MassIVE MSV000080792

Imported Reanalysis Dataset Public PXD004273

LFQ comparison of total cell lysate, lysosomal and plasma membrane fraction of WT and NPC1 KO cells

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Description

Superparamagnetic iron oxide nanoparticles (SPIONs) have so far mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains largely underexploited. We engineered surface functionalized SPIONs (Ø 10 nm) that target very distinct subcellular compartments. Anionic dimercaptosuccinic acid-coated SPIONs are efficiently internalized and accumulate time-dependently in late endosomes and lysosomes, while cationic aminolipid-coated SPIONs surprisingly strongly reside considerably at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for late endosomes/lysosomes and plasma membranes with as consolidated by biochemical, ultrastructural analyses, and to a yield and purity allowing subsequent proteomic and lipidomic profiling. We validated the strength of our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and HeLa to those of HeLa cells deficient in Niemann-Pick disease type C1 (NPC1) deficient HeLa cells expression. While the plasma membrane composition remaineds largely unaltered, pronounced alterations in several protein and lipid species including cholesterol wereare observed in isolated lysosomes reflecting vesicular transport obstruction jamming and deficient lysosomal turnover resulting from NPC1 deficiency. The technology thus allows high-resolution analysis of proteins and lipids. It also, and provides a major advance step forward in fingerprinting subcellular compartments, with an increased potential to identify subtle alterations in biomolecular compositions of lysosomes and/or plasma membranes. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Niemann-Pick disease type C1 ; lysosomal fraction ; superparamegnetic iron oxide nanoparticles ; label-free quantification

Contact

Principal Investigators:
(in alphabetical order) 		Wim Annaert, Laboratory for Membrane Trafficking Center for Human Genetics (KULeuven) & VIB-Center for the Biology of Disease, N/A
Submitting User: ccms

Publications

Tharkeshwar AK, Trekker J, Vermeire W, Pauwels J, Sannerud R, Priestman DA, Te Vruchte D, Vints K, Baatsen P, Decuypere JP, Lu H, Martin S, Vangheluwe P, Swinnen JV, Lagae L, Impens F, Platt FM, Gevaert K, Annaert W.
A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency.
Sci Rep. Epub 2017 Jan 30.

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