KUKU

KU ScholarWorks

  • myKU
  • Email
  • Enroll & Pay
  • KU Directory
    • Login
    View Item 
    •   KU ScholarWorks
    • Chemistry
    • Chemistry Scholarly Works
    • View Item
    •   KU ScholarWorks
    • Chemistry
    • Chemistry Scholarly Works
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    A general protease digestion procedure for optimal protein sequence coverage and PTM analysis of recombinant glycoproteins: Application to the characterization of hLOXL2 glycosylation

    Thumbnail
    View/Open
    Rebecchi_AnalChem_2011.pdf (2.168Mb)
    Issue Date
    2011-10-27
    Author
    Rebecchi, Kathryn
    Go, Eden P.
    Xu, Li
    Woodin, Carrie L.
    Mure, Minae
    Desaire, Heather
    Publisher
    American Chemistry Society
    Type
    Article
    Article Version
    Scholarly/refereed, author accepted manuscript
    Rights
    © 2011 American Chemical Society
    Metadata
    Show full item record
    Abstract
    Using recombinant DNA technology for expression of protein therapeutics is a maturing field of pharmaceutical research and development. As recombinant proteins are increasingly utilized as biotherapeutics, improved methodologies ensuring the characterization of post-translational modifications (PTMs) are needed. Typically, proteins prepared for PTM analysis are proteolytically digested and analyzed by mass spectrometry. To assure full coverage of the PTMs on a given protein, one must obtain complete sequence coverage of the protein, which is often quite challenging. The objective of the research described here is to design a protocol that maximizes protein sequence coverage and enables detection of post-translational modifications, specifically N-linked glycosylation. To achieve this objective, a highly efficient proteolytic digest protocol using trypsin was designed by comparing the relative merits of denaturing agents (urea and Rapigest™ SF), reducing agents (dithiothreitol, DTT, and tris(2-carboxyethyl)phophine, TCEP), and various concentrations of alkylating agent (iodoacetamide, IAM). After analysis of human apo-transferrin using various protease digestion protocols, ideal conditions were determined to contain 6 M urea for denaturation, 5 mM TCEP for reduction, 10 mM IAM for alkylation, and 10 mM DTT, to quench excess IAM before the addition of trypsin. This method was successfully applied to a novel recombinant protein, human lysyl oxidase-like 2 (hLOXL2). Furthermore, the glycosylation PTMs were readily detected at two glycosylation sites in the protein. These digestion conditions were specifically designed for PTM analysis of recombinant proteins and biotherapeutics, and the work described herein fills an unmet need in the growing field of biopharmaceutical analysis.
    URI
    http://hdl.handle.net/1808/23561
    DOI
    https://doi.org/10.1021/ac2017037
    Collections
    • Chemistry Scholarly Works [626]
    Citation
    Rebecchi, K. R., Go, E. P., Xu, L., Woodin, C. L., Mure, M., & Desaire, H. (2011). A general protease digestion procedure for optimal protein sequence coverage and PTM analysis of recombinant glycoproteins: Application to the characterization of hLOXL2 glycosylation. Analytical Chemistry, 83(22), 8484–8491. http://doi.org/10.1021/ac2017037

    Items in KU ScholarWorks are protected by copyright, with all rights reserved, unless otherwise indicated.


    We want to hear from you! Please share your stories about how Open Access to this item benefits YOU.


    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

    Browse

    All of KU ScholarWorksCommunities & CollectionsThis Collection

    My Account

    Login

    Statistics

    View Usage Statistics

    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

    The University of Kansas
      Contact KU ScholarWorks
    Lawrence, KS | Maps
     
    • Academics
    • Admission
    • Alumni
    • Athletics
    • Campuses
    • Giving
    • Jobs

    The University of Kansas prohibits discrimination on the basis of race, color, ethnicity, religion, sex, national origin, age, ancestry, disability, status as a veteran, sexual orientation, marital status, parental status, gender identity, gender expression and genetic information in the University’s programs and activities. The following person has been designated to handle inquiries regarding the non-discrimination policies: Director of the Office of Institutional Opportunity and Access, IOA@ku.edu, 1246 W. Campus Road, Room 153A, Lawrence, KS, 66045, (785)864-6414, 711 TTY.

     Contact KU
    Lawrence, KS | Maps