Absolute Quantification of Lysosomal Proteins by Multiple Reaction Monitoring Mass Spectrometry and QconCAT Protein Standards

Abstract

ABSTRACTLysosomes are membrane-enclosed organelles that play a crucial role in the degradation of intra- and extracellular substrates and the regulation of metabolic signaling. These functions are carried out by a variety of proteins, of which > 150 are currently known to be located in the lysosomal lumen or to be embedded in its membrane. These proteins are typically low abundant, necessitating organelle enrichment experiments to enable their investigation by unbiased mass spectrometry-based proteomics analyses. Enrichment strategies have been applied in a plethora of studies to gain a deeper understanding of lysosomal composition and relative changes of lysosomal proteome abundance in a variety of pathological conditions. Such experiments are restricted, however, to selected cell lines and tissues and do not allow a direct analysis of the lysosomal proteome from whole cell or tissue lysates. Furthermore, they do not provide absolute quantities. We developed a multiple reaction monitoring mass spectrometry-based assay for the highly sensitive and reproducible absolute quantification of 143 mouse lysosomal proteins from any type of sample following the QconCAT strategy. We applied our approach to the investigation of mouse embryonic fibroblast whole cell lysates and lysosome-enriched fractions, providing absolute copy numbers per cell/lysosome for lysosomal hydrolases and membrane proteins. These data reveal a dynamic range of more than three orders of magnitude in protein expression and strong differences in the subcellular distribution of individual lysosomal proteins. Furthermore, we applied our strategy to the investigation of four primary cell types (macrophages, lung fibroblasts, osteoblasts, and osteoclasts), demonstrating pathway-specific heterogeneity of individual lysosomal protein classes and indicating protein-specific post-transcriptional regulation of expression levels.