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

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Wim Annaert  

PROVIDER: MSV000080792 | MassIVE | Wed Mar 29 15:26:00 BST 2017

SECONDARY ACCESSION(S): PXD004273

REPOSITORIES: MassIVE