Research

Protein-protein Interactions

Confident identification and quantitation of system-level or large-scale protein interaction networks are very challenging. One of the most common techniques for studying protein interaction comprehensively is AP-MS/CoIP-MS. This technique enriches a protein from a complex mixture along with its interacting partners by using an antibody (co-immunoprecipitation) or by engineering an affinity tag, which serves as a recognition site for further purification of that protein by affinity chromatography. Co-IP/AP-MS can efficiently identify stable interactions with the target proteins. Unfortunately, transient and weak interactions play a significant role in a cell’s signaling cascade. However, the strong purification conditions applied in the isolation steps remove most of these interactions. Additionally, this technique provides information about protein interactions at the cellular level but very little information about the protein domain involved in the interactions.

To overcome these limitations, a chemistry-focused technique combined with mass spectrometry has appeared to capture nearby proteins utilizing their reactive amino acids. The method exploits a reactive compound called crosslinkers, which can attach two nearby proteins or protein complexes by chemical reactions before or after cell lysis. Crosslinkers can be two types, traditional non-cleavable or cleavable. A crosslinking reaction produces a covalently bound protein complexes so stable, transient, and weak interactions are fixed; therefore, strong purification conditions are unable to detach them. After proteolysis, crosslinking products (intra, inter, and dead end), especially inter crosslinked peptides (two peptides connected with the crosslinkers) provide information about protein interactions. In addition, depending on the reactive groups, these crosslinkers hydrolyze and produce dead-end crosslinking on the proteins. This information also provides the surface topologies of the proteins in their native biological conditions. Overall, this crosslinking method can provide confidence in identifying large-scale protein-to-protein interactions as well as structural information of protein complexes in their native conditions.