Pinho Joao Paulo Costa, Bell-Temin Harris, Liu Bin, Stevens Stanley M
Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 E Fowler Ave, ISA 2015, Tampa, FL, 33620, USA.
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Methods Mol Biol. 2017;1598:295-312. doi: 10.1007/978-1-4939-6952-4_15.
Stable isotope labeling by amino acids in cell culture (SILAC) is a versatile mass spectrometry-based proteomic approach that can achieve accurate relative protein quantitation on a global scale. In this approach, proteins are labeled while being synthesized by the cell due to the presence of certain amino acids exclusively as heavier mass analogs than their regular (light) counterparts. This differential labeling allows for the identification of heavy and light forms of each peptide corresponding to two or more different experimental groups upon mass spectrometric analysis, the intensities of which reflect their abundance in the sample analyzed. Relative quantitation is straightforward when SILAC labeling efficiency is high (>99%) and the same cell proteome is used as the quantitation reference, which is typically the case for immortalized cell lines. However, the SILAC methodology for the proteomic analysis of primary cells isolated after in vivo experimentation is more challenging given the low labeling efficiency that would be achieved post-isolation. Alternatively, a stable-isotope-labeled cell line representing the cell type can be used as an internal standard (spike-in SILAC); however, adequate representation of the primary cell proteome with the stable-isotope-labeled internal standard may limit overall protein quantitation, especially for cell types that exhibit a broad range of phenotypes such as microglia, the resident immune cells in the brain. Here, we present a way to circumvent this limitation by combining multiple phenotypes of a single-cell type (the immortalized mouse BV2 microglial cell line) into a single spike-in standard using primary mouse microglia as our model system. We describe the preparation of media, incorporation of labels, induction of four different activation states (plus resting), isolation of primary microglia from adult mice brains, preparation of lysates for analysis, and general guidelines for data processing.
细胞培养中氨基酸稳定同位素标记法（SILAC）是一种基于质谱的通用蛋白质组学方法，可在全球范围内实现准确的相对蛋白质定量。在这种方法中，由于存在某些氨基酸，其质量比常规（轻）对应物更重，蛋白质在细胞合成时被标记。这种差异标记使得在质谱分析时能够识别对应于两个或更多不同实验组的每种肽的重质和轻质形式，其强度反映了它们在所分析样品中的丰度。当SILAC标记效率高（>99%）且使用相同的细胞蛋白质组作为定量参考时，相对定量很简单，对于永生化细胞系通常就是这种情况。然而，对于体内实验后分离的原代细胞进行蛋白质组分析的SILAC方法更具挑战性，因为分离后标记效率较低。或者，可以使用代表细胞类型的稳定同位素标记细胞系作为内标（掺入式SILAC）；然而，用稳定同位素标记的内标充分代表原代细胞蛋白质组可能会限制整体蛋白质定量，特别是对于表现出广泛表型的细胞类型，如脑内常驻免疫细胞小胶质细胞。在这里，我们提出一种方法来规避这一限制，即使用原代小鼠小胶质细胞作为我们的模型系统，将单一细胞类型（永生化小鼠BV2小胶质细胞系）的多种表型组合成一个单一的掺入式标准。我们描述了培养基的制备、标记的掺入、四种不同激活状态（加上静息状态）的诱导、从成年小鼠脑中分离原代小胶质细胞、制备用于分析的裂解物以及数据处理的一般指南。
