Fasimoye, R. | bioRxiv Science

bioRxiv2024

Tagless LysoIP method for molecular profiling of lysosomal content in clinical samples

Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models allowed major discoveries in the field, however studying lysosomal dysfunction in human patients remains challenging. Here, we report the development of the "tagless LysoIP method" to enable rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g. induced Pluripotent Stem Cell (iPSCs) derived neurons). Isolated lysosomes are intact and suitable for subsequent multimodal omics analyses. To validate our approach, we employed the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells (PBMCs) derived from fresh blood from patients with CLN3 disease, a neurodegenerative LSD. Metabolic profiling of isolated lysosomes showed massive accumulation of glycerophosphodiesters (GPDs) in patients lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify novel biomarkers and discover human-relevant disease mechanisms.

cell biology↗

bioRxiv2022

Golgi-IP, a novel tool for multimodal analysis of Golgi molecular content

The Golgi is a membrane-bound organelle that is essential for protein and lipid biosynthesis. It represents a central trafficking hub that sorts proteins and lipids to various destinations or for secretion from the cell. The Golgi has emerged as a docking platform for cellular signalling pathways including LRRK2 kinase whose deregulation leads to Parkinson disease. Golgi dysfunction is associated with a broad spectrum of diseases including cancer, neurodegeneration, and cardiovascular diseases. To allow the study of the Golgi at high resolution, we report a rapid immunoprecipitation technique (Golgi-IP) to isolate intact Golgi mini-stacks for subsequent analysis of their content. By fusing the Golgi resident protein TMEM115 to three tandem HA epitopes (GolgiTAG), we purified the Golgi using Golgi-IP with minimal contamination from other compartments. We then established an analysis pipeline using liquid chromatography coupled with mass spectrometry to characterize the human Golgi proteome, metabolome and lipidome. Subcellular proteomics confirmed known Golgi proteins and identified novel ones. Metabolite profiling established the first known human Golgi metabolome and revealed the selective enrichment of uridine-diphosphate (UDP) sugars and their derivatives, which is consistent with their roles in protein and lipid glycosylation. Furthermore, targeted metabolomics validated SLC35A2 as the subcellular transporter for UDP-hexose. Finally, lipidomics analysis showed that phospholipids including phosphatidylcholine, phosphatidylinositol and phosphatidylserine are the most abundant Golgi lipids and that glycosphingolipids are enriched in this compartment. Altogether, our work establishes a comprehensive molecular map of the human Golgi and provides a powerful method to study the Golgi with high precision in health and disease states. SignificanceThe Golgi is central to protein and lipid processing. It senses and responds to diverse cell states to allow trafficking of macromolecules based on cellular demands. Traditional techniques for purifying the Golgi shaped our understanding of its functions, however such methods are too slow to preserve the labile Golgi metabolome and transient protein interactions. Here, we overcome this issue through the development of a method for the rapid capture of intact Golgi from human cells using organelle-specific immunoprecipitation (Golgi-IP). Using high resolution mass spectrometry, we demonstrate that our approach allows the unbiased characterization of the Golgi proteome, metabolome and lipidome. Thus, we believe that the Golgi-IP will be useful for the study of the Golgi in health and disease states.

cell biology↗