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

Area of Science:

• Cell Biology
• Biochemistry
• Proteomics

Background:

• Lysosomes are crucial organelles in cellular functions and disease pathogenesis.
• Low lysosome abundance necessitates enrichment for effective downstream analysis, particularly proteomics.
• Existing lysosome isolation methods lack systematic, multimodal performance comparisons.

Purpose of the Study:

• To systematically benchmark four common lysosome enrichment techniques.
• To evaluate methods based on yield, purity, membrane integrity, reproducibility, scalability, and cross-contamination.
• To provide guidance for selecting optimal lysosome enrichment strategies for research and clinical applications.

Main Methods:

• Benchmarking of four lysosome enrichment techniques (gradient-based, filter-based, bead-based, subcellular fractionation) using THP-1 cells.
• Multimodal evaluation using nanoparticle tracking analysis, electron microscopy, flow cytometry, Western blotting, and mass spectrometry-based proteomics.
• Assessment of key performance metrics including yield, purity, integrity, reproducibility, scalability, and contamination.

Main Results:

• Gradient-based and bead-based methods demonstrated superior lysosomal enrichment and proteomic purity.
• Subcellular fractionation yielded higher lysosome numbers but exhibited increased variability and contamination.
• Filter-based methods allowed rapid processing but primarily isolated non-intact lysosomes with significant cross-contamination.

Conclusions:

• Substantial performance differences exist among lysosome enrichment methods, impacting downstream analyses.
• Gradient-based and bead-based methods are recommended for high-purity lysosomal proteomics.
• The study underscores the importance of rigorous validation for lysosomal research, guiding method selection based on specific objectives.