What are benefits & dangers of using bifunctional crosslinkers when 1st attempting to identify the native components of a multi-protein complex.

Hi,

The use of cross-linkers in protein complex characterization is a broad topic that would require a detailed discussion depending on the specific system and downstream application desired. But just to give you a general overview, there are two steps in which we have used cross-linkers for characterizing native protein complexes: 1) To stabilize the complex at the time of cell breakage and solubilization, in which case we have usually followed the method published by Subbotin & Chait (DOI: 10.1074/mcp.M114.041095) where a small concentration of crosslinker was used to stabilize the complex before isolation; 2) To characterize the connectivity and proximity of the different components of the complex once it is already isolated using cross-linking and mass spectrometry (doi: 10.1074/mcp.M114.041673; doi: 10.1038/nmeth.3617; DOI: 10.1016/j.tibs.2015.10.008). In both cases the advantages are that the use of the cross-linkers could help stabilize the complex and make it less prone to disassembly, specially in the case of labile components, and also provide a wealth of data about the arrangement of the components when combined with MS. The dangers could be decrease of the purification yields, difficulties processing the sample and potential artifacts mainly when the cross-linking regime is not properly titrated and defined (over cross-linking). This is a key step that requires to spend some time defining the conditions (time, temperature, concentration, quenching, etc) that would be optimal for your system. The references above could be a good starting point to help identify conditions that could be applied to your system. Another pitfall is that successful and reliable mass spectrometry requires expertise that could be difficult to find or expensive.

I hope this helps. Best.