Biochemistry II, Theodor Boveri-Institute, University of Würzburg, Würzburg, Germany.
CIBSS Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
Methods Mol Biol. 2023;2643:13-31. doi: 10.1007/978-1-0716-3048-8_2.
Peroxisomes are ubiquitous organelles with essential functions in numerous cellular processes such as lipid metabolism, detoxification of reactive oxygen species, and signaling. Knowledge of the peroxisomal proteome including multi-localized proteins and, most importantly, changes of its composition induced by altering cellular conditions or impaired peroxisome biogenesis and function is of paramount importance for a holistic view on peroxisomes and their diverse functions in a cellular context. In this chapter, we provide a spatial proteomics protocol specifically tailored to the analysis of the peroxisomal proteome of baker's yeast that enables the definition of the peroxisomal proteome under distinct conditions and to monitor dynamic changes of the proteome including the relocation of individual proteins to a different cellular compartment. The protocol comprises subcellular fractionation by differential centrifugation followed by Nycodenz density gradient centrifugation of a crude peroxisomal fraction, quantitative mass spectrometric measurements of subcellular and density gradient fractions, and advanced computational data analysis, resulting in the establishment of organellar maps on a global scale.
过氧化物酶体是普遍存在的细胞器，在许多细胞过程中具有重要功能，如脂质代谢、活性氧物种的解毒和信号转导。了解过氧化物酶体的蛋白质组，包括多定位蛋白，以及最重要的是，改变细胞条件或过氧化物酶体生物发生和功能受损时其组成的变化，对于全面了解过氧化物酶体及其在细胞环境中的多种功能至关重要。在本章中，我们提供了一种专门针对面包酵母过氧化物酶体蛋白质组分析的空间蛋白质组学方案，该方案能够在不同条件下定义过氧化物酶体蛋白质组，并监测蛋白质组的动态变化，包括个别蛋白质向不同细胞区室的重新定位。该方案包括通过差速离心进行亚细胞分级，然后对粗过氧化物酶体级分进行 Nycodenz 密度梯度离心，对亚细胞和密度梯度级分进行定量质谱测量，以及先进的计算数据分析，最终在全球范围内建立细胞器图谱。
