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  • 1
    Keywords: CELLS ; CELL ; COMBINATION ; Germany ; imaging ; GENOME SEQUENCE ; PROTEIN ; PHOSPHORYLATION ; PLASMA ; mass spectrometry ; MASS-SPECTROMETRY ; AMINO-ACIDS ; sensitivity ; GEL-ELECTROPHORESIS ; CAPILLARY LIQUID-CHROMATOGRAPHY ; CHEMISTRY ; RE ; ABLATION ; MS ; SEQUENCE DATABASES ; PHOSPHOPROTEIN ; LIQUID ; CAPILLARIES ; ACCESS ; ABSOLUTE QUANTIFICATION ; CORYNEBACTERIUM-GLUTAMICUM ; P-31 DETECTION ; PEPTIDE-FRAGMENTS ; STOICHIOMETRY
    Abstract: Protein phosphorylation stoichiometry was assessed by two analytical strategies. Both are based on element mass spectrometry (ICPMS, inductively coupled plasma mass spectrometry) and simultaneous monitoring of MP and S-34. One strategy employs a combination of 1D gel electrophoresis, in-gel digestion, and final mu LC-ICPMS analysis (mu LC = capillary liquid chromatography). The other strategy uses the combination of 1D gel electrophoresis, protein blotting, and imLA-ICPMS (imLA = imaging laser ablation). The two methods were evaluated with standard phosphoproteins and were applied to the analysis of the cytoplasmatic proteome of bacterial cells (Corynebacterium glutamicum) and eukaryotic cells (Mus musculus). The eukaryotic proteome was found to exhibit a significantly higher phosphorylation degree (similar to 0.8 mol of P/mol of protein) compared to the bacterial proteome (similar to 0.01 mol of P/mol of protein). Both analytical strategies revealed consistent quantitative results, with the mu LC-ICPMS approach providing the higher sensitivity. In summary, two ICPMS-based methods for quantitative estimation of the phosphorylation degree of a cellular proteome are presented which access the native proteome state and do not require any type of label introduction or derivatization
    Type of Publication: Journal article published
    PubMed ID: 16536437
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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
    Abstract: To study the viscoelastic behavior of antibody films on a surface acoustic wave device, we have measured the influence of multiple layers of antibodies onto the propagation of the wave. Attenuation and sound velocity variation caused by up to 20 layers have been measured. The results can be interpreted with the propagation of a viscously damped Love wave in a film with an extremely low velocity of sound. From our results, we conclude that, for the one- or two-layer films normally used in acoustic wave immunosensing, a pure mass effect is dominant. For thick films, a saturation of the sensor response is observed.
    Type of Publication: Journal article published
    PubMed ID: 9684545
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  • 4
    Keywords: MRI ; NMR-SPECTROSCOPY ; CONTRAST AGENTS ; ORDER ; PHIP ; HYDROGEN INDUCED POLARIZATION ; PARAHYDROGEN-INDUCED POLARIZATION ; PASADENA HYPERPOLARIZATION ; C-13 BIOMOLECULES ; MAGNETIZATION
    Abstract: Signal amplification by reversible exchange (SABRE) of a substrate and parahydrogen at a catalytic center promises to overcome the inherent insensitivity of magnetic resonance. In order to apply the new approach to biomedical applications, there is a need to develop experimental equipment, in situ quantification methods, and a biocompatible solvent. We present results detailing a low-field SABRE polarizer which provides well-controlled experimental conditions, defined spins manipulations, and which allows in situ detection of thermally polarized and hyperpolarized samples. We introduce a method for absolute quantification of hyperpolarization yield in situ by means of a thermally polarized reference. A maximum signal-to-noise ratio of approximately 10(3) for 148 mumol of substance, a signal enhancement of 10(6) with respect to polarization transfer field of SABRE, or an absolute (1)H-polarization level of approximately 10(-2) is achieved. In an important step toward biomedical application, we demonstrate (1)H in situ NMR as well as (1)H and (13)C high-field MRI using hyperpolarized pyridine (d3) and (13)C nicotinamide in pure and 11% ethanol in aqueous solution. Further increase of hyperpolarization yield, implications of in situ detection, and in vivo application are discussed.
    Type of Publication: Journal article published
    PubMed ID: 24397559
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  • 5
    Keywords: CANCER ; DISCOVERY ; RENAL-CELL CARCINOMA ; SOLID TUMORS ; receptor tyrosine kinase ; TREATMENT RESPONSE ; sorafenib ; erlotinib ; MULTIKINASE INHIBITOR ; ERBB FAMILY BLOCKER
    Abstract: Drug efficacy strongly depends on the presence of the drug substance at the target site. As vascularization is an important factor for the distribution of drugs in tissues, we analyzed drug distribution as a function of blood vessel localization in tumor tissue. To explore distribution of the anticancer drugs afatinib, erlotinib, and sorafenib, a combined approach of matrix-assisted laser desorption/ionization (MALDI) drug imaging and immunohistochemical vessel staining was applied and examined by digital image analysis. The following two xenograft models were investigated: (1) mice carrying squamous cell carcinoma (FaDu) xenografts (ntumor = 13) were treated with afatinib or erlotinib, and (2) sarcoma (A673) xenograft bearing mice (ntumor = 8) received sorafenib treatment. MALDI drug imaging revealed a heterogeneous distribution of all anticancer drugs. The tumor regions containing high drug levels were associated with a higher degree of vascularization than the regions without drug signals (p 〈 0.05). When correlating the impact of blood vessel size to drug abundance in the sarcoma model, a higher amount of small vessels was detected in the tumor regions with high drug levels compared to the tumor regions with low drug levels (p 〈 0.05). With the analysis of coregistered MALDI imaging and CD31 immunohistochemical data by digital image analysis, we demonstrate for the first time the potential of correlating MALDI drug imaging and immunohistochemistry. Here we describe a specific and precise approach for correlating histological features and pharmacokinetic properties of drugs at microscopic level, which will provide information for the improvement of drug design, administration formula or treatment schemes.
    Type of Publication: Journal article published
    PubMed ID: 25263480
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  • 6
    Abstract: Gas chromatography using atmospheric pressure chemical ionization coupled to mass spectrometry (GC/APCI-MS) is an emerging metabolomics platform, providing much-enhanced capabilities for structural mass spectrometry as compared to traditional electron ionization (EI)-based techniques. To exploit the potential of GC/APCI-MS for more comprehensive metabolite annotation, a major bottleneck in metabolomics, we here present the novel R-based tool InterpretMSSpectrum assisting in the common task of annotating and evaluating in-source mass spectra as obtained from typical full-scan experiments. After passing a list of mass-intensity pairs, InterpretMSSpectrum locates the molecular ion (M0), fragment, and adduct peaks, calculates their most likely sum formula combination, and graphically summarizes results as an annotated mass spectrum. Using (modifiable) filter rules for the commonly used methoximated-trimethylsilylated (MeOx-TMS) derivatives, covering elemental composition, typical substructures, neutral losses, and adducts, InterpretMSSpectrum significantly reduces the number of sum formula candidates, minimizing manual effort for postprocessing candidate lists. We demonstrate the utility of InterpretMSSpectrum for 86 in-source spectra of derivatized standard compounds, in which rank-1 sum formula assignments were achieved in 84% of the cases, compared to only 63% when using mass and isotope information on the M0 alone. We further use, for the first time, automated annotation to evaluate the purity of pseudospectra generated by different metabolomics preprocessing tools, showing that automated annotation can serve as an integrative quality measure for peak picking/deconvolution methods. As an R package, InterpretMSSpectrum integrates flexibly into existing metabolomics pipelines and is freely available from CRAN ( https://cran.r-project.org/ ).
    Type of Publication: Journal article published
    PubMed ID: 27584561
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  • 7
    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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  • 8
    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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  • 9
    Keywords: carcinoma ; IN-VIVO ; TISSUE ; SPECTROSCOPY ; PROGRESSION ; sensitivity ; MALIGNANCY ; CANCER DIAGNOSIS ; histopathology ; SCATTERING MICROSCOPY
    Abstract: At present, tumor diagnostic imaging is commonly based on hematoxylin and eosin or immunohistochemical staining of biopsies, which requires tissue excision, fixation, and staining with exogenous marker molecules. Here, we report on label-free tumor imaging using confocal spontaneous Raman scattering microspectroscopy, which exploits the intrinsic vibrational contrast of endogenous biomolecular species. We present a chemically specific and quantitative approach to monitoring normal human skin cells (keratinocytes and fibroblasts) as well as the human HaCaT in vitro skin carcinogenesis model and the tumor-derived MET in vivo skin cancer progression model. Mapping the amplitudes of two spectrally well isolated Raman bands at 752 and 785 cm(-1) allowed for direct visualization of the distributions representative of tryptophan-rich proteins and nucleic acids, respectively, with subcellular spatial resolution. Using these Raman markers, it was feasible to discriminate between normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) and to confine all tumorigenic cells from both the NHEK and NHDF. First evidence for the successful application of the proposed intracellular nucleic acid and tryptophan Raman signatures for skin cancer diagnosis was further demonstrated in an organotypic cutaneous squamous cell carcinomas model, allowing for the identification of tumor cells and their surrounding stroma in the tissue context.
    Type of Publication: Journal article published
    PubMed ID: 25984831
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  • 10
    Abstract: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to simultaneously visualize the lateral distribution of different lipid classes in tissue sections, but the applicability of the method to real-life samples is often limited by ion suppression effects. In particular, the presence of abundant phosphatidylcholines (PCs) can reduce the ion yields for all other lipid species in positive ion mode measurements. Here, we used on-tissue treatment with buffer-free phospholipase C (PLC) to near-quantitatively degrade PCs in fresh-frozen tissue sections. The ion signal intensities of mono-, di-, and oligohexosylceramides were enhanced by up to 10-fold. In addition, visualization of Shiga toxin receptor globotriaosylceramide (Gb3Cer) in the kidneys of wild-type and alpha-galactosidase A-knockout (Fabry) mice was possible at about ten micrometer resolution. Importantly, the PLC treatment did not decrease the high lateral resolution of the MS imaging analysis.
    Type of Publication: Journal article published
    PubMed ID: 27212679
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