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.