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  • 1
    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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  • 2
    Keywords: PEPTIDE ; Germany ; DENSITY ; SITE ; SITES ; PROTEIN ; MARKER ; PHOSPHORYLATION ; SEQUENCE ; SEQUENCES ; ACID ; CLEAVAGE ; FORM ; IDENTIFICATION ; COLLISION-INDUCED DISSOCIATION ; ELECTROSPRAY ; TANDEM MASS-SPECTROMETRY ; REGION ; REGIONS ; LOCALIZATION ; FRAGMENTS ; BEHAVIOR ; serine ; CLUSTERS ; AFFINITY-CHROMATOGRAPHY ; CLUSTER ; SELECTIVE DETECTION ; GAS-PHASE ; protein phosphorylation ; CHEMISTRY ; CHAIN ; RE ; RESIDUES ; AMINO-ACID ; PHOSPHORYLATION SITES ; analysis ; USA ; ATOMS ; correlation ; FRAGMENT ; BONDS ; 〈M-H〉(-) ANIONS ; BACKBONE CLEAVAGES ; CAPTURE DISSOCIATION ; DEPROTONATED PEPTIDES
    Abstract: Pinpointing of phosphorylation sites by positive ion collision-induced dissociation (CID) in phosphopeptides containing consecutive Ser/Thr residues (Ser/Thr clusters) is frequently hampered by the lack of backbone cleavage between adjacent Ser/Thr or pSer/pThr sites. In this study, we demonstrate that in negative ion collision-induced dissociation phosphorylated and unmodified residues of Ser/Thr clusters exhibit a very selective behavior toward cleavage of their N-C-alpha bonds. Ser/Thr clusters were defined as two and more consecutive serine or threonine residues in phosphopeptide sequences. Dissociation reactions at pSer are significantly more abundant than those of unmodified sites. Thr residues exhibit the same effect, but the cleavages occurring at pThr are generally less prominent than those at pSer. The correlation observed between the facility of the amine backbone bond dissociation of phosphopeptides and the presence of the phosphate group on the side chain residues of Ser and Thr is attributed to the different magnitudes of electron density on the C-alpha atoms of the amino acid in phosphorylated and unmodified forms. The results of this study indicate that the intensity ratio of the fragments generated by N-C-alpha bond cleavage within the phosphopeptide Ser/Thr clusters represents a reliable and general marker for pinpointing of phosphorylation sites. The presented data illustrate that negative ion electrospray CID is superior over the standard positive ion mode approach for the localization of protein phosphorylation inside Ser/Thr clusters
    Type of Publication: Journal article published
    PubMed ID: 17388569
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  • 3
    Keywords: SPECTRA ; Germany ; PROTEIN ; EFFICIENCY ; PHOSPHORYLATION ; IDENTIFICATION ; NUMBER ; COLLISION-INDUCED DISSOCIATION ; mass spectrometry ; IONIZATION ; MASS-SPECTROMETRY ; PEPTIDES ; SELECTIVE DETECTION ; RE ; LEVEL ; LOSSES ; MOBILE ; SCANS ; COMPLEX-MIXTURES ; LARGE MOLECULES ; MS/MS
    Abstract: The nanoelectrospray product ion spectra of multiply charged phosphopeptide anions reveal the occurrence of phosphate-specific high-mass fragment ions of the type [M - nH - 79]((n-1)-). These so far unrecognized fragments, which are observed for phosphoserine-, phosphothreonine-, and phosphotyrosine-containing peptides, are the counterparts of the established inorganic phosphopeptide marker ion found at m/z 79 = [PO3](-). The high-mass marker ions are formed with high efficiency at moderate collision offset values and are particularly useful for sensitive recognition of pSer-, pThr-, and pTyr-peptides due to the low background level in MS/MS spectra at m/z values above those of the precursor ions. By virtue of this feature, the detection of the new phosphorylation-specific fragment ions appears to be more sensitive than the detection of the low-mass phosphate marker ion at m/z 79, where a higher interference by nonspecific background signals is generally observed. The number of phosphate groups within a phosphopeptide can also be estimated on the basis of the [M - nH - 79]((n-1)-) ions, since these exhibit an effective, sequential neutral loss of H3PO4 of the residing phosphate groups. A mechanistic explanation for the formation of the [M - nH - 79]((n-1)) ions from multiply charged phosphopeptides is given. The high-mass marker ions are proposed to originate from phosphopeptide anions, which carry two negative charges located at the phosphate group. A new search tool denominated "variable m/z gain analysis", which utilizes these newly recognized high-mass fragments for spotting of phosphopeptides in a negative ion parent scan, is proposed. The findings strengthen the value of negative ion ESI-MS/MS for analysis of protein phosphorylation
    Type of Publication: Journal article published
    PubMed ID: 16478119
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  • 4
    Keywords: PROTEINS ; MECHANISM ; METABOLITES ; TANDEM MASS-SPECTROMETRY ; BEHAVIOR ; CONFORMATIONAL-CHANGES ; deuterium exchange ; ELECTROSPRAY-IONIZATION ; RESONANCE ; H/D EXCHANGE
    Abstract: Out for the count: Hydrogen-deuterium exchange (HDX) performed in ESI droplets can distinguish chemically distinct forms of labile hydrogen. NanoESI exchanged mainly O-bound hydrogen atoms, whereas ESI also exchanged a subset of N-bound hydrogen atoms. The exchange behavior could be predicted. Thus, the combined use of nanoESI- and ESI-HDX can be applied to count chemically different forms of labile hydrogen atoms.
    Type of Publication: Journal article published
    PubMed ID: 23852916
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  • 5
    Keywords: PEPTIDE ; SPECTRA ; CELL ; Germany ; MODEL ; PATHWAY ; DISEASE ; SITES ; MECHANISM ; IONS ; CONTRAST ; PHOSPHORYLATION ; ACID ; IDENTIFICATION ; COLLISION-INDUCED DISSOCIATION ; fragmentation ; MASS-SPECTROMETRY ; PEPTIDES ; REVEALS ; PHOSPHOPEPTIDES ; SELECTIVE DETECTION ; GAS-PHASE ; neutral loss ; PROTEIN-PHOSPHORYLATION ; phosphoproteins ; CHEMISTRY ; PHOSPHORYLATION SITES ; USA ; EXTENT ; OCCURS ; PRODUCT IONS ; ASSISTED-LASER-DESORPTION/IONIZATION ; WELL ; AMIDE ; PHOSPHORYLATED PEPTIDES ; QUADRUPOLE ; TYROSINE
    Abstract: Phosphotyrosine-containing peptide monoanions [M - H](-) exhibit extensive neutral loss of phosphoric acid (98 Da) upon quadrupole time-of-flight and ion-trap collision-induced dissociation (CID). In contrast, a neutral loss of metaphosphoric acid HPO3 (80 Da) is negligible from the deprotonated phosphotyrosine peptides. The efficient H3PO4 release is unexpected, given the structure of phosphotyrosine. Our study reveals that the abundant [M - H - 98](-) product ions of pTyr-peptides are not a result of consecutive losses of HPO3 and H2O but, rather, are induced by an intramolecular interaction of the phosphotyrosine phosphate with deprotonated peptide functions such as hydroxyl, carboxyl, and to a small extent, amide. As a result, an internal phosphotyrosine phosphate shift occurs, and the obtained phosphorylated functionalities undergo elimination of H3PO4 to give rise to the [M - H - 98](-) fragments. The mechanism proposed for the phosphoric acid neutral loss is based on extensive CID studies of Ala-substituted model phosphorylated peptides and oxygen-18 labeling. The proposed mechanistic pathway explains the fact that the pTyr phosphate transfer and the subsequent H3PO4 neutral loss are not observed for multiply charged anions of pTyr-peptides. Monoanions of pSer-containing peptides undergo the intramolecular phosphate shift as well, although its efficiency is much lower compared to the aromatic phosphorylation sites. These observations facilitate correct identification of pSer-, pThr-, and pTyr-peptides in CID studies. This work demonstrates that the established phosphate-specific neutral loss fragmentation rules of protonated pTyr-peptides cannot be applied to the CID spectra of their [M - H](-) ions
    Type of Publication: Journal article published
    PubMed ID: 19402683
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  • 6
    Keywords: PEPTIDE ; COMBINATION ; Germany ; SITES ; PROTEINS ; ACCURACY ; PHOSPHORYLATION ; ACID ; IDENTIFICATION ; mass spectrometry ; TANDEM MASS-SPECTROMETRY ; MASS-SPECTROMETRY ; TIME-OF-FLIGHT ; PEPTIDES ; STATES ; phosphoproteins ; MASSES ; molecular ; RE ; RESIDUES ; INTERFERENCE ; SODIUM ; CHARGE
    Abstract: Formation of S-carbamidomethylmethionine (camMet) occurs as a side reaction during cysteine alkylation with iodoacetamide (IAA). In collision-induced dissociation, peptides with camMet show an abundant neutral loss of 2-(methylthio)acetamide (C3H7NOS=105.025 Da) at moderate collision offset values which are similar to those optimal for loss of phosphoric acid (H3PO4 = 97.977 Da). Neutral loss analysis is used for spotting of phosphopeptides which contain phosphoserine (pSer) or phosphothreonine (pThr) residues. In the case where precursor ions cannot be accurately assigned in the survey spectrum (e.g. due to low ion abundance or signal overlap), the mass accuracy of a neutral loss tandem mass spectrometry (MS/MS) analysis depends on the precursor ion isolation window. For the charge states 2+, 3+ or 4+, a typical 3.5 Da precursor isolation window results in neutral loss windows of 7, 10.5 or 14 Da, respectively. Consequently, neutral loss of 105 Da from alkylated methionine residues can mimic the phosphoserine/phosphothreonine-specific neutral loss of 98 Da. In the evaluation of quadrupole time-of-flight (QTOF) parent ion scan data for neutral loss of H3PO4, this interference was frequently observed. It is illustrated in this study using the analysis of ovalbumin phosphorylation as an example. The +80 Da molecular weight shift connected with phosphorylation at serine or threonine may also be mimicked by carbamidomethylation of methionine through a combination with sodium adduction (+57 Da+22 Da = +79 Da). For highly sensitive neutral loss analysis of serine and threonine phosphorylation, careful data inspection is recommended if reduction and alkylation by IAA is employed. Copyright (c) 2005 John Wiley & Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 15912474
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  • 7
    Keywords: CELL ; PROTEIN ; PROTEINS ; MECHANISM ; PHOSPHORYLATION ; IDENTIFICATION ; ELECTROSPRAY ; fragmentation ; DISSOCIATION ; posttranslational modification ; TYROSINE SULFATION ; Negative ion CID ; Phosphotyrosine ; Sulfotyrosine ; THREONINE
    Abstract: Unambiguous differentiation between isobaric sulfated and phosphorylated tyrosine residues (sTyr and pTyr) of proteins by mass spectrometry is challenging, even using high resolution mass spectrometers. Here we show that upon negative ion mode collision-induced dissociation (CID), pTyr- and sTyr-containing peptides exhibit entirely different modification-specific fragmentation patterns leading to a rapid discrimination between the isobaric covalent modifications using the tandem mass spectral data. This study reveals that the ratio between the relative abundances of [M-H-80](-) and [M-H-98](-) fragment ions in ion-trap CID and higher energy collision dissociation (HCD) spectra of singly deprotonated +80 Da Tyr-peptides can be used as a reliable indication of the Tyr modification group nature. For multiply deprotonated +80 Da Tyr-peptides, CID spectra of sTyr- and pTyr-containing sequences can be readily distinguished based on the presence/absence of the [M-nH-79]((n-1)-) and [M-nH-79-NL]((n-1)-) (n=2, 3) fragment ions (NL=neutral loss).
    Type of Publication: Journal article published
    PubMed ID: 21952787
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  • 8
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Chemical Physics 51 (1980), S. 77-88 
    ISSN: 0301-0104
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Chemistry and Pharmacology , Physics
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Chemical Physics 8 (1975), S. 27-36 
    ISSN: 0301-0104
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Chemistry and Pharmacology , Physics
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Chemical Physics 30 (1978), S. 353-359 
    ISSN: 0301-0104
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Chemistry and Pharmacology , Physics
    Type of Medium: Electronic Resource
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