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  • 1
    Keywords: CANCER ; Germany ; QUANTIFICATION ; TOOL ; RISK ; GENE ; SAMPLE ; SAMPLES ; TUMORS ; PATIENT ; DNA ; RISK-FACTORS ; MUTATION ; risk factors ; leukemia ; DNA methylation ; RISK FACTOR ; DERIVATIVES ; PARAMETERS ; FLUORESCENCE ; CYTOSINE METHYLATION ; CD38 EXPRESSION ; HYPERMETHYLATION ; 5-METHYLCYTOSINE CONTENT ; TUMORIGENESIS ; HEAVY ; chronic lymphocytic leukemia ; MUTATION STATUS ; 5-methylcytosine ; CPG ISLANDS ; capillary electrophoresis - laser induced fluorescence ; CAPILLARY-ELECTROPHORESIS ; GENOMIC HYPOMETHYLATION ; immunoglobulin variable heavy chain gene homology
    Abstract: Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples
    Type of Publication: Journal article published
    PubMed ID: 15188237
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  • 2
    Keywords: EXPRESSION ; IN-VITRO ; AGENTS ; CELL ; Germany ; human ; PATHWAY ; PATHWAYS ; THERAPY ; TOOL ; PROTEIN ; PROTEINS ; DRUG ; METABOLISM ; cell line ; LINES ; MECHANISM ; prognosis ; mechanisms ; BIOLOGY ; CELL-LINES ; treatment ; VARIANTS ; IDENTIFICATION ; resistance ; NUMBER ; SPECTROMETRY ; CELL-LINE ; chemotherapy ; LINE ; MELANOMA ; MASS-SPECTROMETRY ; PHENOTYPE ; MALIGNANT-MELANOMA ; CISPLATIN ; malignant melanoma ; POOR-PROGNOSIS ; drug resistance ; DRUG-RESISTANCE ; MULTIDRUG-RESISTANCE ; HIGH-LEVEL ; PROTEOMICS ; ANTICANCER DRUGS ; ACQUIRED DRUG-RESISTANCE ; ETOPOSIDE ; FOTEMUSTINE ; 2-DIMENSIONAL GEL-ELECTROPHORESIS ; basic pH gradients ; chemoresistance ; HEAT-SHOCK PROTEINS ; IMMOBILIZED PH GRADIENTS ; INCREASED EXPRESSION ; multidrug resistance ; POLYACRYLAMIDE GELS ; PROTEIN IDENTIFICATION ; proteome analysis ; SMALL STRESS-PROTEINS ; two-dimensional electrophoresis
    Abstract: Malignant melanomas have poor prognosis since treatment with anti-neoplastic agents is mostly ineffective. The biological mechanisms of this strong intrinsic therapy resistance are unknown. In order to identify new molecular factors potentially associated with the drug-resistant phenotype of malignant melanoma, a panel of human melanoma cell variants exhibiting low and high levels of resistance to four commonly used anticancer drugs in melanoma treatment, i.e., vindesine, etoposide, cisplatin, and fotemustine, was characterized using proteomic tools (two-dimensional electrophoresis for protein fractionation and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry for protein identification). In the neutral and weak acidic milieu (pH 4.0-8.0) a total number of 14 proteins showed alterations in expression whereas 20 proteins were differentially expressed in the basic milieu (pH 8.0-11.0). Besides proteins with unknown physiologic function, several factors were identified that show chaperone activity. Moreover, proteins involved in drug detoxification, metabolism, and regulation of apoptotic pathways could be identified. The possible role of these proteins in the development of chemoresistance is discussed, although detailed functional tests with these proteins have still to be performed. Nevertheless, it is clear that this proteomic approach for studying chemoresistance phenomena is a prerequisite before further investigation can yield insight into the biology and development of drug resistance in malignant melanoma
    Type of Publication: Journal article published
    PubMed ID: 12874874
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  • 3
    Keywords: PEPTIDE ; Germany ; MODEL ; PROTEINS ; RESOLUTION ; ACID ; DIFFERENCE ; ELECTROSPRAY ; EFFICIENT ; PEPTIDES ; Jun ; STRATEGIES ; ELECTROPHORESIS ; IMMOBILIZED PH GRADIENTS ; CHEMISTRY ; RE ; RESIDUES ; PH ; EXTRACTION ; analysis ; methods ; SEPARATION ; function ; UNIT ; MS/MS ; POINT ; ESTERS ; 1ST-DIMENSION ; IEF ; phosphopeptide enrichment ; SHOTGUN PROTEOMICS
    Abstract: IEF is introduced as a new principle for enrichment and separation of phosphopeptides as obtained after digestion of phosphoproteins by trypsin. Tryptic peptides and phosphopeptides exhibit pI values, which overlap in the range of about 4-6. However, after methyl esterification of all carboxyl functions, the pl values of tryptic peptides and phosphopeptides regroup in discrete dusters. In addition, mono- and diphosphorylated peptides show different but very homogeneous pI values, with variations when internal Arg, Lys, or His residues are present. Experimentally, this new concept was applied for separation of model peptides on IPG strips pH 3-10 as used in the first dimension of 2-DE. After IEF of methyl-esterified peptides, the IPG strip was cut into pieces followed by peptide extraction, desalting and MS analysis by nanoESI-MS. Phosphopeptides were found to focus in good agreement with their calculated pI values. This analytical strategy showed a resolution of about 0.2 pI units, and thus turned out to be capable of detecting minor differences in pI values, such as those occurring between pSer, pThr and pTyr residues. Using IPG strips with a pI range of 3-10, methyl esterified nonphosphorylated tryptic peptides are concentrated in the basic part of the IPG strip or even leave the strip. Thus, efficient enrichment of phosphopeptides and their subfractionation according to p I is obtained in one step. Minor hydrolytic side reactions including deamidation of Asn and partial hydrolysis of methyl esters are observed. The results show that IEF opens attractive avenues for the further advancement of analytical phosphoproteomics
    Type of Publication: Journal article published
    PubMed ID: 17523138
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  • 4
    Keywords: CELLS ; RNA ; DNA ; DNA methylation ; METHYLATION ; 5-methylcytosine ; CAPILLARY-ELECTROPHORESIS ; BODIPY ; INDUCED FLUORESCENCE DETECTION ; CE ; DNA METHYLATION LEVEL ; LIF ; N-6-methyladenine
    Abstract: 2'-Deoxy-N-6-methyladenosine (N(6)mdA) is frequently found in prokaryotic and unicellular eukaryotic genomes. Although methylated bases represent only a minor fraction of the genome, they, however, exhibit strong biological effects. Here, we report a fast and sensitive method for the quantification of global adenine methylation in DNA. The method is based on a recently developed procedure consisting of fluorescence labeling of deoxyribonucleotides with BODIPY FL EDA and analysis by CE with LIF. An oligodeoxy-ribonucleotide site specifically modified with N(6)mdA was used for peak assignment, to establish separation conditions and to determine the LOD. The method yielded a LOD for N(6)mdA of 280 pM (1.4 amol), which is equivalent to similar to 1 N(6)mdA per 10(4) normal nucleotides (0.01%) using 1 mu g of DNA as the matrix. After calibration with completely dam methylated lambda DNA, the assay was applied to the analysis of various DNAs
    Type of Publication: Journal article published
    PubMed ID: 20925053
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  • 5
    Abstract: Strong, sequence-specific gas-phase bindings between proline-rich peptides and alkaline earth metal ions in nanoESI-MS experiments were reported by Lehmann et al. (Rapid Commun. Mass Spectrom. 2006, 20, 2404-2410), however its relevance for physiological-like aqueous phase is uncertain. Therefore, the complexes should also be studied in aqueous solution and the relevance of the MS method for binding studies be evaluated. A mobility shift ACE method was used for determining the binding between the small peptide GAPAGPLIVPY and various metal ions in aqueous solution. The findings were compared to the MS results and further explained using computational methods. While the MS data showed a strong alkaline earth ion binding, the ACE results showed nonsignificant binding. The proposed vacuum state complex also decomposed during a molecular dynamic simulation in aqueous solution. This study shows that the formed stable peptide-metal ion adducts in the gas phase by ESI-MS does not imply the existence of analogous adducts in the aqueous phase. Comparing peptide-metal ion interaction under the gaseous MS and aqueous ACE conditions showed huge difference in binding behavior.
    Type of Publication: Journal article published
    PubMed ID: 26627117
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  • 6
    Keywords: QUANTIFICATION ; PROTEIN ; PROTEINS ; mass spectrometry ; SPECT ; PROTEIN-PHOSPHORYLATION ; fibrinogen ; phosphoproteins ; CAPILLARY LIQUID-CHROMATOGRAPHY ; element mass spectrometry ; inductively coupled plasma ; laser ablation ; phosphorus-31 ; protein phosphorylation
    Abstract: Laser ablation inductively coupled plasma-mass spectrometry (ICP-MS) with P-31 detection has been used for spotting of phosphoproteins after one-dimensional polyacrylamide gel electrophoresis (1-D PAGE) and membrane transfer. By analyzing a mixture of myoglobin, alpha-casein and reduced fibrinogen it is demonstrated that phosphoproteins are specifically recognized by this method. A special washing step was found to be necessary to remove phosphate noncovalently bound to proteins. The P-31 signal was found to contain quantitative information both with respect to relative and absolute amounts of phosphorus present in phosphoproteins. Normalizing the P-31 signal from a single laser ablation trace by the total amount of phosphoprotein applied to the gel, a detection limit of 5 pmol of phosphorus is estimated
    Type of Publication: Journal article published
    PubMed ID: 12707922
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  • 7
    Keywords: APOPTOSIS ; CANCER ; EXPRESSION ; IN-VITRO ; TUMOR-CELLS ; carcinoma ; CELL ; COMBINATION ; Germany ; human ; THERAPY ; VITRO ; PROTEIN ; PROTEINS ; DRUG ; cell line ; LINES ; MECHANISM ; mechanisms ; CELL-LINES ; VARIANTS ; GLUTATHIONE ; resistance ; CELL-LINE ; LINE ; PHENOTYPE ; MULTIDRUG-RESISTANCE ; protein expression ; PROTEOMICS ; HEAT-SHOCK PROTEINS ; POLYACRYLAMIDE GELS ; SMALL STRESS-PROTEINS ; 2-DIMENSIONAL ELECTROPHORESIS ; chemoresistance,mass spectrometry,pancreatic carcinoma,proteome analysis,proteomics,thermoresistance
    Abstract: In order to find candidate proteins that are potentially associated with the thermoresistant phenotype in combination with drug resistance, we analyzed the differential protein expression in vitro in the human pancreatic cancer cell line EPP85-181-P and classical and atypical multidrug-resistant variants and their thermoresistant counterparts using proteomics. This study identifies sets of proteins that may lead to the development of thermoresistance. These results provide a fundamental basis to elucidate the molecular mechanism of thermoresistance and chemoresistance phenomena that may assist the therapy of inoperable cancers
    Type of Publication: Journal article published
    PubMed ID: 14730581
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  • 8
    Keywords: measurement ; CANCER ; CELLS ; tumor ; TUMOR-CELLS ; CELL ; Germany ; QUANTIFICATION ; SAMPLE ; SAMPLES ; DNA ; NEOPLASIA ; ASSAY ; INSTABILITY ; FLUORESCENCE ; isolation ; 5-METHYLCYTOSINE CONTENT ; HYPOMETHYLATION ; CAPILLARY-ELECTROPHORESIS ; cancer,capillary electrophoresis,laser-induced fluorescence,DNA adducts,DNA methylation,5-methylcyto ; POTENTIALS
    Abstract: An analytical method to determine the genome-wide DNA methylation in only 100 ng DNA is presented. The analysis is based on DNA isolation and hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with a fluorescence dye (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyI ethylene diamine hydrochloride, Bodipy FL EDA). The separation of the derivatives was carried out by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. To calculate the methylation level, the derivatization factor and the quantum yields of the Bodipy conjugates of 2'-desoxycytidine-3'-monophosphate (dCMP) and 2'-desoxy-5-methylcytidine-3'-monophosphate (5m-dCMP) were determined by measurement of methylated Lambda DNA. The assignment was made by cochromatography with the synthesized and characterized standard compound 5mdCMP After optimization of the method it was possible to determine the methylation level in 100-ng DNA samples with a standard deviation of less than 5%
    Type of Publication: Journal article published
    PubMed ID: 15004844
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  • 9
    Keywords: Germany ; DNA adducts ; TUMORS ; DNA ; ASSAY ; DNA methylation ; DAMAGE ; NUCLEOTIDES ; DNA-DAMAGE ; Jun ; ADDUCTS ; FLUORESCENCE ; METHYLATION ; HYPOMETHYLATION ; STANDARD ; RE ; OLIGONUCLEOTIDE ; 5-methylcytosine ; DNA damage ; BODIPY ; BODIPY postlabeling ; capillary electrophoresis ; capillary electrophoresis laser-induced fluorescence ; deoxynucleotides ; IMI
    Abstract: We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monorphosphate, 2'-deoxyadenosine-5'monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive, Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation
    Type of Publication: Journal article published
    PubMed ID: 15934055
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  • 10
    Keywords: EXPRESSION ; Germany ; SYSTEM ; SYSTEMS ; DISEASE ; GENE ; GENES ; PROTEIN ; PROTEINS ; RNA ; ACCURACY ; TIME ; DNA ; BIOLOGY ; DISCOVERY ; ACID ; NUCLEIC-ACIDS ; AMPLIFICATION ; NUMBER ; REPRODUCIBILITY ; PCR ; PURIFICATION ; systems biology ; CALIBRATION ; GEL-ELECTROPHORESIS ; CHEMISTRY ; PROTEIN-ANALYSIS ; miniaturization ; high throughput ; HIGH-THROUGHPUT ; multiplex ; SIZE ; TECHNOLOGY ; SEPARATION ; gene-screening ; high-throughput analysis ; lab-on-a-chip ; microfluidics ; protein purification ; RNA quality
    Abstract: On-chip electrophoresis can provide size separations of nucleic acids and proteins similar to more traditional slab gel electrophoresis. Lab-on-a-chip (LoaC) systems utilize on-chip electrophoresis in conjunction with sizing calibration, sensitive detection schemes, and sophisticated data analysis to achieve rapid analysis times (〈 120 s). This work describes the utility of LoaC systems to enable and augment systems biology investigations. RNA quality, as assessed by an RNA integrity number score, is compared to existing quality control (QC) measurements. High-throughput DNA analysis of multiplex PCR samples is used to stratify gene sets for disease discovery. Finally, the applicability of a high-throughput LoaC system for assessing protein purification is demonstrated. The improvements in workflow processes, speed of analysis, data accuracy and reproducibility, and automated data analysis are illustrated
    Type of Publication: Journal article published
    PubMed ID: 16136523
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