使用超LOPIT高分辨率绘制酿酒酵母空间蛋白质组图谱。
Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT.
作者信息
Nightingale Daniel J H, Oliver Stephen G, Lilley Kathryn S
机构信息
Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK.
Cambridge Systems Biology Centre, Department of Biochemistry, University of Cambridge, Cambridge, UK.
出版信息
Methods Mol Biol. 2019;2049:165-190. doi: 10.1007/978-1-4939-9736-7_10.
The subcellular localization of proteins is a posttranslational modification of paramount importance. The ability to study subcellular and organelle proteomes improves our understanding of cellular homeostasis and cellular dynamics. In this chapter, we describe a protocol for the unbiased and high-throughput study of protein subcellular localization in the yeast Saccharomyces cerevisiae: hyperplexed localization of organelle proteins by isotope tagging (hyperLOPIT), which involves biochemical fractionation of Saccharomyces cerevisiae and high resolution mass spectrometry-based protein quantitation using TMT 10-plex isobaric tags. This protocol enables the determination of the subcellular localizations of thousands of proteins in parallel in a single experiment and thereby deep sampling and high-resolution mapping of the spatial proteome.
蛋白质的亚细胞定位是一种极为重要的翻译后修饰。研究亚细胞和细胞器蛋白质组的能力有助于我们更好地理解细胞内稳态和细胞动态。在本章中，我们描述了一种用于在酿酒酵母中对蛋白质亚细胞定位进行无偏差且高通量研究的方法：通过同位素标记进行细胞器蛋白质的超多重定位（hyperLOPIT），该方法涉及酿酒酵母的生化分级分离以及使用TMT 10重等压标签基于高分辨率质谱的蛋白质定量。此方法能够在单个实验中并行测定数千种蛋白质的亚细胞定位，从而实现对空间蛋白质组的深度采样和高分辨率图谱绘制。
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