Miniaturized Lysosome Enrichment through Selective Plasma Membrane Destabilization

Organelle enrichment presents a prerequisite for the unbiased biochemical analysis of cellular subcellular compartments. The performance of this step is decisive for the experiments 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. HighlightsO_LIRelease of intact lysosomes negatively correlates with cell concentration. C_LIO_LICombination of detergent-based plasma membrane destabilization and mechanical homogenization increases lysosomal intactness from low cell numbers. C_LIO_LIEnrichment columns require a minimum sample input. C_LIO_LIImmunoprecipitation of intact lysosomes enables enrichment from lower cell numbers. C_LIO_LIProteomics of low input lysosome enriched fractions identifies superior performance. C_LI MotivationModern mass spectrometry (MS)-based proteomics strategies facilitate the detection and quantification of peptides and proteins with unprecedented sensitivity, enabling the analysis of low input samples down to single-cells. However, subcellular fractionation experiments typically require tens of millions of cells to achieve sufficient yield and purity, presenting a strong contrast. This restricts the application of organelle profiling to cell lines that can be grown in sufficient amounts, excluding many physiologically relevant species which are only available in small quantities. To be able to miniaturize subcellular fractionation experiments, it is of crucial importance to overcome this limitation and to miniaturize sample preparation strategies.