Two-Step Enrichment Facilitates Background Reduction for Proteomic Analysis of Lysosomes

Lysosomes constitute the main degradative compartment of most mammalian cells and are involved in various cellular functions. Most of them are catalyzed by lysosomal proteins, which typically are low abundant, complicating their analysis by mass spectrometry-based proteomics. To increase analytical performance and to enable profiling of lysosomal content, lysosomes are often enriched. Two approaches have gained popularity in recent years, namely, superparamagnetic iron oxide nanoparticles (SPIONs) and immunoprecipitation from cells overexpressing a 3xHA-tagged version of TMEM192 (TMEM-IP). The effect of these approaches on the lysosomal proteome has not been investigated to date. We addressed this topic through a combination of both techniques and proteomic analysis of lysosome-enriched fractions. For SPIONs treatment, we identified altered cellular iron homeostasis and moderate changes of the lysosomal proteome. For overexpression of TMEM192, we observed more pronounced effects in lysosomal protein expression, especially for lysosomal membrane proteins and those involved in protein trafficking. Furthermore, we established a combined strategy based on the sequential enrichment of lysosomes with SPIONs and TMEM-IP. This enabled increased purity of lysosome-enriched fractions and, through TMEM-IP-based lysosome enrichment from SPIONs flow-through and eluate fractions, additional insights into the properties of individual approaches. All data are available via ProteomeXchange with PXD048696.溶酶体（Lysosomes）是多数哺乳动物细胞的主要降解区室，参与多种细胞功能。这类过程大多由溶酶体蛋白催化，而此类蛋白通常丰度较低，给基于质谱的蛋白质组学分析带来了极大挑战。为提升分析性能并实现溶酶体内容物的组学表征，通常需对溶酶体进行富集分离。近年来有两种富集方法得到广泛应用，分别是超顺磁性氧化铁纳米颗粒（superparamagnetic iron oxide nanoparticles, SPIONs）法，以及基于过表达3xHA标签版TMEM192的细胞进行免疫沉淀的TMEM-IP法。迄今为止，尚无研究探究这两种方法对溶酶体蛋白质组的影响。本研究通过联合两种富集技术，并对溶酶体富集组分开展蛋白质组学分析，对该问题展开了探究。针对SPIONs处理组，我们发现细胞铁稳态发生改变，同时溶酶体蛋白质组出现轻度变化。针对TMEM192过表达组，我们观察到溶酶体蛋白表达出现更为显著的变化，尤其是溶酶体膜蛋白以及参与蛋白质转运的蛋白。此外，我们构建了一套基于SPIONs与TMEM-IP两步富集溶酶体的联合策略。该策略可提升溶酶体富集组分的纯度，且通过对SPIONs流出组分与洗脱组分开展TMEM-IP法溶酶体富集，我们进一步解析了两种单一富集方法各自的特性。所有数据均可通过ProteomeXchange数据库获取，登录号为PXD048696。