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.
Liang
Xiquan
X
Life Sciences R&D, Protein Analysis, Invitrogen, Carlsbad, California 92008, USA.
Zhao
Jenson
J
Hajivandi
Mahbod
M
Wu
Rina
R
Tao
Janet
J
Amshey
Joseph W
JW
Pope
R Marshall
RM
D000368:Aged; D000595:Amino Acid Sequence; D001940:Breast / Q000737:chemistry / Q000378:metabolism; D001943:Breast Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002277:Carcinoma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D002853:Chromatography, Liquid; D015536:Down-Regulation; D005260:Female; D006801:Humans; D007120:Immunochemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010455:Peptides; D020543:Proteome