Utilization of fluorescent probes for the quantification and identification of subcellular proteomes and biological...

Oxidative modifications to cellular proteins are critical in mediating redox-sensitive processes such as autophagy, the antioxidant response, and apoptosis. The proteins that become modified by reactive species are often compartmentalized to specific organelles or regions of the cell. Here, we detail protocols for identifying the subcellular protein targets of lipid oxidation and for linking protein modifications with biological responses such as autophagy. Fluorophores such as BODIPY-labeled arachidonic acid or BODIPY-conjugated electrophiles can be paired with organelle-specific probes to identify specific biological processes and signaling pathways activated in response to oxidative stress. In particular, we demonstrate "negative" and "positive" labeling methods using BODIPY-tagged reagents for examining oxidative modifications to protein nucleophiles. The protocol describes the use of these probes in slot immunoblotting, quantitative Western blotting, in-gel fluorescence, and confocal microscopy techniques. In particular, the use of the BODIPY fluorophore with organelle- or biological process-specific dyes and chromophores is highlighted. These methods can be used in multiple cell types as well as isolated organelles to interrogate the role of oxidative modifications in regulating biological responses to oxidative stress.