Systematic comparison of strategies for the enrichment of lysosomes by data independent acquisition

In mammalian cells, the lysosome is the main organelle for the degradation of macromolecules and the recycling of their building blocks. Correct lysosomal function is essential, and mutations in every known lysosomal hydrolase result in so-called lysosomal storage disorders, a group of rare and often fatal inherited diseases. Furthermore, it is becoming more and more apparent, that lysosomes play also decisive roles in other diseases, such as cancer and common neurodegenerative disorders. This leads to an increasing interest in the proteomic analysis of lysosomes, for which enrichment is a prerequisite. In this study, we compared the four most common strategies for the enrichment of lysosomes using data independent acquisition. We performed centrifugation at 20,000 x g to generate an organelle enriched pellet, two-step sucrose density gradient centrifugation, enrichment by superparamagnetic iron oxide nanoparticles (SPIONs), and immunoprecipitation using a 3xHA tagged version of the lysosomal membrane pro...