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silac–based quantitative MS analysis for protein-mediated cell-cell interactions

SILAC–based quantitative MS analysis for protein-mediated c...

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中国生物化学与分子生物学会2016年全国学术会议

作者： Xixi Wang Pengbo Yang Shufang Liang State Key Laboratory of Biotherapy and Cancer Center West China HospitalSichuan University / National

In tumor microenvironment, interactions among multiple cell types are critical for cancer progression. To understand the molecular mechanisms of these complex interplays, the secreted protein analysis between malignant cancer cells and the surrounding nonmalignant stroma is a good viewpoint to investigate cell-cell interactions. Here, we develop two methods termed ‘spike-in silac’ and ‘triple-silac’ to quantify the changes of secretome in a co-cultured system of CT26 and Ana-1 cells(Figure 1). As results, compared with mono-cultured cells, 188 and 56 secreted proteins were identified to change in the co-cultured system by ‘spike-in silac’ and ‘triple-silac’ methods respectively. The Figure 2 was shown three representative both-identified proteins(Galectin-1, Cathepsin L1 and Thrombospondin-1) by silac-based mass spectra(MS), in which pairs of isotope labeling peaks reflected the expression change of a protein in the co-cultured cell system. To validate the accuracy of MS analysis, cell supernatant was collected to detect the secretion level of the three proteins by Western blotting, and the results matched very well(Figure 3). So far the ‘spike-in silac’ and ‘triple-silac’ based quantitative MS approaches are sensitive to measure protein secretion changes due to cell-cell interactions of two different types of cells in vitro.

关键词： Secretome Co-culture Quantitative proteomics spike-in silac triple-silac tumor microenvironment

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