Abstract
Lysosomes are the main degradative organelles of cells and involved in a variety of processes including the recycling of macromolecules, storage of compounds, and metabolic signaling. Despite an increasing interest in the proteomic analysis of lysosomes, no systematic study of sample preparation protocols for lysosome enriched fractions has been performed to date. In the current study, we used samples enriched for lysosomes by paramagnetic nanoparticles and systematically evaluated experimental parameters for the analysis of the lysosomal proteome. This includes different approaches for the concentration of lysosome-containing fractions; desalting of samples by solid phase extraction; fractionation of peptide samples; and different gradient lengths for LC-MS/MS analyses of unfractionated samples by data dependent and data independent acquisition. Furthermore, we evaluated four different digestion methods including filter aided sample preparation (FASP), in-gel digestion, and in-solution digestion using either RapiGest or urea. Using the combined data, we generated a benchmark lysosomal proteome data set for mouse embryonic fibroblasts as well as a spectral library for the analysis of lysosomes by data independent acquisition.
Highlights
Background & SummaryLysosomes are the main degradative compartments of mammalian cells and contain a variety of hydrolases which catalyze the breakdown of virtually any cellular macromolecule
Malfunctions of hydrolases leads to the accumulation of their respective substrate, resulting in so-called lysosomal storage disorders (LSDs), a group of about 50 genetically different but phenotypically connected severe diseases[1]
While no mechanisms for the regulation of lysosomal hydrolases are known, it is becoming more and more apparent that lysosomes play an important role in the distribution and regulation of cellular metabolites, and that they are significantly involved in cellular signaling, which is regulated e.g. by phoshorylation[3]
Summary
Background & SummaryLysosomes are the main degradative compartments of mammalian cells and contain a variety of hydrolases which catalyze the breakdown of virtually any cellular macromolecule. We analyzed LC gradient lengths, solid phase extraction resins, peptide fractionation, and concentration approaches for lysosomes and lysosomal proteins in combination with protocols for proteolytic digestion (Fig. 1a, Table 1).
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