SILAC-based quantitative MS approach for real-time recording protein-mediated cell-cell interactions.
SILAC-based quantitative MS approach for real-time recording protein-mediated cell-cell interactions.
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基于 SILAC 的定量 MS 方法用于实时记录蛋白质介导的细胞间相互作用
DOI：
10.1038/s41598-018-26262-2
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发表时间：
2018-05-31
影响因子：
4.6
通讯作者：
Liang S
中科院分区：
文献类型：
--
作者：
Wang X;He Y;Ye Y;Zhao X;Deng S;He G;Zhu H;Xu N;Liang S
在肿瘤微环境中，多种细胞类型之间的相互作用对肿瘤的进展至关重要。为了了解这些复杂相互作用的分子机制，恶性癌细胞与周围非恶性间质之间的分泌蛋白分析是研究细胞-细胞相互作用的一个很好的观点。在这里，我们开发了两种基于细胞培养（SILAC）的稳定同位素标记氨基酸的质谱(MS)/MS方法，称为峰值SILAC和三重SILAC，以量化细胞共培养系统中蛋白质分泌水平的变化。在CT26和Ana-1细胞共培养系统中，峰值SILAC和三重SILAC质谱方法对定量测量蛋白分泌变化非常敏感。两种基于silac的质谱方法定量的3种代表性蛋白（Galectin-1， Cathepsin L1和Thrombospondin-1）通过Western blotting进一步验证，结果与SILACs的结果吻合较好。我们进一步将这两种SILAC应用于人类细胞系NCM460和HT29共培养体系，评估其可行性，证实了峰值和三倍SILAC能够监测人类细胞系分泌蛋白的变化。考虑到这两种方法在耗时、样品复杂性和蛋白质组覆盖率方面的差异，三重silac方法在实时记录肿瘤微环境中分泌蛋白水平方面表现出更高的效率和经济性。
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 developed two stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry (MS)/MS approaches termed spike-in SILAC and triple-SILAC to quantify changes of protein secretion level in a cell co-cultured system. Within the co-culture system of CT26 and Ana-1 cells, the spike-in SILAC and triple-SILAC MS approaches are sensitive to quantitatively measure protein secretion changes. Three representative quantified proteins (Galectin-1, Cathepsin L1 and Thrombospondin-1) by two SILAC-based MS methods were further validated by Western blotting, and the coming result matched well with SILACs’. We further applied these two SILACs to human cell lines, NCM460 and HT29 co-culture system, for evaluating the feasibility, which confirmed the spike-in and triple SILAC were capable of monitoring the changed secreted proteins of human cell lines. Considering these two strategies in time consuming, sample complexity and proteome coverage, the triple-SILAC way shows more efficiency and economy for real-time recording secreted protein levels in tumor microenvironment.
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影响因子：
3.7
作者：
Brindle NR;Joyce JA;Rostker F;Lawlor ER;Swigart-Brown L;Evan G;Hanahan D;Shchors K
通讯作者：
Shchors K
三席拉克（Triple Silac）确定Wnt途径中的刺激特定相互作用。
影响因子：
4.4
作者：
Hilger, Maximiliane;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
14.8
作者：
Ong, Shao-En;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
14.8
作者：
Geiger, Tamar;Wisniewski, Jacek R.;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
--
作者：
Chen B;Zeng X;He Y;Wang X;Liang Z;Liu J;Zhang P;Zhu H;Xu N;Liang S
通讯作者：
Liang S