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

Abstract

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.

Quantitative comparison of IMAC and TiO2 surfaces used in the study of regulated, dynamic protein phosphorylation

“…Unfortunately, the specific phosphoresidues in these peptides could not be assigned precisely due to the low intensity of internal fragments in the MS/MS spectra. On the other hand, the peptide at m/z 2322.1 was identified as the heavy, [37]. The signal intensity ratio of EGF-stimulated phosphopeptide to unstimulated phosphopeptide was greater than 20:1, which is consistent with the result obtained by MALDI-TOF-TOF analysis.…”

“…There is increasing evidence in the literature that SILAC is an excellent approach to study differential protein expression and posttranslational modification, especially phosphorylation [29,[33][34][35][36][37]. In this study, we combined SILAC with MMC and MMO to quantify the dynamic changes of tyrosine phosphorylation in response to EGF stimulation in HeLa cells.…”

A Multiplexed Quantitative Strategy for Membrane Proteomics

“…Alternatively SILAC offers more options for selection of isotopically labeled amino acids such as arginine, leucine, lysine, serine, methionine, and tyrosine (12). Although some successes have been reported in analysis of the membrane proteome (13,14), the major drawback of SILAC is that it is most applicable to cell culture rather than tissue or body fluids.…”

La protéomique quantitative par la méthode SILAC

“…Il a par exemple été utilisé avec succès pour identifier des biomarqueurs spécifiques de cellules cancéreuses en comparant lignées de cellules pancréatiques [37] et mammaires [38] normales et tumorales. Le SILAC a également été utilisé pour évaluer des réponses pharmacologiques comme celle déclenchée par l'inhibition du récepteur ERBB2 (ou HER2/neu, human epidermal growth factor receptor 2) dans le traitement de certains cancers du sein [39].…”