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  • 1
    Keywords: CELLS ; EXPRESSION ; carcinoma ; CELL ; Germany ; human ; GENE ; GENE-EXPRESSION ; GENES ; PROTEIN ; PROTEINS ; MOLECULES ; COMPLEX ; COMPLEXES ; KERATINOCYTES ; BOVINE PAPILLOMAVIRUS ; DOWN-REGULATION ; GROWTH-FACTOR RECEPTOR ; papillomavirus ; MOLECULE ; MHC ; TRANSPORT ; IDENTIFICATION ; LESIONS ; gene expression ; DESIGN ; DIFFERENCE ; PLASMA ; MEMBRANE ; STRESS ; SPECTROMETRY ; human papillomavirus ; TYPE-16 ; MASS-SPECTROMETRY ; SURFACE ; CLASS-I ; EPITHELIAL-CELL LINE ; GOLGI-APPARATUS ; HUMAN-PAPILLOMAVIRUS ; MHC class I ; MHC CLASS-I ; CARCINOMAS ; DIFFERENTIAL EXPRESSION ; MEMBRANE PROTEIN ; HaCaT ; MEMBRANE-PROTEIN ; GEL-ELECTROPHORESIS ; MASSES ; E5 PROTEIN ; CERVICAL NEOPLASIA
    Abstract: Membrane proteins differentially expressed in human papillomavirus type 16 (HPV-16) E5-transfected HaCaT cells have been identified. Membrane proteins were isolated and separated by two-dimensional gel electrophoresis. Spots showing quantitative differences between E5-transfected and control cells were extracted and the proteins were identified by nanoelectrospray ionization mass spectrometry. A total of 24 spots was analysed. Among the proteins showing differential expression, a decreased amount of calnexin and increased expression of hsp70, proteins both involved in maturation and transport of MHC class I complexes to the plasma membrane, were noticed. These findings correlate with the decreased surface expression of MHC class I molecules described in E5-expressing cells, HPV-positive cervical lesions and cervical carcinomas. These results stress the value of the proteomic approach, as used here in the experimental design, which allows the correlation of changes in host gene expression with biological functions of viral genes
    Type of Publication: Journal article published
    PubMed ID: 15166425
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  • 2
    Keywords: CANCER ; KINASE ; SITE ; SITES ; PROTEIN ; ACTIVATION ; DNA ; MECHANISM ; INDUCTION ; mechanisms ; PHOSPHORYLATION ; PROTEIN-KINASE ; LESIONS ; STRESS ; p53 ; DAMAGE ; SIGNALING PATHWAYS ; DNA-DAMAGE ; DEGRADATION ; MDM2 ; DOUBLE-STRAND BREAKS ; ATAXIA-TELANGIECTASIA ; signaling ; RE ; DNA damage ; KINASES ; ATM ; DNA damage response ; protein degradation
    Abstract: Maintenance of genomic stability depends on the DNA damage response, an extensive signaling network that is activated by DNA lesions such as double-strand breaks (DSBs). The primary activator of the mammalian DSB response is the nuclear protein kinase ataxia-telangiectasia, mutated (ATM), which phosphorylates key players in various arms of this network. The activation and stabilization of the p53 protein play a major role in the DNA damage response and are mediated by ATM-dependent posttranslational modifications of p53 and Mdm2, a ubiquitin ligase of p53. p53's response to DNA damage also depends on Mdm2-dependent proteolysis of Mdmx, a homologue of Mdm2 that represses p53's transactivation function. Here we show that efficient damage-induced degradation of human Hdmx depends on functional ATM and at least three sites on the Hdmx that are phosphorylated in response to DSBs. One of these sites, S403, is a direct ATM target. Accordingly, each of these sites is important for Hdm2-mediated ubiquitination of Hdmx after DSB induction. These results demonstrate a sophisticated mechanism whereby ATM fine-tunes the optimal activation of p53 by simultaneously modifying each player in the process
    Type of Publication: Journal article published
    PubMed ID: 15788536
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  • 3
    Keywords: PEPTIDE ; RECEPTOR ; SPECTRA ; COMBINATION ; Germany ; PROTEIN ; SERA ; DOMAIN ; PAIRS ; IONS ; SEQUENCE ; RECOGNITION ; antibodies ; CLEAVAGE ; ACQUISITION ; hormone ; IDENTIFICATION ; SPECTROMETRY ; MASS-SPECTROMETRY ; PRODUCT ; PEPTIDES ; STRATEGIES ; SPECT ; MASSES ; SERUM ; RESIDUES ; UPDATE
    Abstract: Tyrosine-O-sulfated peptides were studied by nanoESI Q-TOF mass spectrometry and were found to exhibit an abundant loss of SO3 in positive ion mode under the usually nonfragmenting conditions of survey spectrum acquisition. A new strategy for the detection of tyrosine-O-sulfated peptides in total protein digests was designed based on exhaustive product ion scanning at the collision offset conditions typical for the recording of survey spectra (minimum collision offset). From these data, Q-TOF neutral loss scans for loss of 80/z and Q-TOF precursor ions scans were extracted. The specificity of this approach for analysis of tyrosine-O-sulfation was tested using a tryptic digest of bovine serum albumin spiked with sulfated hirudin (1:1 and 1000:1 molar ratio of BSA to sulfated hirudin, respectively) and using an in-solution digest of the recombinant extracellular domain of thyroid stimulating hormone receptor (ECD-TSHr). For both examples, the combination of in silico neutral loss scans for 80/z and subsequent in silico precursor ion scans resulted in a specific identification of sulfated peptides. In the analysis of recombinant ECD-TSHr, a doubly sulfated peptide could be identified in this way. Surprisingly, similar to1/4 of the product ion spectra acquired from the tryptic digest of ECD-TSHr at minimum collision offset exhibited sequence-specific ions suitable for peptide identification. Complementary ion pairs were frequently observed, which either were b(2)/y((max-2)) pairs or were induced by cleavage N-terminal to proline. MS/MS analysis at minimum collision offset followed by extraction of neutral loss and precursor ion scans is ideally suited for highly sensitive detection of analyte ions which exhibit facile gas-phase decomposition reactions
    Type of Publication: Journal article published
    PubMed ID: 15373453
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  • 4
    Keywords: PEPTIDE ; Germany ; SITE ; SITES ; RESOLUTION ; MARKER ; IDENTIFICATION ; COLLISION-INDUCED DISSOCIATION ; ELECTROSPRAY ; mass spectrometry ; tandem mass spectrometry ; MASS-SPECTROMETRY ; TIME-OF-FLIGHT ; PEPTIDES ; MASSES ; RE ; HIGH-RESOLUTION ; BOVINE ; diiodo-tyrosine ; immonium ion ; mass-deficiency ; monoiodo-tyrosine ; thyroglobulin ; tyrosine iodination
    Abstract: Peptides containing a monoiodo- or diiodo-tyrosine residue (monoiodo-Y, diiodo-Y) were found Received: March 31, 2004 to generate abundant immonium ions following collision-induced dissociation at m/z 261.97 Accepted: June 30, 2004 and 387.87 Da, respectively. These residue-specific marker ions are between about 140 mDa (monoiodo-Y) and 300 mDa (diiodo-Y) mass deficient relative to any other peptide fragment ions of unmodified peptides, qualifying them as highly specific marker ions for tyrosine iodination when analyzed by high resolution tandem mass spectrometry (MS/MS). Two new iodination sites (Y-364 and Y-2165) were pinpointed in bovine thyroglobulin by MS/MS using these iodotyrosine-specific marker ions and combined tryptic/chymotryptic digestion
    Type of Publication: Journal article published
    PubMed ID: 15627961
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  • 5
    Keywords: EXPRESSION ; Germany ; IN-VIVO ; PATHWAY ; PATHWAYS ; VIVO ; PROTEIN ; FAMILY ; DOMAIN ; BINDING ; ASSAY ; PLASMA ; PLASMA-MEMBRANE ; DEGRADATION ; INVOLVEMENT ; KINASE-C ; DOMAINS ; INTERCELLULAR COMMUNICATION ; CARDIAC MYOCYTES ; ASSAYS ; in vivo ; PROLINE ; JUNCTION ; connexin ; EPITHELIAL NA+ CHANNEL ; GAMMA-ENAC ; gap junction ; GAP-JUNCTION PROTEIN ; PY motif ; ubiquitylation ; WW DOMAINS
    Abstract: Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization
    Type of Publication: Journal article published
    PubMed ID: 16931598
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  • 6
    Keywords: RECEPTOR ; IN-VITRO ; CELL ; KINASE ; PATHWAY ; VITRO ; NEW-YORK ; GENE ; PROTEIN ; PROTEINS ; transcription ; DIFFERENTIATION ; ACTIVATION ; T cell ; T-CELL ; BINDING ; PHOSPHORYLATION ; ASSOCIATION ; ACID ; TARGET ; ELEMENT ; MUTANT ; MUTATION ; SIGNAL-TRANSDUCTION ; FRANCE ; RECRUITMENT ; leukocyte ; TCR ; TYROSINE PHOSPHORYLATION ; INTERLEUKIN-2 ; signaling ; INTERFERENCE ; T-CELL-ACTIVATION ; interaction ; GENE-TRANSCRIPTION ; TYROSINE KINASES ; ANTIGEN RECEPTORS ; progenitor ; USA ; UNIT ; immunology ; KINASE-1 ; interleukin ; MEDICINE ; ADAPTER PROTEIN ; LAT ; T-CELL-RECEPTOR ; ZAP-70
    Abstract: The SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3 epsilon and.. proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Interestingly, either S376A mutation or HPK-1 knockdown resulted in increased TCR-induced tyrosine phosphorylation of SLP-76 and phospholipase C-gamma 1. Moreover, an SLP-76-S376A mutant induced higher interleukin 2 gene transcription than wild-type SLP-76. These data reveal a novel negative feedback loop involving HPK-1-dependent serine phosphorylation of SLP-76 and 14-3-3 protein recruitment, which tunes T cell activation
    Type of Publication: Journal article published
    PubMed ID: 17353368
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  • 7
    Abstract: For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation-derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC-MS(3) -based targeted detection of low-abundant human leukocyte antigen (HLA) class-I-presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen-derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA-peptide complexes, while keeping high isolation yields of low-abundant target peptides. The subsequent targeted LC-MS(3) detection allowed for increased sensitivity, which resulted in successful detection of the known HLA-A2-restricted epitope E711-19 and ten additional E7-derived peptides on the surface of HPV16-transformed cells. T-cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low-abundant candidate epitopes to be true immunotherapy targets.
    Type of Publication: Journal article published
    PubMed ID: 29603667
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  • 8
    Keywords: PEPTIDE ; RECEPTOR ; SPECTRA ; GROWTH ; GROWTH-FACTOR ; FACTOR RECEPTOR ; PATHWAYS ; PROTEIN ; ENRICHMENT ; PHOSPHORYLATION ; IDENTIFICATION ; fragmentation ; PRODUCT ; TIME-OF-FLIGHT ; PEPTIDES ; SPECT ; AFFINITY-CHROMATOGRAPHY ; PHOSPHOPEPTIDES ; PHOSPHOPROTEOME ; SELECTIVE DETECTION
    Abstract: A novel highly sensitive strategy is introduced for analysis of tyrosine phosphorylation in previously identified proteins channelling for this aim all analytical and sequence information available. Nanoelectrospray high-resolution MS/MS analysis is targeted to precalculated m/z values corresponding to phosphotyrosine-containing tryptic peptides. Identification of these peptides is supported by the occurrence of the phosphotyrosine immonium ion at m/z 216, neutral loss of 79.97/z (= loss of HPO3), and similarity of the fragmentation patterns of phosphotyrosine-containing peptides with their nonphosphorylated analogues. This tyrosine-targeted tandem mass spectrometry strategy is demonstrated for epidermal growth factor receptor showing that phosphotyrosine-containing tryptic peptides invisible in the survey spectrum can be safely identified
    Type of Publication: Journal article published
    PubMed ID: 12948142
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  • 9
    Keywords: PEPTIDE ; SPECTRA ; Germany ; PATHWAY ; INFORMATION ; MOLECULES ; IONS ; PHOSPHORYLATION ; SEQUENCE ; SEQUENCES ; RECOGNITION ; ACID ; ACIDS ; IDENTIFICATION ; DIFFERENCE ; REQUIRES ; COLLISION-INDUCED DISSOCIATION ; ELECTROSPRAY ; mass spectrometry ; SPECTROMETRY ; tandem mass spectrometry ; TANDEM MASS-SPECTROMETRY ; fragmentation ; MASS-SPECTROMETRY ; PEPTIDES ; FRAGMENTS ; SERIES ; AMINO-ACIDS ; DISSOCIATION ; PROTONATED PEPTIDES ; NOMENCLATURE ; accurate mass measurement ; GAS-PHASE ; METHIONINE ; neutral loss ; peptide sequencing
    Abstract: The widespread occurrence of the neutral loss of one to six amino acid residues as neutral fragments from doubly protonated tryptic peptides is documented for 23 peptides with individual sequences. Neutral loss of amino acids from the N-terminus of doubly charged tryptic peptides results in doubly charged y-ions, forming a ladder-like series with the ions [M + 2H](2+) = y(max)(2+), y(max-1)(2+), y(max-2)(2+), etc. An internal residue such as histidine, proline, lysine or arginine appears to favor this type of fragmentation, although it was sometimes also observed for peptides without this structure. For doubly protonated non-tryptic peptides with one of these residues at or near the N-terminus, we observed neutral loss from the C-terminus, resulting in a doubly charged b-type ion ladder. The analyses were performed by Q-TOF tandem mass spectrometry, facilitating the recognition of neutral loss ladders by their 2+ charge state and the conversion of the observed mass differences into reliable sequence information. It is shown that the neutral loss of amino acid residues requires low collision offset values, a simple mechanistic explanation based on established fragmentation rules is proposed and the utility of this neutral loss fragmentation pathway as an additional source for dependable peptide sequence information is documented. Copyright (C) 2003 John Wiley Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 14648821
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  • 10
    Keywords: PEPTIDE ; SPECTRA ; Germany ; PROTEIN ; PROTEINS ; data mining ; PHOSPHORYLATION ; SEQUENCE ; VARIANTS ; ACID ; CLEAVAGE ; IDENTIFICATION ; ELECTROSPRAY ; mass spectrometry ; tandem mass spectrometry ; fragmentation ; DATABASE ; MASS-SPECTROMETRY ; PEPTIDES ; STRATEGIES ; MASSES ; HIGH-RESOLUTION ; EXTRACTION ; intensity ; PHOSPHORYLATION SITES ; covalent modification ; N-terminal acetylation ; POSITIVE-ION MODE ; sequence isoforms
    Abstract: Protein analysis by database search engines using tandem mass spectra is limited by the presence of unexpected protein modifications, sequence isoforms which may not be in the protein databases, and poor quality tandem mass spectrometry (MS/MS) of low abundance proteins. The analysis of expected protein modifications can be efficiently addressed by precursor ion scanning. However, it is limited to modifications that show such a characteristic loss in a peptide independent manner. We observed that proline and aspartic acid induced backbone fragmentation is accompanied by a low intensity signal for loss of H3PO4 for several pSer- or pThr-phosphopeptides. We describe here the use of peptide-specific fragments that can be used after a protein was identified to allow in-depth characterization of modifications and isoforms. We consider high abundance fragments formed by cleavage at the C-terminal side of aspartic acid, at the N-terminal side of proline and low mass ions such as a(2), b(2), b(3), y(1), y(2), and y(3). The MS/MS dataset is filtered for each sequence tag of interest by an in silico precursor ion scan. The resulting extracted ion traces are then combined by multiplication to increase specificity. Since the strategy is based on common peptide segments which are shared by different isoforms of peptides it can be applied to the analysis of any post-translational modification or sequence variants of a protein. This is demonstrated for the cases of serine and threonine phosphorylation, histone H1 acetylation and the spotting of multiple H1 isoforms
    Type of Publication: Journal article published
    PubMed ID: 15714472
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