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

We describe a comparison of individual approaches for the enrichment of lysosomes comparing four different strategies using data independent acquisition (DIA). As a benchmark, we chose a crude enrichment strategy, which is the centrifugation of the PNS at 20,000 x g termed organelle enriched pellet (OEP), a two-step density gradient centrifugation (SDGC) approach employing sucrose, histodenz, and percoll, the SPIONs approach, which is based on the endocytosis of dextran coated nano-particles and their delivery to the lysosomal compartment, and the TMEM192-HA immunoprecipitation a 3 x HA tagged version of the lysosomal membrane protein TMEM192 used for lysosome isolation by anti-HA antibodies immobilized on magnetic beads. We evaluated the abundance and distributions of lysosomal, as well total proteins, allowing for the determination of lysosomal enrichment among the different strategies.