Proteomics of the Lysosome
The lysosome is the central lytic organelle of mammalian cells. After it has been regarded for decades as a static and unregulated “cellular waste bag”, it is becoming more and more apparent, that the lysosome is a dynamic and regulated organelle, which is playing a central role in cellular metabolism. This is further underlined by the fact, that malfunctions of lysosomal hydrolases result in a group of >70 rare inherited diseases, so-called lysosomal storage disorders, and that lysosomes can play a decisive role in more common conditions such as cancer and neurodegenerative diseases. We are investigating the composition of lysosomes as well as the lysosomal response to various disease pathologies using mass spectrometry based proteomics. In particular, we optimized strategies for lysosomal enrichment, the proteomic characterization of lysosomal and lysosome-interacting proteins, and the identification of novel lysosomal proteins. Using Quantification Concatemers (QConCats), we generated absolutely quantified stable isotope labeled internal standards for the absolute quantification of lysosomal protein copy numbers and developed a multiple reaction monitoring (MRM) assay covering ~400 peptides from 143 proteins. Analysis of cell lines, isolated lysosomes, and mouse tissues revealed a dynamic range of several orders of magnitude for individual lysosomal proteins and organ-specific abundance profiles. For a better understanding of lysosomal protein-protein interactions and structures, we performed cross linking mass spectrometry experiments with isolated lysosomes. We were able to identify ~4300 cross links from >1300 proteins including 882 interactions for lysosomal and lysosome-associated proteins. This enabled us to identify novel interaction partners, to validate known structures, and to propose novel structures for lysosomal proteins and protein complexes. Taken together, these data provide us with a better view of lysosomal composition, providing valuable information for a better understanding of lysosomal function.