We are investigating the lysosome, the central lytic organelle of mammalian cells, with mass spectrometry-based proteomics and biochemistry/molecular biology approaches. For this purpose, we are developing novel mass spectrometry strategies for the enrichment of lysosomes and the characterization of lysosomal proteins. These methods include the relative and absolute quantification of proteins and posttranslational modifications, the identification of novel lysosomal proteins, the investigation of cell type- and organelle-specific proteins, the identification of protein-protein interactions, and the structural characterization of lysosomal proteins and protein complexes. We apply these techniques to the investigation of cell- and organ-specific differences in lysosomal protein composition, transcriptional regulation of lysosomal proteins, and the effect of pathological conditions on lysosomal composition and signaling.