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.
Highlights
The ability of a given epithelial cell type to adhere to the basement membrane and to associate in the appropriate geometry with neighboring cells of the same type is dependent upon the expression of appropriate plasma membrane proteins governing cell-cell interactions and adhesion
A total of four EGFR peptides were observed, all with high SEQUEST XCorr scores. These results demonstrate our ability to detect and identify plasma membrane proteins expressed at low abundance and representing several functionally important classes
The most common approach is 2D PAGE for separation coupled with mass spectrometry for protein identification; 2D PAGE analysis of membrane proteins is associated with significant technical difficulties related to the size, hydrophobicity, and heterogeneity via glycosylation of most membrane proteins
Summary
The ability of a given epithelial cell type to adhere to the basement membrane and to associate in the appropriate geometry with neighboring cells of the same type is dependent upon the expression of appropriate plasma membrane proteins governing cell-cell interactions and adhesion. Invasion of basement membranes, and the ability to survive as anchorageindependent cells are likely to involve changes in the expression of key membrane-associated proteins [24]. D.L. Springer et al / Characterization of plasma membrane proteins from ovarian cancer cells tic significance [10]. Springer et al / Characterization of plasma membrane proteins from ovarian cancer cells tic significance [10] This need is compelling in ovarian carcinoma, as presently there are no good diagnostic markers for early stage detection [6]. Over 75% of the women presenting with ovarian cancer have stage III or IV disease, for which the morbidity is still unacceptably high; 95% of these women will die of their cancer within five years of diagnosis [14,15]
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