Identification of mitochondrial proteins associated with cyanine 5 using a combination of affinity-based photocrosslinking with bio-orthogonal supramolecular latching

Visualization of mitochondria with cyanine dyes has been exploited to study the functions of mitochondria. However, proteins interacting with cyanine dyes have not been investigated clearly. Here, we report a chemical proteomics approach for the identification of mitochondrial proteins associated with cyanine dyes. This approach uses affinity-based photocrosslinking of Cy5-diazirine-alkyne (Cy5-DAyne) in combination with selective enrichment of target proteins by bio-orthogonal supramolecular latching based on unique host-guest interaction between cucurbit[7]uril (CB[7]) and its selected guest such as adamantylammonium (AdA). The enriched proteins then were analyzed using LC-MS/MS to provide a list of proteins closely associated with the Cy5 motif. This study suggests that the proteomic environment around the Cy5 motif is highly associated with energy production processes, including the respiratory chain, ATP synthesis, and fatty acid oxidization. Moreover, it demonstrates the potential of this chemical proteomic approach for the investigation of other organellar proteins using different selective dyes.