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  1. Article: Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy

    Tsai, Hsing-Fen / He-Hsuan Hsiao

    Analytica chimica acta. 2017 Mar. 01, v. 956

    2017  

    Abstract: A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic 16O/18O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum ... ...

    Abstract A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic 16O/18O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol–1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per μl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease.
    Keywords acetone ; analytical chemistry ; arginine ; biomarkers ; blood serum ; detection limit ; hepatitis B ; hepatitis B antigens ; Hepatitis B virus ; humans ; isotope labeling ; lysine ; molecular weight ; monitoring ; proteomics ; spectrometers ; synthetic peptides ; tandem mass spectrometry ; trichloroacetic acid ; trypsin
    Language English
    Dates of publication 2017-0301
    Size p. 32-39.
    Publishing place Elsevier B.V.
    Document type Article
    ZDB-ID 1483436-4
    ISSN 1873-4324 ; 0003-2670
    ISSN (online) 1873-4324
    ISSN 0003-2670
    DOI 10.1016/j.aca.2016.12.015
    Database NAL-Catalogue (AGRICOLA)

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  2. Article: Stage-frit: A straightforward sub-2 μm nano-liquid chromatography column fabrication for proteomic analysis

    Hsieh, Ming-Yueh / He-Hsuan Hsiao

    Analytica chimica acta. 2015 July 30, v. 886

    2015  

    Abstract: In this work we demonstrated a facile method for the fabrication of C18 coordination polymer gel in a capillary, called stage-frit, which was efficiently applied to pack sub-2 μm C18 beads into the capillary by a high pressure bomb for the online ... ...

    Abstract In this work we demonstrated a facile method for the fabrication of C18 coordination polymer gel in a capillary, called stage-frit, which was efficiently applied to pack sub-2 μm C18 beads into the capillary by a high pressure bomb for the online separation of proteolytic peptides. The back pressure of the column with 10 cm × 75 μm i.d. is regularly lower than 170 bar at a flow rate of 300 nl/min, which could be operated on a common nanoLC system instead of nanoUPLC system due to the good permeability, low back pressure and high mechanical stress of the frit that will totally reduce the cost for the purchase of instrument. The stage-frit allows long-term continuous flow of the solvent and no significant beads loss or pressure instability was observed during the period. The repeatability of retention time for fifteen BSA tryptic peaks was found to be less than 1.08% (RSD) in six time nanoLC-ESI-MS/MS experiments. The average full width at half maximum (FWHM) of peptide peaks is 5.87 s. The sub-2 μm stage-frit nanoLC column showed better sensitivity than the commercial available for large scale proteomic analysis of total tissue proteins from human spleen. The number of identified peptides is approximately 0.4-fold and 0.2-fold higher than that obtained by utilizing commercial columns packed with 3 μm and 1.8 μm C18 materials, respectively. In the field of analytical chemistry, particularly the use of nanoLC systems, stage-frit nanoLC column offers a great potential for the separation of complex mixtures.
    Keywords analytical chemistry ; chromatography ; coordination polymers ; cost effectiveness ; gels ; humans ; mechanical stress ; peptides ; permeability ; proteins ; proteolysis ; proteomics ; solvents ; spleen
    Language English
    Dates of publication 2015-0730
    Size p. 200-206.
    Publishing place Elsevier B.V.
    Document type Article
    ZDB-ID 1483436-4
    ISSN 1873-4324 ; 0003-2670
    ISSN (online) 1873-4324
    ISSN 0003-2670
    DOI 10.1016/j.aca.2015.06.023
    Database NAL-Catalogue (AGRICOLA)

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  3. Article ; Online: The human cytomegalovirus DNA polymerase processivity factor UL44 is modified by SUMO in a DNA-dependent manner.

    Elisa Sinigalia / Gualtiero Alvisi / Chiara V Segré / Beatrice Mercorelli / Giulia Muratore / Michael Winkler / He-Hsuan Hsiao / Henning Urlaub / Alessandro Ripalti / Susanna Chiocca / Giorgio Palù / Arianna Loregian

    PLoS ONE, Vol 7, Iss 11, p e

    2012  Volume 49630

    Abstract: During the replication of human cytomegalovirus (HCMV) genome, the viral DNA polymerase subunit UL44 plays a key role, as by binding both DNA and the polymerase catalytic subunit it confers processivity to the holoenzyme. However, several lines of ... ...

    Abstract During the replication of human cytomegalovirus (HCMV) genome, the viral DNA polymerase subunit UL44 plays a key role, as by binding both DNA and the polymerase catalytic subunit it confers processivity to the holoenzyme. However, several lines of evidence suggest that UL44 might have additional roles during virus life cycle. To shed light on this, we searched for cellular partners of UL44 by yeast two-hybrid screenings. Intriguingly, we discovered the interaction of UL44 with Ubc9, an enzyme involved in the covalent conjugation of SUMO (Small Ubiquitin-related MOdifier) to cellular and viral proteins. We found that UL44 can be extensively sumoylated not only in a cell-free system and in transfected cells, but also in HCMV-infected cells, in which about 50% of the protein resulted to be modified at late times post-infection, when viral genome replication is accomplished. Mass spectrometry studies revealed that UL44 possesses multiple SUMO target sites, located throughout the protein. Remarkably, we observed that binding of UL44 to DNA greatly stimulates its sumoylation both in vitro and in vivo. In addition, we showed that overexpression of SUMO alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both virus production and DNA replication, arguing for an important role for sumoylation in HCMV life cycle and UL44 function(s). These data report for the first time the sumoylation of a viral processivity factor and show that there is a functional interplay between the HCMV UL44 protein and the cellular sumoylation system.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2012-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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