Biochemistry

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    Negative Ion Mode nanoLC-ESI-MS/MS Analyses of Permethylated Sulfated Glycans
    Authors:  Shin-Yi Yu, Chu-Wen Cheng and Kay-Hooi Khoo, date: 05/20/2020, view: 1032, Q&A: 0
    [Abstract] We have developed enabling techniques for sulfoglycomics based on MALDI-MS mapping and MS/MS sequencing of permethylated sulfated glycans. We then extended further the analytical workflow to C18 reverse phase (RP)-nanoLC-nanoESI-MS/MS analyses of permethylated sulfated glycans in the negative ion mode. The advantages are that extra sulfates on ...
    Permethylation and Microfractionation of Sulfated Glycans for MS Analysis
    Authors:  Shin-Yi Yu, Sergei Snovida and Kay-Hooi Khoo, date: 05/20/2020, view: 1114, Q&A: 0
    [Abstract] Sulfated glycans are barely detectable in routine mass spectrometry (MS)-based glycomic analysis due to ion suppression by the significantly more abundant neutral glycans in the positive ion mode, and sialylated non-sulfated glycans in the negative ion mode, respectively. Nevertheless, the negative charge imparted by sulfate can be advantageous ...
    Glucosidase Inhibition to Study Calnexin-assisted Glycoprotein Folding in Cells
    Authors:  Hao Wang and Qingyu Wu, date: 06/05/2019, view: 2112, Q&A: 0
    [Abstract] Calnexin is a chaperone protein that plays a critical role in glycoprotein folding in the endoplasmic reticulum (ER). The function of calnexin depends on its binding to monoglucosylated oligosaccharides on nascent glycoproteins, whereas the generation of monoglucosylated oligosaccharides depends on the activity of α-glucosidases I and II, which ...
    PNGase Sensitivity Assay to Study the Folding Status of Proteins
    Authors:  Satoshi Ninagawa and Kazutoshi Mori, date: 10/05/2016, view: 5340, Q&A: 0
    [Abstract] This protocol aims to evaluate folding status of proteins, utilizing peptide:N-glycanase (PNGase) sensitivity. In the cytosol, PNGase works as a deglycosylation-enzyme. N-glycans on unfolded/misfolded proteins are more susceptible to PNGase than N-glycans on folded proteins because of the preference of PNGase to non-native proteins. PNGase is ...
    LC/MS-based Detection of Hydroxyproline O-galactosyltransferase Activity
    Authors:  Mari Ogawa-Ohnishi and Yoshikatsu Matsubayashi, date: 01/20/2016, view: 5483, Q&A: 0
    [Abstract] Arabinogalactan proteins (AGPs) are plant-specific extracellular glycoproteins regulating a variety of processes during growth and development. AGP biosynthesis involves O-galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. The initial Hyp O-galactosylation is mediated ...
    Preparation and Analysis of Crude Autolytic Enzyme Extracts from Staphylococcus aureus
    Authors:  Filipa Vaz and Sérgio R. Filipe, date: 12/20/2015, view: 9362, Q&A: 0
    [Abstract] The metabolism of the cell surface during bacterial cell division involves synthesis and degradation of peptidoglycan (PGN), the major component of the bacterial cell wall. Bacteria have to ensure that their surface remains capable of withstanding high turgor pressures and, simultaneously, that the PGN at their surface is concealed from receptors ...
    Lectin Binding Analysis of Streptococcus mutans Glycoproteins
    Authors:  Alejandro Avilés-Reyes, José A. Lemos and Jacqueline Abranches, date: 04/05/2015, view: 6709, Q&A: 0
    [Abstract] Bacterial glycoproteins are of increasing interest due to their abundance in nature and importance in health and infectious diseases. However, only a very small fraction of bacterial glycoproteins have been characterized and its post-translational modification machinery identified. While analysis of glycoproteins can be achieved through various ...
    Dimethylmethylene Blue Assay (DMMB)
    Authors:  Vivien Jane Coulson- Thomas and Tarsis Ferreira Gesteira, date: 09/20/2014, view: 45285, Q&A: 8
    [Abstract] Glycosaminoglycans (GAGs) are long unbranched polysaccharides consisting of repeating disaccharide units composed of a hexosamine (glucosamine or galactosamine) and a hexuronic acid (glucuronic or iduronic acid). Depending on the disaccharide unit the GAGs can be organized into five groups: chondroitin sulfate, dermatan sulfate, heparan sulfate, ...
    Determination of D-galactofuranose Content of Galactomannoproteins in Aspergillus nidulans
    [Abstract] Galactofuranose (Galf) is a component of several polysaccharides and glycoconjugates in certain species of filamentous fungi. Galf residues are frequently found in Aspergillus glycoproteins, including N-glycans and O-mannose glycans that modify many cell wall proteins and extracellular enzymes. It is ...
    Seed Coat Ruthenium Red Staining Assay
    Authors:  Heather E. McFarlane, Delphine Gendre and Tamara L. Western, date: 04/05/2014, view: 14793, Q&A: 0
    [Abstract] The goal of this protocol is to assay for defects in synthesis/secretion/release of seed coat mucilage by ruthenium red staining of mature whole seeds.

    The mucilage secretory cells of the Arabidopsis seed coat synthesize and secrete a large quantity of primarily pectinaceous mucilage to a ring-shaped apical domain during their ...



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