Global cellular proteo-lipidomic profiling of diverse lysosomal storage disease mutants using nMOST.

Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multi-omic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in NPC1 ^-/- and NPC2 ^-/- mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lyso-phosphatidylcholine species and multi-lamellar structures visualized by cryo-electron tomography. Loss of mitochondrial cristae, MICOS-complex components, and OXPHOS components rich in iron-sulfur cluster proteins in NPC2 ^-/- cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.
Competing Interests: DECLARATION OF INTERESTS J.W.H. is a consultant and founder of Caraway Therapeutics (a wholly owned subsidiary of Merck & Co, Inc) and is a member of the scientific advisory board for Lyterian Therapeutics. B.A.S. is a co-founding scientific advisory board member of Interline Therapeutics and on the scientific advisory boards of Biotheryx and Proxygen. J.M.P. holds a position on the advisory board of Thermo Fisher Scientific. J.J.C. is a consultant for Thermo Fischer Scientific. Other authors declare no competing interests. S.P.G. is on the advisory board for Thermo Fisher Scientific, Cedilla Therapeutics, Casma Therapeutics, Cell Signaling Technology, and Frontier Medicines.