Fajardo-Callejon, S.; Winter, D.

Organelle enrichment presents a prerequisite for the unbiased biochemical analysis of cellular subcellular compartments. The performance of this step is decisive for the experiment's success, and despite the high sensitivity of downstream analytical strategies, such as enzymatic assays, western blotting, or mass spectrometry (MS)-based OMICs approaches, typically tens of millions of cells are required, standing in stark contrast. Here, we demonstrate that selective rupture of the plasma membrane constitutes a limiting factor for the reduction of cell numbers and present an approach to overcome this limitation through detergent-based plasma membrane destabilization, followed by mechanical homogenization. By combination of this strategy with two common methods for lysosome enrichment, namely superparamagnetic iron oxide nanoparticles (SPIONs) and immunoprecipitation via 3xHA-tagged TMEM192 (TMEM IP), we scale down lysosome enrichment to only half a million cells and demonstrate that reduced input cell numbers yield superior results with respect to sample purity and organelle proteome characterization.