Affiliations: Biological Sciences Division, Pacific Northwest
National Laboratories, Richland, WA 99352, USA | Analytical Chemistry Group, Pacific Northwest National
Laboratories, Richland, WA 99352, USA
Note: [] Corresponding author: David L. Springer, Ph.D., Biological
Sciences Division, Protein Function Group, K4-12, Pacific Northwest National
Laboratory Richland, WA 99352, USA. Tel.: +1 509 372 6762; Fax: +1 509 372
6544; E-mail: [email protected]
Abstract: To determine how the repertoire of plasma membrane proteins change
with disease state, specifically related to cancer, several methods for
preparation of plasma membrane proteins were evaluated. Cultured cells derived
from stage IV ovarian tumors were grown to 90% confluence and harvested in
buffer containing CHAPS detergent. This preparation was centrifuged at low
speed to remove insoluble cellular debris resulting in a crude homogenate.
Glycosylated proteins in the crude homogenate were selectively enriched using
lectin affinity chromatography. The crude homogenate and the lectin purified
sample were prepared for mass spectrometric evaluation. The general procedure
for protein identification began with trypsin digestion of protein fractions
followed by separation by reversed phase liquid chromatography that was coupled
directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap). Mass
and fragmentation data for the peptides were searched against a human proteome
data base using the informatics program SEQUEST. Using this procedure 398
proteins were identified with high confidence, including receptors,
membrane-associated ligands, proteases, phosphatases, as well as structural and
adhesion proteins. Results indicate that lectin chromatography provides a
select subset of proteins and that the number and quality of the
identifications improve as does the confidence of the protein identifications
for this subset. These results represent the first step in development of
methods to separate and successfully identify plasma membrane proteins from
advanced ovarian cancer cells. Further characterization of plasma membrane
proteins will contribute to our understanding of the mechanisms underlying
progression of this deadly disease and may lead to new targeted interventions
as well as new biomarkers for diagnosis.
Keywords: plasma membrane, mass spectrometry, ovarian cancer, glycosylation
Journal: Disease Markers, vol. 19, no. 4-5, pp. 219-228, 2003,2004
