Plasma membrane proteomes from leukemia cell lines

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
electrophoresis.
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
streptavidin-AP.