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Comprehensive proteomic analysis of mouse embryonic fibroblast lysosomes by mass spectrometry

ABSTRACT: The lysosome is the main degradative organelle in eukaryotic cells. It is involved in diverse cellular functions from intracellular digestion to metabolic signaling. Despite various efforts to enrich for lysosomes and analyze their proteome with mass spectrometry based proteomics approaches, no systematic evaluation of sample preparation parameters for the analysis of lysosome enriched fractions has been performed. Using samples enriched for lysosomes with paramagnetic nanoparticles, we conducted a systematic evaluation of the protocol for the analysis of the lysosomal proteome. We compared centrifugation and protein precipitation for the concentration of lysosome enriched fractions and tested four different digestion methods in combination with each of these approaches. This included filter aided sample preparation (FASP), in-gel digestion, and in solution digestion using either RapiGest or Urea. Furthermore, we evaluated three formats for sample desalting, four gradient lengths for LC-MSMS analysis of unfractionated samples, as well as fractionation by strong anion exchange tip columns into three or six fractions. Using the combined data, we generated a draft map of lysosomal proteome for mouse embryonic fibroblasts as well as a spectral library for the analysis of lysosomes by data independent acquisition (DIA) and evaluated different LC gradient lengths for DIA.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Jasjot Singh

LAB HEAD: Dominic Winter

PROVIDER: PXD014576 | Pride | 2020-02-17

REPOSITORIES: Pride

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Project description:Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in parasitic protists. Whereas the mannose-6 phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide independent sorting was reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, protein targeting and involvement of lysosomes in hydrolase secretion. The organelles were purified using conventional Percoll and OptiPrep density gradient centrifugation, and a novel purification protocol based on phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in the lysosomal proteome of 462 proteins, which were sorted into 21 functional classes. Hydrolases represent the largest functional class and include proteases, lipases, phosphatases, glycosidases, and other hydrolases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate high dynamics of phagolysosomal compartment. Several lysosomal proteins including the cysteine protease TvCP2 were previously shown to be secreted. Our experiment showed that secretion of TvCP2 was strongly inhibited by chloroquine that increased intralysosomal pH and thus indicated TvCP2 secretion through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified in the phagolysosomal proteome divergent homologs of M6P receptor TvMPR, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP that possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as recognition marker for lysosomal hydrolases. Whether TvMPR or other possible receptors are involved in this process and what is the precise structure of the lysosomal recognition marker need to be clarified in future studies.

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Comprehensive proteomic analysis of mouse embryonic fibroblast lysosomes by mass spectrometry

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