Investigation of the Lysosomal Proteome and the Phosphorylation Dynamics of Transcription Factor EB Using Mass Spectrometry

Abstract

Lysosomes, the organelles central for degradation, are involved in a variety of cellular pathways including immune response, metabolic signaling, and exocytosis. To date, there are ca. 400 proteins known to be located in the lysosomal lumen, its membrane, or being lysosomal membrane-associated, including ~60 lysosomal hydrolases. These hydrolases are responsible for the degradation of lysosomal substrates and a malfunction of any of them can result in lysosomal storage disorders (LSDs), a group of ~70 rare hereditary diseases. Due to the low abundance of lysosomal proteins, their unbiased proteomic analysis typically requires the prior enrichment of lysosomes. To circumvent this bottleneck, we designed a parallel reaction monitoring (PRM) assay targeting 340 lysosomal proteins and compared its performance to data-independent acquisition (DIA) runs of differently complex samples. Regarding the lysosomal proteins, PRM exhibited superior quantification with lower variability than DIA, especially in higher-complex samples. In a spike-in experiment, PRM more accurately quantified changes in lysosomal protein intensities, underlining its suitability for studying low-abundant proteins, even in highly complex backgrounds. We further applied PRM to investigate post-translational modifications (PTMs) of the transcription factor EB (TFEB), often described as the master regulator of autophagy and lysosomal biogenesis. After identifying 42 phosphorylation sites using untargeted MS, 16 of them being novel, we created three PRM assays targeting 34 phosphorylation sites. We identified S121 as a priming site for the subsequent phosphorylation of S108 and S113. We additionally examined TFEB’s subcellular localization, interactome, and PTM profile in response to metabolic alterations. Lipid starvation induced a non-canonical mTORC1 pathway, sequestering TFEB in the cytosol, possibly via interactions with RNA stress granules and the RNA nuclear exosome complex. Aiming to combine the advantages of PRM and DIA in one, we designed a method in which the two approaches are coupled to each other, thereby merging the sensitivity and quantitative accuracy of PRM with the unbiased proteome-wide analysis of DIA. This reduced both the required measurement time and sample amount by 50%. We applied DIA-PRM to a mouse model of metachromatic leukodystrophy, an LSD in which sulfatide accumulates in lysosomes, and revealed a progressive, age-dependent upregulation of distinct classes of lysosomal proteins, as well as systematic changes in proteins related to myelin and cellular metabolism. <br/> In summary, this work underlines the advantages of PRM for the investigation of low-abundant proteins and PTMs. It further demonstrates the strength of the combined DIA-PRM method, especially in the context of disease-related proteomics. The presented methods expand the analytical toolbox for lysosomal research and provide the first assay to comprehensively map TFEB PTMs and their interdependencies.