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  • 1
    Keywords: CELLS ; GENERATION ; microarray ; MONOCLONAL-ANTIBODY ; MICE ; ANTIGEN ; ANTIGENS ; BINDING ; ALPHA ; antibodies ; antibody ; MOUSE ; IDENTIFICATION ; ASSAY ; microarrays ; FUSION ; MONOCLONAL-ANTIBODIES ; SELECTION ; TARGETS ; IMMUNIZATION ; SINGLE ; LIBRARIES ; CORE ; high throughput ; HIGH-THROUGHPUT ; NEED ; AFFINITY MATURATION ; hybridoma production ; monoclonal antibodies ; monoclonal antibody ; multiplexing
    Abstract: Recent advances in proteornics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification. Monoclonal antibodies were raised to different targets in single batch runs of 6-10wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated monoclonal antibodies against 68 of the targets (85%), within 6 wk of the primary immunization
    Type of Publication: Journal article published
    PubMed ID: 16254927
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  • 2
    Keywords: CELLS ; GROWTH-FACTOR ; BLOOD ; CELL ; Germany ; human ; VIVO ; EXPOSURE ; PROTEIN ; PROTEINS ; COMPLEX ; COMPLEXES ; DONOR ; BIOMARKERS ; BODY-WEIGHT ; INTERVENTION ; BIOLOGY ; MOLECULAR-BIOLOGY ; BREAST-CANCER ; ACID ; GLYCOPROTEIN ; PLASMA ; NUMBER ; STRESS ; MEN ; ATHEROSCLEROSIS ; DIETARY ; BODY ; OXIDATIVE STRESS ; PERIPHERAL-BLOOD ; CONSUMPTION ; LIGNANS ; PROTEOMICS ; METABOLITE ; proteome analysis ; BODIES ; molecular biology ; molecular ; RE ; EX-VIVO ; WEIGHT ; CARDIOVASCULAR-DISEASE ; body weight ; RENAL-DISEASE ; LEVEL ; biomarker ; analysis ; methods ; PLASMA-LEVELS ; ENTEROLACTONE ; COMPOUND ; RISK-FACTOR ; in vivo ; peripheral blood mononuclear cells ; PLASMA ENTEROLACTONE ; TIME-RESOLVED FLUOROIMMUNOASSAY ; SERUM ENTEROLACTONE ; STEADY-STATE ; GLYCOPROTEIN-IIB/IIIA ; HYPERCHOLESTEROLEMIC ATHEROSCLEROSIS ; INTESTINAL MICROFLORA
    Abstract: Flaxseed is one of the richest sources of lignans that are converted to enterolactone by the intestinal microflora. Enterolactone has been suggested to be the prime active compound mediating atherosclerosis-protective effects that were shown for flaxseed. The effects of a 1-wk intervention with 0.4 g of flaxseed/kg body weight per day on enterolactone plasma levels in seven healthy men revealed that all participants (PAs) responded with enhanced enterolactone plasma levels. Proteome analysis of peripheral blood mononuclear cells (PBMC) from donors before, during, and after the intervention showed that flaxseed consumption affected significantly the steady-state levels of 16 proteins of which four were altered in a similar manner when blood mononuclear cells were exposed ex vivo to enterolactone. Enhanced levels of peroxiredoxin and reduced levels of the long-chain fatty acid beta-oxidation multienzyme complex may be taken as indicators of a reduced oxidative stress whereas reduced levels of glycoprotein IIIa/II could indicate improved protection from thrombotic and inflammatory processes. In conclusion, the blood mononuclear cell proteome responds to dietary flaxseed intake with changes in a number of atherosclerosis-relevant proteins that may be taken as biomarkers of exposure and some of these changes observed can be attributed to the action of the lignan metabolite enterolactone
    Type of Publication: Journal article published
    PubMed ID: 17708591
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  • 3
    Keywords: 2-DE singular DY-maleimide dye protein labeling, 2-DIMENSIONAL ELECTROPHORESIS, BIOLOGY, COMPLEX, CO
    Abstract: Sensitive differential proteomic analysis is challenging and often limited by distinct labeling or tagging strategies. In this study, we have examined the sensitivity, linearity, and photophysical properties of novel protein labeling DY-maleimide dyes (DY-505-MAL, DY-555-MAL and DY-635-MAL). All MS compatible DY-maleimide dyes exhibited excellent emission spectra, high sensitivity, and high linearity, when applied to standard 1-DE protein analysis. Correspondingly, 2-DE analysis of DY-635-MAL or DY-505-MAL maximal-labeled human keratinocyte proteins displayed remarkably high sensitivity. Compared with a standard fluorescent protein stain, DY-635-MAL or DY-505-MAL 2-DE analysis demonstrated equally high spot quality with an overall increase in the number of spots detectable (up to threefold higher; 〉1000 spots/gel). However, as determined with a FLA-5100 imaging system, comparative MultiGauge, and Delta2D analysis, not all DY-maleimide dyes possessed DIGE compatible fluorescent emission properties. However, DY-505-MAL and DY-635-MAL were found to be suitable for more complex, time and gel intensive, focused multiplexing analyses. Notably - as demonstrated with allergen-stimulated human skin proteins - defined, singular DY-maleimide dye protein labeling (SDPL) allows high quality, time saving, simple, and reliable differential proteomic examination
    Type of Publication: Journal article published
    PubMed ID: 19693804
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  • 4
    Keywords: EXPRESSION ; Germany ; QUANTIFICATION ; PROTEIN ; PROTEINS ; BIOLOGY ; ACID ; TARGET ; PEPTIDES ; PROTEOMICS ; MS ; methods ; TECHNOLOGY ; FRAGMENT ; MIXTURES ; QUANTITATIVE PROTEOMICS ; STRATEGY ; TIMES ; SILAC ; Type ; ACCURATE QUANTITATION ; MIPA ; Sequence permutation
    Abstract: A novel type of isobaric internal peptide standard for quantitative proteomics is described. The standard is a synthetic peptide derived from the target peptide by positional permutation of two amino acids. This type of internal standard is denominated minimally permutated peptide analog (MIPA). MIPA can be differentiated from their target analytes by LC-MS due to individual retention times and/or by MS/MS due to specific fragment ions. Both quantification methods are demonstrated using peptide mixtures of low and high complexity
    Type of Publication: Journal article published
    PubMed ID: 20104620
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  • 5
    Keywords: KINASE ; CATALYTIC SUBUNIT ; PEPTIDES ; SELECTIVE DETECTION ; phosphoproteins ; CAPILLARY LIQUID-CHROMATOGRAPHY ; EXTRACTION ; PRECURSOR ; PHOSPHOPEPTIDE ANALYSIS ; PHOSPHATASES
    Abstract: Using the combination of in-gel elastase digestion, immobilized metal affinity chromatography and high resolution electrospray tandem mass spectrometry, the phosphorylation sites of two phosphoproteins were determined. Complete coverage of all phosphorylation sites (Ser10, Ser139, Thr197, Ser338) of the model phosphoprotein protein kinase A C(alpha)-subunit could be achieved by this strategy in the low picomole range. In addition, three previously unknown phosphorylation sites of the human transcription initiation factor TIF-IA (Ser44, Ser170, Ser172) were determined in this way. Both phosphoproteins could be identified in a protein database on the basis of their elastase generated phosphopeptides alone. The data of seven phosphopeptides were used for identification of protein kinase A, and those of two phosphopeptides for TIF-IA, respectively. The accurate mass data of the electrospray mass spectra recorded at high resolution are extremely useful for sequencing of the elastase generated phosphopeptides and for protein identification by database searching.
    Type of Publication: Journal article published
    PubMed ID: 12124936
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  • 6
    Abstract: PURPOSE: The majority of gastric cancers are diagnosed at advanced stages, characterized by robust therapy resistance. The oncoprotein hypoxia-inducible factor 1 (HIF-1) is associated with therapy resistance, partly via activation of the DNA damage response. We have noted a robust ability of gastric cancer cells to functionally compensate the loss of HIF-1 in vitro. The purpose of this study was to identify molecular pathways that underlie this compensation. EXPERIMENTAL DESIGN: We performed 2DE to compare the nuclear proteome of wild-type and HIF-1-deficient gastric cancer cells. Differently expressed protein spots were identified via MS). After bioinformatic evaluation, functional validation of selected identified pathways was performed. RESULTS: 2DE displayed a total of 2523 protein spots, from which 87 were identified as regulated by HIF-1. Seventy of the identified spots were different proteins and 17 were isoforms. Bioinformatic analyses revealed that a significant amount of the identified proteins were related to cellular survival pathways. Specifically, members of the proteasome pathway were found upregulated upon loss of HIF-1. Combined inhibition of HIF-1 and the proteasome inflicted significant DNA damage, supporting the hypothesis that the proteasome is of functional importance to compensate the loss of HIF-1. CONCLUSIONS AND CLINICAL RELEVANCE: Our data show robust and functional changes of the nuclear proteome upon inactivation of the HIF-1 oncoprotein in gastric cancer cells. We propose that 2DE-MS represents a useful tool to functionally dissect resistance mechanisms to targeted therapy and to identify novel targets for antiproliferative combination therapy.
    Type of Publication: Journal article published
    PubMed ID: 24307263
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  • 7
    Abstract: The slowing down or stalling of replication forks is commonly known as replication stress and arises from multiple causes such as DNA lesions, nucleotide depletion, RNA-DNA hybrids, and oncogene activation. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancers that exhibit high levels of replication stress. However, the cellular signaling induced by replication stress and the substrate spectrum of ATR has not been systematically investigated. In this study, we employed quantitative MS-based proteomics to define the cellular signaling after nucleotide depletion-induced replication stress and replication fork collapse following ATR inhibition. We demonstrate that replication stress results in increased phosphorylation of a subset of proteins, many of which are involved in RNA splicing and transcription and have previously not been associated with the cellular replication stress response. Furthermore, our data reveal the ATR-dependent phosphorylation following replication stress and discover novel putative ATR target sites on MCM6, TOPBP1, RAD51AP1, and PSMD4. We establish that ATR inhibition rewires cellular signaling networks induced by replication stress and leads to the activation of the ATM-driven double-strand break repair signaling.
    Type of Publication: Journal article published
    PubMed ID: 26572502
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  • 8
    Abstract: Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe-IMAC column chromatography and subjected to LC-MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI-TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC-MS/MS systems was comparable to that of using alternative proteases such as Asp-N, Arg-C, chymotrypsin, Glu-C and Lys-C on just one LC-MS/MS instrument. Notably, MALDI-TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites ( approximately 20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC-MALDI MS/MS can be a useful complement to LC-nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides.
    Type of Publication: Journal article published
    PubMed ID: 26990019
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  • 9
    Keywords: PROTEINS ; PHOSPHORYLATION ; PERFORMANCE ; MASS-SPECTROMETRY ; ABSOLUTE QUANTIFICATION ; PROTON AFFINITIES
    Abstract: A novel type of peptide standard is introduced that consists of two peptides combined in one synthetic molecule and separated by a proteolytic cleavage site. Upon enzymatic digestion, the two peptides are released in a molar one-to-one ratio. This method enables the generation of exact equimolar mixtures of two peptides of any nature and origin, thereby providing a valuable tool for the investigation of fundamental phenomena in MS. The applicability of the method is exemplified by the analysis of the effect of peptide sequence variations on the relative ionization efficiency in ESI- and MALDI-MS.
    Type of Publication: Journal article published
    PubMed ID: 23090848
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  • 10
    Keywords: EXPRESSION ; evaluation ; Germany ; SUPPORT ; SYSTEM ; SYSTEMS ; DISEASE ; microarray ; PROTEIN ; STORAGE ; COMPLEX ; COMPLEXES ; MESSENGER-RNA ; IMPACT ; ANTIGEN ; ANTIGENS ; DISCOVERY ; antibodies ; antibody ; microarrays ; CANCER-CELLS ; SURFACE ; STABILITY ; FUNCTIONAL GENOMICS ; IMMOBILIZATION ; cross-linker ; glass slide ; IMMUNOASSAYS ; LINKED-IMMUNOSORBENT-ASSAY ; PROTEOMICS ; surface modification
    Abstract: Antibody microarrays could have an enormous impact on the functional analysis of cellular activity and regulation, especially at the level of protein expression and protein- protein interaction, and might become an invaluable tool in disease diagnostics. The array surface is bound to have a tremendous influence on the findings from such studies. Apart from the basic issue of how to attach antibodies optimally without affecting their function, it is also important that the cognate antigens, applied within a complex protein mixture, all bind to the arrayed antibodies irrespective of their enormous variety in structure. In this study, various factors in the production of antibody microarrays on glass support were analysed: the modification of the glass surface; kind and length of cross-linkers; composition and pH of the spotting buffer; blocking reagents; antibody concentration and storage procedures, in order to evaluate their effect on array performance. Altogether, data from more than 700 individual array experiments were taken into account. In addition to home- made slides, commercially available systems were also included in the analysis
    Type of Publication: Journal article published
    PubMed ID: 12627378
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