Quantification of Membrane and Membrane-Bound Proteins in Normal and Malignant Breast Cancer Cells Isolated from the Same Patient with Primary Breast Carcinoma

More than 50% of all major drug targets are membrane proteins, and their role in cell−cell interaction
and signal transduction is a vital concern. By culturing normal and malignant breast cancer cells with
light or heavy isotopes of amino acids (SILAC), followed by cell fractionation, 1D gel separation of
crude membrane proteins, and analysis of the digests using nanoelectrospray LC−MS/MS, we have
quantified 1600 gene products that group into 997 protein families with approximately 830 membrane
or membrane-associated proteins; 100 unknown, unnamed, or hypothetical proteins; and 65 protein
families classified as ribosomal, heat shock, or histone proteins. A number of proteins show increased
expression levels in malignant breast cancer cells, such as autoantigen p542, osteoblast-specific factor
2 (OSF-2), 4F2 heavy chain antigen, 34 kDa nucleolar scleroderma antigen, and apoptosis inhibitor 5.
The expression of other proteins, such as membrane alanine aminopeptidase (CD13), epididymal protein,
macroglobulin alpha2, PZP_HUMAN, and transglutaminase C, decreased in malignant breast cancer
cells, whereas the majority of proteins remained unchanged when compared to the corresponding
nonmalignant samples. Downregulation of CD13 and upregulation of OSF-2 were confirmed by
immunohistochemistry using human tissue arrays with breast carcinomas. Furthermore, at least half
the gene products displaying an expression change of 5-fold or higher have been described previously
in the literature as having an association with cancerous malignancy. These results indicate that SILAC
is a powerful technique that can be extended to the discovery of membrane-bound antigens that may
be used to phenotype diseased cells.
Keywords: Breast Cancer • Quantitative Proteomics • SILAC • Biomarkers • Membrane Proteins • Immunohistochemistry (IHC)