Sadick Jessica S, Boutin Molly E, Hoffman-Kim Diane, Darling Eric M
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA.
Center for Biomedical Engineering, Brown University, Providence, RI, USA.
Sci Rep. 2016 Sep 26;6:33999. doi: 10.1038/srep33999.
Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular β-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated.
细胞异质性在大多数人体组织中是固有的，这使得对特定细胞类型的研究具有挑战性。在此，我们描述了一种基于固定/细胞内靶点的新型分选和蛋白质提取方法，以对细胞亚群进行准确的蛋白质表征。分别使用均一的神经细胞系和异质的大鼠脑细胞进行了验证和可行性测试。将感兴趣的细胞内蛋白质用荧光抗体标记，用于荧光激活细胞分选。从新鲜和固定样本中进行可重复的蛋白质提取需要含有高浓度Tris-HCl和十二烷基硫酸钠的裂解缓冲液，以及高温处理。对于固定、分选后的样本，未观察到蛋白质数量或质量的下降。在可行性实验中，基于细胞内β-III微管蛋白的高信号选择了大鼠神经元细胞的一个主要亚群。这些细胞在特定（磷酸化tau）和非特异性（总tau）蛋白质靶点上显示出与未分选群体明显不同的蛋白质表达差异。我们的方法允许直接从感兴趣的细胞类型中确定更准确的蛋白质谱，并提供了一种平台技术，可对任何细胞亚群进行生化研究。
