Plasma membrane proteomes from leukemia cell lines

L. Bransgrove, S. Cordwell, M. Baker, R. Christopherson

    Research output: Contribution to journalMeeting abstractpeer-review


    Historically, much cancer research has involved a search for differences
    between normal and neoplastic tissue. While there are very few cancerspecific
    antigens, cancers certainly express different repertoires of surface
    proteins. Similarly, the plasma membrane proteomes of leukaemias
    should be different to normal leukocytes. We have investigated the proteomes
    of plasma membranes from leukaemia cells, in particular proteins
    differentially expressed by each of the four classes: acute lymphoblastic
    leukaemia (ALL), acute myeloid leukaemia (AML), chronic lymphoblastic
    leukaemia (CLL) and chronic myeloid leukaemia (CML). Patterns of expression
    of plasma membrane proteins have been determined by 2D gel
    There is little data available on plasma membrane proteomes due to
    difficulties in preparation, separation and identification of very hydrophobic
    proteins particularly low abundance proteins. Many studies have used
    whole-membrane (plasma and internal) protein purification kits that fail to
    identify low abundance surface proteins. Alternatively, density gradient
    centrifugation has been employed to isolate plasma membranes but these
    preparations may be contaminated with cytoskeletal proteins and internal
    membranes. We have specifically labelled cell surface proteins with biotin
    before cellular disruption followed by affinity purification on a monomeric
    avidin-agarose column to capture biotinylated surface proteins, subsequently
    eluted with 100 mM glycine (pH 2.8) and subjected to 2D electrophoresis
    on an 8–18% SDS-PAGE gel. Protein spots were visualized with
    Sypro Ruby or Coomassie Blue and protein maps compared between the
    five cell lines: CCRF-CEM (T-ALL), Raji (B-cell lymphoma), HL60 (promyelocytic
    leukaemia), MEC-1 and MEC-2 (B-CLL). Proteins of interest were
    excised from the gels, digested with trypsin and identified by peptide
    mass fingerprinting. These cell lines differentially expressed a number
    of proteins in plasma membranes. Proteins were blotted onto PVDF
    membranes and those that were biotinylated were detected with
    Original languageEnglish
    Article number13.15
    Pages (from-to)S103-S103
    Number of pages1
    JournalMolecular and Cellular Proteomics
    Issue number8 suppl 1
    Publication statusPublished - Aug 2005


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