Formolo Catherine A, Mintz Michelle, Takanohashi Asako, Brown Kristy J, Vanderver Adeline, Halligan Brian, Hathout Yetrib
Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC, USA.
Methods Mol Biol. 2011;694:365-77. doi: 10.1007/978-1-60761-977-2_22.
This chapter provides a detailed description of a method used to study temporal changes in the endoplasmic reticulum (ER) proteome of fibroblast cells exposed to ER stress agents (tunicamycin and thapsigargin). Differential stable isotope labeling by amino acids in cell culture (SILAC) is used in combination with crude ER fractionation, SDS-PAGE and LC-MS/MS to define altered protein expression in tunicamycin or thapsigargin treated cells versus untreated cells. Treated and untreated cells are harvested at different time points, mixed at a 1:1 ratio and processed for ER fractionation. Samples containing labeled and unlabeled proteins are separated by SDS-PAGE, bands are digested with trypsin and the resulting peptides analyzed by LC-MS/MS. Proteins are identified using Bioworks software and the Swiss-Prot database, whereas ratios of protein expression between treated and untreated cells are quantified using ZoomQuant software. Data visualization is facilitated by GeneSpring software.
本章详细描述了一种用于研究暴露于内质网（ER）应激剂（衣霉素和毒胡萝卜素）的成纤维细胞内质网蛋白质组随时间变化的方法。细胞培养中氨基酸的差异稳定同位素标记（SILAC）与粗内质网分级分离、SDS-PAGE和LC-MS/MS相结合，以确定衣霉素或毒胡萝卜素处理的细胞与未处理的细胞中蛋白质表达的变化。在不同时间点收获处理过的和未处理的细胞，以1:1的比例混合并进行内质网分级分离处理。含有标记和未标记蛋白质的样品通过SDS-PAGE分离，条带用胰蛋白酶消化，所得肽段通过LC-MS/MS分析。使用Bioworks软件和Swiss-Prot数据库鉴定蛋白质，而使用ZoomQuant软件定量处理过的和未处理的细胞之间蛋白质表达的比率。GeneSpring软件有助于数据可视化。
