Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult-Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model
Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult-Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model
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DOI：
10.1002/pmic.201800469
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发表时间：
2019-06-01
期刊：
影响因子：
3.4
通讯作者：
Stevens, Stanley M., Jr.
中科院分区：
文献类型：
--
作者：
Guergues, Jennifer;Zhang, Ping;Stevens, Stanley M., Jr.
小胶质细胞作为脑内的免疫细胞，可以表现出广泛的激活表型，与多种中枢神经系统疾病有关。目前被广泛研究的小胶质细胞系主要来源于新生啮齿类动物的脑，这限制了它们与成年脑内的稳态功能和疾病相关的神经免疫反应的相关性。最近，一个成年小鼠脑源性小胶质细胞系已经建立，然而，一个全面的蛋白质组数据集仍然缺乏。在这里，描述了一种灵敏和快速的小胶质细胞蛋白质组分析的优化方法，该方法包括悬浮捕捉法(S陷阱)，用于从预计从成年小鼠脑中获得的有限数量的小胶质细胞(大约300000个)中高效和可重复地提取蛋白质。利用75 cm UPLC色谱柱上的2 h梯度和改进的数据依赖采集方法，在四极轨道混合质谱仪上共鉴定了4855个蛋白质，其中4698个蛋白质可通过无标记定量进行定量，中位变异系数为6.7%，平均变异系数为10.6%。这个数据集突出了成人来源的小胶质蛋白质组的高度蛋白质组覆盖率和相关的定量精度，包括与免疫反应相关的几个关键途径相关的蛋白质。数据可通过标识符为PXD012006的ProteomeXchange获得。
Microglia, as the resident brain immune cells, can exhibit a broad range of activation phenotypes, which have been implicated in a multitude of central nervous system disorders. Current widely studied microglial cell lines are mainly derived from neonatal rodent brain that can limit their relevance to homeostatic function and disease-related neuroimmune responses in the adult brain. Recently, an adult mouse brain-derived microglial cell line has been established; however, a comprehensive proteome dataset remains lacking. Here, an optimization method for sensitive and rapid quantitative proteomic analysis of microglia is described that involves suspension trapping (S-Trap) for efficient and reproducible protein extraction from a limited number of microglial cells expected from an adult mouse brain (approximate to 300000). Using a 2-h gradient on a 75-cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole-Orbitrap mass spectrometer, 4855 total proteins have been identified where 4698 of which are quantifiable by label-free quantitation with a median and average coefficient of variation (CV) of 6.7% and 10.6%, respectively. This dataset highlights the high depth of proteome coverage and related quantitation precision of the adult-derived microglial proteome including proteins associated with several key pathways related to immune response. Data are available via ProteomeXchange with identifier PXD012006.