Benchmarking Lysosome Enrichment Methods: A Guide for Research and Clinical Translation.

Lysosomes, essential organelles involved in diverse cellular processes, are increasingly recognized as central players in the pathogenesis of numerous diseases. Due to their low abundance in whole-cell extracts, enrichment strategies are required for downstream analyses such as proteomics. Despite the availability of various lysosome isolation methods, including density gradient-based separation, filter-based approaches, magnetic bead-based isolation, and subcellular fractionation, a systematic, multimodal comparison of their performance is lacking. Here, four widely used lysosome enrichment techniques are benchmarked using the THP-1 monocytic cell line as a model. Each method has been evaluated for yield, purity, membrane integrity, reproducibility, scalability, and cross-contamination, employing nanoparticle tracking analysis, electron microscopy, flow cytometry, Western blotting, and mass spectrometry-based proteomics. Data reveal substantial differences: gradient-based and bead-based methods provide the highest lysosomal enrichment and proteomic purity, whereas the subcellular fractionation approach yields greater numbers of lysosomes but with increased variability and contamination. Finally, the filter-based method enables rapid processing, but mainly nonintact lysosomes are obtained with significant cross-contamination. These findings provide practical guidance for selecting the appropriate lysosome enrichment strategy, tailored to specific research or clinical objectives. The results also emphasize the need for rigorous validation to ensure the robustness of lysosomal studies in both basic and clinical research settings.