Membrane Protein Binding Interactions Studied in Live Cells via Diethylpyrocarbonate Covalent Labeling Mass Spectrometry

Membrane proteins are vital in the human proteome for their cellular functions and make up a majority of drug targets in the U.S. However, characterizing their higher-order structures and interactions remains challenging. Most often membrane proteins are studied in artificial membranes, but such artificial systems do not fully account for the diversity of components present in cell membranes. In this study, we demonstrate that diethylpyrocarbonate (DEPC) covalent labeling mass spectrometry can provide binding site information for membrane proteins in living cells using membrane-bound tumor necrosis factor alpha (mTNF alpha) as a model system. Using three therapeutic monoclonal antibodies that bind TNF alpha, our results show that residues that are buried in the epitope upon antibody binding generally decrease in DEPC labeling extent. Additionally, serine, threonine, and tyrosine residues on the periphery of the epitope increase in labeling upon antibody binding because of a more hydrophobic microenvironment that is created. We also observe changes in labeling away from the epitope, indicating changes to the packing of the mTNF alpha homotrimer, compaction of the mTNF alpha trimer against the cell membrane, and/or previously uncharacterized allosteric changes upon antibody binding. Overall, DEPC-based covalent labeling mass spectrometry offers an effective means of characterizing structure and interactions of membrane proteins in living cells.

Automated summary

Membrane proteins are essential components of the human proteome and major drug targets. This study demonstrates the use of DEPC covalent labeling mass spectrometry to characterize the binding sites of membrane proteins in living cells. The results show changes in labeling extent and location upon antibody binding, indicating alterations in the structure and interactions of the membrane protein. Overall, DEPC-based covalent labeling mass spectrometry offers an effective method for studying membrane protein properties in a cellular context.