Protein cross-linking of the multi-protein complex of S100A8 and S100A9 with TLR4/MD2: Considerations regarding chemistry and efficiency

摘要

Calcium-binding proteins S100A8 and S100A9 are important in inflammation and have been identified as endogenous ligands of TLR4 [1]. In order to elucidate the interaction site, cross-linking experiments in combination with mass spectrometry were performed. Due to the facts that TLR4 (70 kDa) is always accompanied by MD2 protein and the S100A8/A9 proteins form active multimers, the cross-linked complex can consist of up to four proteins so that the resulting peptide mixture is highly complex. Several types of bifunctional cross-linkers were used with varying success due to side reactions and low yields.