This in situ strategy opens the door to mapping dynamic macromolecular machines directly in their cellular context, and hints that much has been missed by previous AP-MS studies of the interactome.

Using PhIX-MS combined with cryo-EM and integrative modeling, we discovered how the ubiquitination enzyme UBE3C, the reductase TXNL1, and the assembly factor PSMD5 bind to the proteasome, providing important information on their functions.

Many proteasome binders are transient or low-affinity and are very difficult to visualize with conventional methods. To overcome this, we developed PhIX-MS (Photo-induced In situ Crosslinking Mass Spectrometry) to map native protein interactions directly inside cells.