Thelen Melanie, Winter Dominic, Braulke Thomas, Gieselmann Volkmar
Institute for Biochemistry and Molecular Biology, Rheinische-Friedrich-Wilhelms-University, Bonn, Germany.
Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Methods Mol Biol. 2017;1594:1-18. doi: 10.1007/978-1-4939-6934-0_1.
Mass spectrometry-based proteomics of lysosomal proteins has led to significant advances in understanding lysosomal function and pathology. The ever-increasing sensitivity and resolution of mass spectrometry in combination with labeling procedures which allow comparative quantitative proteomics can be applied to shed more light on the steadily increasing range of lysosomal functions. In addition, investigation of alterations in lysosomal protein composition in the many lysosomal storage diseases may yield further insights into the molecular pathology of these disorders. Here, we describe a protocol which allows to determine quantitative differences in the lysosomal proteome of cells which are genetically and/or biochemically different or have been exposed to certain stimuli. The method is based on stable isotope labeling of amino acids in cell culture (SILAC). Cells are exposed to superparamagnetic iron oxide particles which are endocytosed and delivered to lysosomes. After homogenization of cells, intact lysosomes are rapidly enriched by passing the cell homogenates over a magnetic column. Lysosomes are eluted after withdrawal of the magnetic field and subjected to mass spectrometry.
基于质谱的溶酶体蛋白质组学在理解溶酶体功能和病理学方面取得了重大进展。质谱不断提高的灵敏度和分辨率，结合允许进行比较定量蛋白质组学的标记程序，可用于更深入地了解溶酶体功能不断扩大的范围。此外，对许多溶酶体贮积病中溶酶体蛋白质组成变化的研究可能会进一步深入了解这些疾病的分子病理学。在这里，我们描述了一种方案，该方案可以确定在遗传和/或生物化学上不同或受到某些刺激的细胞的溶酶体蛋白质组中的定量差异。该方法基于细胞培养中氨基酸的稳定同位素标记（SILAC）。细胞暴露于超顺磁性氧化铁颗粒，这些颗粒被内吞并输送到溶酶体。细胞匀浆后，通过将细胞匀浆通过磁柱快速富集完整的溶酶体。撤去磁场后洗脱溶酶体，并进行质谱分析。
