SILAC–based quantitative MS analysis for protein-mediated cell-cell interactions
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- 英文论文题名：SILAC–based quantitative MS analysis for protein-mediated cell-cell interactions
- 论文作者：Xixi Wang ; Pengbo Yang ; Shufang Liang
- 英文论文作者：Xixi Wang ; Pengbo Yang ; Shufang Liang ; State Key Laboratory of Biotherapy and Cancer Center ; West China Hospital ; Sichuan University / National ; Collaborative Innovation Center for Biotherapy
- 年：2016
- 作者机构：State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,Sichuan University / National;Collaborative Innovation Center for Biotherapy;
- 英文论文关键词：Secretome ; Co-culture ; Quantitative proteomics ; Spike-in SILAC ; triple-SILAC ; tumor microenvironment
- 会议召开时间：2016-10-20
- 会议录名称：中国生物化学与分子生物学会2016年全国学术会议论文集
- 语种：英文
- 分类号：R730.2
- 学会代码：IGSS
- 会议名称：中国生物化学与分子生物学会2016年全国学术会议
- 会议地点：中国浙江杭州
- 主办单位：中国生物化学与分子生物学会
- 学会名称：中国生物化学与分子生物学会
- 页数：1
- 文件大小：6866k
- 原文格式：D
- 会议级别：全国
摘要
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.
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.
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.
引文