Magnetic Separation of Autophagosomes from Mammalian Cells Using Magnetic–Plasmonic Hybrid Nanobeads

[Image: see text] Developments in subcellular fractionation strategies have provided the means to analyze the protein and lipid composition of organelles by proteomics. Here, we developed ultrasmall magnetic–plasmonic hybrid nanobeads and applied them to the isolation of autophagosomes by applying a magnetic field. The beads were chemically synthesized and comprised an Ag/FeCo/Ag core/shell/shell structure with a mean diameter of 15 nm. The Ag core and the FeCo shell conferred imaging and magnetic separation capabilities, respectively. The nanobeads were transfected into mammalian cells by lipofection. Thirty minutes after lipofection, the nanobeads colocalized with Vps26 and subsequently with LC3. Cell lysates were prepared at the appropriate time points and were subjected to magnetic separation. The separated fraction contained LC3-II, transferrin receptor, and LAMP2, but not LC3-I, suggesting that autophagosomes engulfing endosomal origin had been isolated. The magnetic separation process was completed in less than 30 min, providing a rapid method for isolation of autophagosomes. The present organelle isolation technique using the hybrid nanobeads with imaging and magnetic separation capabilities is highly promising for isolation of other types of organelles such as endosomes and endosome-related organelles.