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Biomedical subjects

Christian G Huber

Publications and source records attributed to Christian G Huber.

At least 19 recordsLinked to original sources
PubMed2007

Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS.

A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 microg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis.

Amino Acid Sequence↗
PubMed2006

Capillary scale monolithic trap column for desalting and preconcentration of peptides and proteins in one- and two-dimensional separations.

Monolithic columns based on poly-(styrene-divinylbenzene) (PS-DVB) were utilized both for preconcentration (in 10 mm x 0.20 mm I.D. format) and analytical separation (in 60 mm x 0.20 and 0.10 mm I.D. format) of peptides and proteins in column switching micro-scale high-performance liquid chromatography. A special holder for short monolithic preconcentration columns was designed and pressure durability tests approved long-term stability up to 400 bar. An 11-20% decrease in the average peak widths of nine peptides was obtained upon combining a preconcentration column with an analytical column as compared with a setup using an analytical column only. Trapping efficiency, especially for small and hydrophilic peptides, was optimized by using 0.10% heptafluorobutyric acid instead of 0.050% trifluoroacetic acid as solvent additive during sample loading. Using a 10 mm x 0.20 mm I.D. preconcentration column, loadabilities between 0.5 and 1.6 microg were determined by frontal analysis of proteins and bioactive peptides, respectively. A 100-fold concentration followed by direct on-line intact mass determination is demonstrated for diluted (3 micromolL(-1)) protein solutions. The applicability of the monolithic preconcentration column for multidimensional chromatography was tested by off-line two-dimensional separation, combining strong cation-exchange chromatography and ion-pair reversed-phase chromatography. Peptide identification data from digested protein mixtures demonstrated reproducibilities of 46-75% in triplicate analyses, and confident peptide identifications of low abundant peptides even in the presence of a 650-fold molar excess of high abundant peptides.

Analytic Sample Preparation Methods↗
PubMed2006

Monolithic column HPLC separation of intact proteins analyzed by LC-MALDI using on-plate digestion: An approach to integrate protein separation and identification.

A method is developed to integrate a protein separation by monolithic capillary reversed-phase high-performance liquid chromatography to on-probe tryptic digestion for subsequent analyses by MALDI-TOF MS and MALDI-TOF/TOF MS. The method provides a means of directly interfacing separations to MALDI-MS, reducing the amount of time required for traditional procedures involving in-solution enzymatic digestion and sample cleanup prior to MALDI-MS analysis. When used with pI-based fractionation as a first dimension, it provides a means of analyzing complex mixtures of proteins with minimal sample handling and cleanup. The use of monolithic capillary columns sufficiently resolved intact proteins so that peptide mass fingerprinting analysis by MALDI-TOF MS resulted in the identification of close to 40 unique proteins from 120 ng of sample obtained from a prefractionated MCF10 cell line at pH 6.34, where the identifications of several of these proteins were also confirmed by intact MW and tandem mass spectrometric analysis. The reproducibility of this method has been demonstrated to be sufficient for the purpose of protein identifications. Experimental values of protein intact MW are obtained and compared to that expected for each protein identified.

Amino Acid Sequence↗
PubMed2006

Accurate determination of allelic frequencies in mitochondrial DNA mixtures by electrospray ionization time-of-flight mass spectrometry.

The mitochondrial locus 16519T/C was used as a model for the evaluation of the benefits of ion-pair reversed-phase high-performance liquid chromatography on-line hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS assay) for the determination of allelic frequencies of single nucleotide polymorphisms. This marker has gained interest in forensic science owing to its ability to increase the discrimination power of mitochondrial DNA testing as a consequence of its high variability across various populations. In a first set of experiments, artificial mitochondrial DNA mixtures prepared from all four theoretically possible 16519 alleles served as samples. Any allele occurring at a frequency of as low as 1-5% was unequivocally detectable irrespective of the kind of allelic mixture. Measured and expected allelic frequencies correlated well following correction of observed experimental bias, which was most probably attributable to differential PCR amplification and/or preferential ionization. For thirteen different T/C mixtures with C contents in the range 1.0-99.0%, an average error of 1.2% and a maximum error of 2.2% were observed. Furthermore, ICEMS was applied to the quantitative genotyping of eight selected individuals of which four were heteroplasmic with C contents in the range 1.9-34.1%. To check the reliability of these results, allelic proportions were additionally determined by a cloning assay. The results of the two assays correlated well (R (2)=0.9971). In all cases, deviations were obtained that were smaller than 5.4%. The overall observed assay performance suggests that the described mass spectrometric technique represents one of the most powerful assays for the determination of allelic frequencies available today.

DNA, Mitochondrial↗
PubMed2006

Glycosylation patterns of human chorionic gonadotropin revealed by liquid chromatography-mass spectrometry and bioinformatics.

Due to their extensive structural heterogeneity, the elucidation of glycosylation patterns in glycoproteins such as the subunits of human chorionic gonadotropin (hCG), hCG-alpha, and hCG-beta, remains one of the most challenging problems in the proteomic analysis of post-translational modifications. In consequence, glycosylation is usually studied after decomposition of the intact proteins to the proteolytic peptide level. However, by this approach all information about the combination of the different glycopeptides in the intact protein is lost. In this study we have, therefore, attempted to combine the results of glycan identification after tryptic digestion with molecular mass measurements on the native starting material of the new first WHO Reference Reagents (RR) for hCG-alpha (99/720) and hCG-beta (99/650). Despite the extremely high number of possible combinations of the glycans identified in the tryptic peptides by HPLC-MS (>1000 for hCG-alpha and >10 000 for hCG-beta), the mass spectra of intact hCG-alpha and hCG-beta revealed only a limited number of glycoforms present in hCG preparations from pools of pregnancy urines. Peak annotations for hCG-alpha were performed with the help of a bioinformatic algorithm that generated a database containing all possible modifications of the proteins, including modifications possibly introduced during sample preparation such as oxidation or truncation, for subsequent searches for combinations fitting the mass difference between the polypeptide backbone and the measured molecular masses. Fourteen different glycoforms of hCG-alpha, containing biantennary, partly sialylized hybrid-type glycans, including methionine-oxidized and N-terminally truncated forms, were identified. Mass spectra of high quality were also obtained for hCG-beta, however, a database search mass accuracy of +/-5 Da was insufficient to unambiguously assign the possible combinations of post-translational modifications. In summary, mass spectrometric fingerprints of intact molecules were shown to be highly useful for the characterization of glycosylation patterns of different hCG preparations such as the new first WHO RR for immunoassays and could be the first step in establishing biophysical reference methods for hCG and related molecules.

Chorionic Gonadotropin↗
PubMed2006

Automated integration of monolith-based protein separation with on-plate digestion for mass spectrometric analysis of esophageal adenocarcinoma human epithelial samples.

A unique approach of automating the integration of monolithic capillary HPLC-based protein separation and on-plate digestion for subsequent MALDI-MS analysis has been developed. All liquid-handling procedures were performed using a robotic module. This automated high-throughput method minimizes the amount of time and extensive labor required for traditional in-solution digestion followed by exhaustive sample cleanup and analysis. Also, precise positioning of the droplet from the capillary HPLC separation onto the MALDI plate allows for preconcentration effects of analytes for improved sensitivity. Proteins from primary esophageal Barrett's adenocarcinoma tissue were prefractionated by chromatofocusing and analyzed successfully by this automated configuration, obtaining rapid protein identifications through PMF and sequencing analyses with high sequence coverage. Additionally, intact protein molecular weight values were obtained as a means to further confirm protein identification and also to identify potential sequence modifications of proteins. This simple and rapid method is a highly versatile and robust approach for the analysis of complex proteomes.

Adenocarcinoma↗
PubMed2006

Absolute myoglobin quantitation in serum by combining two-dimensional liquid chromatography-electrospray ionization mass spectrometry and novel data analysis algorithms.

To measure myoglobin, a marker for myocardial infarction, directly in human serum, two-dimensional liquid chromatography in combination with electrospray ionization mass spectrometry was applied as an analytical method. High-abundant serum proteins were depleted by strong anion-exchange chromatography. The myoglobin fraction was digested and injected onto a 60 mm x 0.2 mm i.d. monolithic capillary column for quantitation of selected peptides upon mass spectrometric detection. The addition of known amounts of myoglobin to the serum sample was utilized for calibration, and horse myoglobin was added as an internal standard to improve reproducibility. Calibration graphs were linear and facilitated the reproducible and accurate determination of the myoglobin amount present in serum. Manual data evaluation using integrated peak areas and an automated multistage algorithm fitting two-dimensional models of peptide elution profiles and isotope patterns to the mass spectrometric raw data were compared. When the automated method was applied, a myoglobin concentration of 460 pg/microL serum was determined with a maximum relative deviation from the theoretical value of 10.1% and a maximum relative standard deviation of 13.4%.

Algorithms↗
PubMed2006

Proteome analysis of Myxococcus xanthus by off-line two-dimensional chromatographic separation using monolithic poly-(styrene-divinylbenzene) columns combined with ion-trap tandem mass spectrometry.

Myxobacteria are potent producers of secondary metabolites exhibiting diverse biological activities and pharmacological potential. The proteome of Myxococcus xanthus DK1622 was characterized by two-dimensional chromatographic separation of tryptic peptides from a lysate followed by tandem mass spectrometric identification. The high degree of orthogonality of the separation system employing polymer-based strong cation-exchange and monolithic reversed-phase stationary phases was clearly demonstrated. Upon automated database searching, 1312 unique peptides were identified, which were associated with 631 unique proteins. High-molecular polyketide synthetases and nonribosomal peptide synthetases, known to be involved in the biosynthesis of various secondary metabolites, were readily detected. Besides the identification of gene products associated with the production of known secondary metabolites, proteins could also be identified for six gene clusters, for which no biosynthetic product has been known so far.

Amino Acid Sequence↗
PubMed2005

Identification of bacteria by polymerase chain reaction followed by liquid chromatography-mass spectrometry.

Bloodstream infections are an important cause of serious morbidity and mortality. Rapid detection and identification of specific pathogens from blood or other clinical specimens could improve the rational use of antimicrobial therapy in clinical medicine and have a great impact on the outcome of patients with systemic infections. Polymerase chain reaction using generic primers was used to amplify genomic DNA of different bacterial strains. The identification was accomplished by measuring the molecular masses of the PCR products using ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization mass spectrometry. DNA from 10 bacterial species was amplified by PCR, and the resulting amplification products were analyzed. In all cases, the measured molecular masses of the PCR products matched the theoretical value for the species-specific DNA sequence. However, three pairs of bacteria could not be distinguished since the theoretical difference in amplicon molecular mass was < 1.0 Da (different sequence, same base composition of amplicon). Determination of intra- and interday mass reproducibility resulted in relative standard deviations of 0.0030 and 0.018%, respectively. The limit of detection of the presented method was shown to be 0.5 genome equivalents/PCR. The specificity of the method in a human background was successfully tested by amplifying and analyzing 1000-10000 genome equivalents of Staphylococcus aureus spiked into human plasma.

Bacteria↗
PubMed2005

Separation and detection of phosphorylated and nonphosphorylated peptides in liquid chromatography-mass spectrometry using monolithic columns and acidic or alkaline mobile phases.

Efficient chromatographic separation is a prerequisite for the sensitive analysis of complex peptide mixtures using liquid chromatography-mass spectrometry. This is especially true for the analysis of mixtures of unmodified and posttranslationally modified peptides, for example, phosphorylated peptides in the presence of their unmodified analogues. Applying monolithic capillary columns based on poly(styrene/divinylbenzene), the influence of acidic eluents based on trifluoroacetic and heptafluorobutyric acid as well as an alkaline eluent based on triethylamine-acetic acid (pH 9.2) on the separation of synthetic phosphopeptides was evaluated. Heptafluorobutyric acid offered the longest retention times and highest selectivities and, hence, the most effective separation. Application of the alkaline eluent in conjunction with detection in negative ion mode electrospray ionization mass spectrometry, on the other hand, allowed the detection of phosphorylated peptides with significantly lower limits of detection, as compared to acidic eluents in combination with detection in positive ion mode. Pairs of phosphorylated and nonphosphorylated synthetic peptides, ranging from 7- to 16-mers, as well as phosphorylated peptides form a tryptic protein digest could be separated both at acidic and alkaline pH. Utilizing a 60 x 0.20-mm-i.d. capillary column, the limit of detection in negative ion detection mode for a 4-fold phosphorylated peptide in a beta-casein digest was 10 fmol. Together with the capability for fast separation of protein digests, monolithic columns, thus, facilitate the effective and sensitive analysis of this important posttranslational modification.

Chromatography, Liquid↗
PubMed2005

Monolithic capillary-based ion-selective electrodes.

Poly(styrene-co-divinylbenzene)-based monolithic capillaries of an inner diameter of 200 mum and a length of 2-5 mm have been used to construct Ca2+-, Ag+-, and Na+-selective electrodes. The membranes consist of a solution of ionophore and ion exchanger in bis(2-ethylhexyl) sebacate or 2-nitrophenyl octyl ether, which are used as plasticizers in conventional PVC-based membranes. With capillaries of low porosity, the potentiometric responses down to 10(-8)-10(-9) M solutions do not depend on the composition of the internal solution, which indicates a strong suppression of transmembrane ion fluxes. Thus, no tedious optimization of the inner solution is required with monolith ISEs. The lower detection limits of Ag+- and Ca2+-ISEs are comparable to the best ones obtained earlier with optimized inner solutions. Additionally, a monolithic Na+-selective ISE has been obtained exhibiting a lower detection limit of 3 x 10(-8) M Na+. With monolithic capillaries of higher porosity and fused-silica GC capillaries, the transmembrane flux effects are noticeable but still significantly smaller than with conventional PVC membranes.

Calcium↗
PubMed2005

Separation, detection, and identification of peptides by ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry at high and low pH.

Bioactive peptides and tryptic digests of various proteins were separated under acidic and alkaline conditions by ion-pair-reversed-phase high-performance liquid chromatography (RP-HPIPC) in 200 microm I.D. monolithic, poly(styrene-divinylbenzene)-based capillary columns using gradients of acetonitrile in 0.050% aqueous trifluoroacetic acid, pH 2.1, or 1.0% triethylamine-acetic acid, pH 10.6. Chromatographic performances with mobile phases of low and high-pH were practically equivalent and facilitated the separation of more than 50 tryptic peptides of bovine serum albumin within 15-20 min with peak widths at half height between 4 and 10 s. Neither a significant change in retentivity nor efficiency of the monolithic column was observed during 17-day operation at pH 10.6 and 50 degrees C. Upon separation by RP-HPIPC at high-pH, peptide detectabilities in full-scan negative-ion electrospray ionization mass spectrometry (negESI-MS) were about two to three times lower as compared to RP-HPIPC at low-pH with posESI-MS detection. Tandem mass spectra obtained by fragmentation of deprotonated peptide ions in negative ion mode yielded interpretable sequence information only in a few cases of relatively short peptides. However, in order to obtain sequence information for peptides separated with alkaline mobile phases, tandem mass spectrometry (MS/MS) could be performed in positive ion mode. The chromatographic selectivities were significantly different in separations performed with acidic and alkaline eluents, which facilitated the fractionation of a complex peptide mixture obtained by the tryptic digestion of 10 proteins utilizing off-line, two-dimensional RP-HPIPC at high pH x RP-HPIPC at low pH and subsequent on-line identification by posESI-MS/MS.

Chromatography, High Pressure Liquid↗
PubMed2005

Analysis of biological and synthetic ribonucleic acids by liquid chromatography-mass spectrometry using monolithic capillary columns.

Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) has been evaluated as a method for the fractionation and desalting of ribonucleic acids prior to their characterization by electrospray ionization mass spectrometry. Monolithic, poly(styrene-divinylbenzene)-based capillary columns allowed the rapid and highly efficient fractionation of both synthetic and biological ribonucleic acids. The common problem of gas-phase cation adduction that is particularly prevalent in the mass spectrometric analysis of ribonucleic acids was tackled through a combination of chromatographic purification and the addition of ethylenediaminetetraacetic acid to the sample at a concentration of 25 mmol/L shortly before on-line analysis. For RNA molecules ranging in size from 10 to 120 nucleotides, the mass accuracies were typically better than 0.02%, which allowed the characterization and identification of failure sequences and byproducts with high confidence. Following injection of a 500 nL sample onto a 60 x 0.2 mm column, the limit of detection for a 120-nucleotide ribosomal RNA transcript from Escherichia coli was in the 50-80 fmol range. The method was applied to the analysis of synthetic oligoribonucleotides, transfer RNAs, and ribosomal RNA. Finally, sequence information was derived for low picomole amounts of a 32-mer RNA upon chromatographic purification and tandem mass spectrometric investigation in an ion trap mass spectrometer. Complete series of fragment ions of the c- and y-types could be assigned in the tandem mass spectrum. In conclusion, IP-RP-HPLC using monolithic capillary columns represents a very useful tool for the structural investigation and quantitative determination of RNAs of synthetic and biological origin.

Chelating Agents↗
PubMed2005

Comparing monolithic and microparticular capillary columns for the separation and analysis of peptide mixtures by liquid chromatography-mass spectrometry.

A mixture of ten proteins was trypsinized and injected onto poly-(styrene-divinylben-zene) monolithic columns (60 x 0.20 or 0.10 mm ID) and a column packed with C18 silica particles (75 x 0.075 mm ID), respectively. The columns were eluted at 200-2000 nL/min with gradients of ACN in 0.050% TFA. Eluting peptides were detected by ESI-MS/MS and subsequently identified by database searching. The 100 microm ID monolithic column showed the highest cumulative Mowse scores based on the highest ion scores for the peptides and the largest number of identified peptides. It is shown that the number of identified peptides strongly depends on the dynamic range within the peptide mixture. In consequence, all proteins were identified in a mixture of relatively balanced analyte amounts (12.5-80 fmol) whereas only peptides for six out of ten proteins were found in a sample of high-dynamic range (0.65-270 fmol). The 100 microm monolithic column showed the highest reproducibility for peptide identifications in three consecutive runs. Depending on sample amount, 57-72% of the identified peptides were detectable in each of the three runs of triplicate analyses. The results demonstrate the high suitability of 100 microm monolithic columns for high-resolution peptide separations in proteomic research.

Amino Acid Sequence↗
PubMed2004

Operational variables in high-performance liquid chromatography-electrospray ionization mass spectrometry of peptides and proteins using poly(styrene-divinylbenzene) monoliths.

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) utilizing monolithic poly(styrene-divinylbenzene) columns was optimized for the coupling to electrospray ionization mass spectrometry (ESI-MS) by the application of various temperatures and mobile phase additives during peptide and protein analysis. Peak widths at half height improved significantly upon increasing the temperature and ranged from 2.0 to 5.4 s for peptide and protein separations at 70 degrees. Selectivity of peptide elution was significantly modulated by temperature, whereas the effect on proteins was only minor. A comparison of 0.10% formic acid (FA), 0.050% trifluoroacetic acid (TFA), and 0.050% heptafluorobutyric acid (HFBA) as mobile phase additives revealed that highest chromatographic efficiency but poorest mass spectrometric detectabilities were achieved with HFBA. Clusters of HFBA, water, and acetonitrile were observed in the mass spectra at m/z values >500. Although the signal-to-noise ratios for the individual peptides diverged considerably both in the selected ion chromatograms and extracted mass spectra, the average mass spectrometric detectabilities varied only by a factor of less than 1.7 measured with the different additives. Limits of detection for peptides with 500 nl sample volumes injected onto a 60 mm x 0.20 mm monolithic column were in the 0.2-13 fmol range. In the analysis of hydrophobic membrane proteins, HFBA enabled highest separation selectivity at the cost of lower mass spectral quality. The use of 0.050% TFA as mobile phase additive turned out to be the best compromise between chromatographic and mass spectrometric performance in the analysis of peptides and proteins by RP-HPLC-ESI-MS using monolithic separation columns.

Chromatography, High Pressure Liquid↗
PubMed2004

Separation and identification of the light harvesting proteins contained in grana and stroma thylakoid membrane fractions.

This paper presents the results of a study performed to develop a rapid and straightforward method to resolve and simultaneously identify the light-harvesting proteins of photosystem I (LHCI) and photosystem II (LHCII) present in the grana and stroma of the thylakoid membranes of higher plants. These hydrophobic proteins are embedded in the phospholipid membrane, and their extraction usually requires detergent and time consuming manipulations that may introduce artifacts. The method presented here makes use of digitonin, a detergent which causes rapid (within less than 3 min) cleavage of the thylakoid membrane into two subfractions: appressed (grana) and non-appressed (stroma) membranes, the former enriched in photosystem II and the latter containing mainly photosystem I. From these two fractions identification of the protein components was performed by separating them by reversed-phase high-performance liquid chromatography (RP-HPLC) and determining the intact molecular mass by electrospray ionization mass spectrometry (ESI-MS). By this strategy the ion suppression during ESI-MS that normally occurs in the presence of membrane phospholipids was avoided, since RP-HPLC removed most phospholipids from the analytes. Consequently, high quality mass spectra were extracted from the reconstructed ion chromatograms. The specific cleavage of thylakoid membranes by digitonin, as well as the rapid identification and quantification of the antenna composition of the two complexes facilitate future studies of the lateral migration of the chlorophyll-protein complexes along thylakoid membranes, which is well known to be induced by high intensity light or other environmental stresses. Such investigations could not be performed by sodium dodecylsulfate-polyacrylamide gel electrophoresis because of insufficient resolution of the proteins having molecular masses between 22,000 and 25,000.

Chromatography, High Pressure Liquid↗