Analysis Of The Secretome Of Breast Cancer Cell Lines

Secretome, the totality of secreted proteins, is viewed as a promising pool of candidate cancer biomarkers. In present work, we established several methods to identify secretome of breast cancer cell lines.First, we established an optimized semi-shotgun method to analyze the secretome of breast cancer cell line MDA-MB-231. We optimized the cell culture condition of MDA-MB-231 in serum free medium to control the cell death rate <3%. The concentration, desalting and fractionation of secretome sample were completed in a single step using the small reverse phase C2 column. We modulated flow speed and volume of graded elution buffer to achieve best protein recovery and better fractionation of samples. Then 5 fractions were digested and analyzed by Liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. A total of 464 proteins were identified. 84 (22.8%) of these proteins were classified as extracellular proteins, and 127 (34.4%) were membrane proteins, including many promising breast cancer biomarkers, which were thought to be correlated with tumorigenesis, tumor development and metastasis. These results demonstrate that our optimized method is a useful strategy for cell line secretome profiling, and could be used to find potential cancer biomarkers.Second, we established method to analyze glycoproteins in the secretome of breast cancer cell lines. To reduce the contamination from intracellular proteins, further fractionation method of secreted proteins is desired. Most secreted proteins are glycosylated, and the glycosylation of proteins are tightly linked to cancer initiation and progression. We used multiple lectins (Con A and WGA) to capture glycoproteins in the concentrated conditioned media of breast cancer cell line MCF-7 and breast epithelial cell line MCF-10A, respectively. The captured glycoproteins were digested and then analyzed by LC-MS/MS. A totality of 320 proteins was identified. Among them, 92.2% were found to be glycoproteins, and about 64.4% were identified as secreted proteins and membrane proteins. We found that more glycoproteins were identified in the secretome of MCF-7, suggesting the glycosylation of cancer cell lines is altered. Furthermore, 140 secreted glycoproteins were found to be up-regulated at least 1.5-fold in MCF-7. These proteins could be the pool of potential biomarkers for breast cancer. Our results indicate the analysis of glycoproteome is an effective way to identify potential biomarker in the conditioned media of cell lines.In summary, we established two methods to analyze the secretome of cell lines. These methods are fundamental and critical for us to further explore the potential cancer biomarkers in the secretome of cell lines.