Mouse osteoclast secretory lysosomes nLC-ESI-MS/MS

To survey the proteome of osteoclast secretory lysosomes, we used superparamagnetic iron oxide nanoparticles (SPIONs) to enrich for these endo-lysosomal-related organelles from murine osteoclast cultures. Briefly, large scale murine bone marrow monocyte (BMM)-derived osteoclast cultures were ‘pulsed’ with SPIONs to encourage uptake into endosomes and then ‘chased’ into secretory lysosomes upon the convergence of SPION-loaded endosomes with lysosomes and secretory pathways. Following the ‘pulse-chase’, osteoclasts were homogenized, SPION-loaded organelles captured-from post-nuclear supernatants using magnetic columns, and enriched organelles eluted and processed for 1D in-gel digestion and mass spectrometry.