bp. The approach may prove particularly useful in the mutational scanning of AT- or GC-rich sequences that are recalcitrant to most other methods.

Chromatography, High Pressure Liquid↗
PubMed2003

Effect of instrument tuning on the detectabilityof biopolymers in electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry of multiply charged biopolymer ions of different molecular size revealed a strong influence of tuning parameters on their detectability in quadrupole ion trap and triple quadrupole mass spectrometers. Hence, after optimizing the ion optical parameters with the signal of the 4- charge state of (dT)(24) (low charge state tuning), a tenfold increase in the signal-to-noise ratio for a mixture of oligodeoxythymidylic acids (n = 12-18) was obtained compared with the results achieved with tune parameters optimized with a synthetic 80-mer oligodeoxynucleotide. By contrast, a detection limit in the upper femtomole region could only be reached for a 104-mer oligodeoxynucleotide utilizing the 24- charge state of the 80-mer (high charge state tuning). The same effect was observed for proteins investigated in the positive ion mode using low and high charge states of cytochrome c and carbonic anhydrase, respectively, for instrument tuning. By comparing the settings for low and high charge state tuning, it became obvious that the most significant difference was observed in the potential applied to the heated metal capillary used to transfer ions from the atmospheric pressure to the vacuum region of the ion source. Taking advantage of the optimized tuning procedure, the molecular mass of a 61 base pair product of polymerase chain reaction was accurately determined by electrospray ionization mass spectrometry on-line interfaced to ion-pair reversed-phase high-performance liquid chromatography.

Base Sequence↗
PubMed2003

Proteomics of light-harvesting proteins in different plant species. Analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem II.

An overview of the intact molecular masses and the hydrophobic properties of the photosystem II (PSII) light-harvesting proteins in 14 different plant species is presented. The protein separation and identification was achieved by means of reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry. The good correspondence of the molecular masses measured by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry with those deduced from the DNA sequence (0.008%-0.016% relative deviation in Arabidopsis) enabled the identification of the different protein types. Utilizing this correlation, it was possible in several cases to spot a gene product for the previously cloned genes. In PSII, all antenna proteins show hydrophobic properties considerably different within the same as well as among various species, in contrast to observations made previously with PSI. These differences might reflect a tuning of protein-protein interactions that play a role in inducing different supramolecular organizations of PSII: within the same species as a consequence of short-term adaptations, and among species for seasonal species adaptation. The relative antenna stoichiometry was readily established on the basis of relative peak areas of the separated proteins in the ultraviolet chromatograms. The correspondence found between the high copy number of genes with the gene products reveals that the genes are not silent in their protein expression. Moreover, the high copy number of gene products as well as protein heterogeneity observed in PSII suggest a possible plant strategy to realize the high degree of organization and interconnection of the light-harvesting systems under any environmental conditions.

Chromatography, Liquid↗
PubMed2002

Monolithic capillary columns for liquid chromatography-electrospray ionization mass spectrometry in proteomic and genomic research.

Peptides, proteins, single-stranded oligonucleotides, and double-stranded DNA fragments were separated with high resolution in micropellicular, monolithic capillary columns prepared by in situ radical copolymerization of styrene and divinylbenzene. Miniaturized chromatography both in the reversed-phase and the ion-pair reversed-phase mode could be realized in the same capillary column because of the nonpolar character of the poly-(styrene/divinylbenzene) stationary phase. The high chromatographic performance of the monolithic stationary phase facilitated the generation of peak capacities for the biopolymers in the range of 50-140 within 10 min under gradient elution conditions. Employing volatile mobile phase components, separations in the two chromatographic separation modes were on-line hyphenated to electrospray ionization (tandem) mass spectrometry, which yielded intact accurate molecular masses as well as sequence information derived from collision-induced fragmentation. The inaccuracy of mass determination in a quadrupole ion trap mass spectrometer was in the range of 0.01-0.02% for proteins up to a molecular mass of 20000, and 0.02-0.12% for DNA fragments up to a molecular mass of 310000. High-performance liquid chromatography-electrospray ionization mass spectrometry utilizing monolithic capillary columns was applied to the identification of proteins by peptide mass fingerprinting, tandem mass spectrometric sequencing, or intact molecular mass determination, as well as to the accurate sizing of double-stranded DNA fragments ranging in size from 50 to 500 base pairs, and to the detection of sequence variations in DNA fragments amplified by the polymerase chain reaction.

Amino Acid Sequence↗
PubMed2002

Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9.

A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC-ESI-MS a potent tool for the routine characterization of SNPs in forensic science.

Base Sequence↗
PubMed2002

A decade of high-resolution liquid chromatography of nucleic acids on styrene-divinylbenzene copolymers.

The introduction of alkylated, nonporous poly-(styrene-divinylbenzene) microparticles in 1992 enabled the subsequent development of denaturing HPLC that has emerged as the most sensitive screening method for mutations to date. Denaturing HPLC has provided unprecedented insight into human origins and prehistoric migrations, accelerated the cloning of genes involved in mono- and polygenic traits, and facilitated the mutational analysis of more than a hundred candidate genes of human disease. A significant step toward increased sample-throughput and information content was accomplished by the recent introduction of monolithic poly(styrene-divinylbenzene) capillary columns. They have enabled the construction of capillary arrays amenable to multiplex analysis of fluorescent dye-labeled nucleic acids by laser-induced fluorescence detection. Hyphenation of denaturing HPLC with electrospray ionization mass spectrometry, on the other hand, has allowed the direct elucidation of the chemical nature of DNA variation and determination of phase of multiple alleles on a chromosome.

Chromatography, High Pressure Liquid↗
PubMed2002

Hydrophobic, pellicular, monolithic capillary columns based on cross-linked polynorbornene for biopolymer separations.

Monolithic capillary columns were prepared by transition metal-catalyzed ring-opening metathesis copolymerization of norborn-2-ene and 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene inside a silanized 200-microm-i.d. fused-silica capillary using a mixture of toluene and 2-propanol as porogen and Cl2(PCy3)2Ru(=CHPh) as initiator. The synthesized columns allowed the rapid and highly efficient separation of single- and double-stranded nucleic acids by ion-pair reversed-phase high-performance liquid chromatography and of proteins by reversed-phase high-performance liquid chromatography. Compared to 3-mm-i.d. analytical columns synthesized from an identical polymerization mixture, a considerable improvement in the peak widths at half-height of oligonucleotides in the order of 60-80% was obtained. Significant differences in morphology between the capillary column, where the surface of the monolith was rather soft and rugulose, and the analytical column, where the surface was very sharp and smooth, were observed, most probably due to differences in polymerization kinetics. The synthesized monoliths were successfully applied to the separation of the diastereomers of phosphorothioate oligodeoxynucleotides. To confirm the identity of the eluting compounds on the basis of their intact molecular masses, the chromatographic separation system was on-line hyphenated to electrospray ionization mass spectrometry.

Biopolymers↗
PubMed2002

Capillary array high-performance liquid chromatography of nucleic acids and proteins.

An array of monolithic poly(styrene/divinylbenzene) capillaries with individual column thermostats was constructed to demonstrate its utility for the separation of nucleic acids, proteins, and tryptic digests in combination with UV absorbance detection. Because of polymerization-related variation in surface area of monolithic columns, the concentration of acetonitrile required for elution of DNA fragments in denaturing HPLC may vary sufficiently to affect the degree of denaturation. Modulation of column temperature offers a convenient way to harmonize elution profiles among columns. Individual regulation of column temperature also provides the means to determine rapidly in a single parallel run the optimum temperature for resolution of biomolecules. Given the high reproducibility of separations among columns and the ease with which poly(styrene/divinylbenzene)-based stationary phases can be modified to accommodate different modes of chromatography, such arrays will find broad applicability in proteogenomics.

Chromatography, High Pressure Liquid↗
PubMed2002

Micropreparative fractionation of DNA fragments on metathesis-based monoliths: influence of stoichiometry on separation.

Applying Grubbs' first generation benzylidene-type catalyst Cl2Ru(PCy3)2(CHPh) in ring opening metathesis polymerization (ROMP) of norborn-2-ene (NBE) and 1,4,5,8,8a-hexahydro-1,4,5,8, exo, endo-dimethanonapthalene (DMN-H6), various monoliths were prepared within the confines of silanized borosilicate columns (100x3 mm I.D.) and investigated for the micropreparative separation of pBR322 DNA-Hae III restriction fragments ranging in size from 51 to 587 base pairs (bp), as a sample of double-stranded (ds) DNA. The approach to good resolution of dsDNA on monolithic columns entailed the modulation of the polymer morphology in terms of structure and porosity to suit such an analysis. Structural variations were achieved by changing the relative ratios of comonomers (NBE+DMN-H6) at the expense of porogens, and by increasing the DMN-H6 to NBE mass ratio. For dsDNA separations, eluents comprised 0.1 M aqueous triethylammonium acetate, pH 7.0, and acetonitrile. Alternatively, methanol was introduced in this study as a less polar gradient former. In terms of column evaluation, each column prepared was first tested in the separation of 5'-phosphorylated oligodeoxythymidylic acids [p(dT)(12-18)], since good separation of oligodeoxynucleotides indicates the potential liability of the column tested for dsDNA analysis, and vice versa. It was noted that monoliths with combinations of 25:25:40:10, 28:28:35:9, and 30:30:32:8 (as weight% of NBE/DMN-H6/2-propanol/toluene) showed good resolution of p(dT)(12-18). Moreover, they demonstrated good separation of the first 12 fragments (51-267 bp) of the pBR322 DNA-Hae III digest; however, reduced resolution in the separation of the last five highest molecular mass fragments (434-587 bp) was experienced. The best separation of these fragments was accomplished on a 25:25:40:10 NBE/DMN-H6/2-propanol/toluene combination at a flow-rate of 2 ml/min, a temperature of 50 degrees C, and a gradient of 4-10% acetonitrile in 1 min, then 10-16% in 14 min. The total amount of pBR322 HaeIII digest that may be fractionated on these systems is 0.5-2.5 microg.

Chromatography, High Pressure Liquid↗