Apoptosis Department and Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, Copenhagen, Denmark.
Proteomics. 2011 Jul;11(14):2830-8. doi: 10.1002/pmic.201000734. Epub 2011 Jun 14.
Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic purification of iron-loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar-type H(+) -ATPase. Quantitative MS-based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal membrane proteome was significantly altered by the ectopic expression of an active form of the ErbB2 oncogene, which renders the cells highly metastatic. The furthermost ErbB2-associated changes included increased levels of CD63, S100A11 and ferritin heavy chain. Overall, our data introduce the antibody-based purification of lysosomes as a suitable method for the characterization of lysosomes from a variety of pathological conditions with altered lysosomal density and stability.
晚期内体和溶酶体（以下简称溶酶体）在细胞大分子和细胞器的周转中发挥着重要作用。它们的生化特性迄今为止依赖于基于密度梯度离心或铁加载细胞器的磁性纯化的纯化方法。由于与溶酶体疾病和癌症相关的溶酶体密度和稳定性的剧烈变化，这些方法对于正常和病理溶酶体的比较不是最佳的。在这里，我们介绍了一种通过针对液泡型 H(+) -ATP 酶的抗体进行磁性免疫沉淀来纯化完整溶酶体的有效方法。获得的溶酶体膜的基于 MS 的定量蛋白质组学分析鉴定了 60 种蛋白质，其中大多数先前与溶酶体区室有关。有趣的是，外源性表达具有活性形式的 ErbB2 癌基因显着改变了溶酶体膜蛋白组，使细胞具有高度转移性。与 ErbB2 相关的最远距离变化包括 CD63、S100A11 和铁蛋白重链的水平增加。总体而言，我们的数据介绍了基于抗体的溶酶体纯化作为一种合适的方法，用于表征具有改变的溶酶体密度和稳定性的各种病理条件下的溶酶体。
