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 α (mTNFα) as a model system. Using three therapeutic monoclonal antibodies that bind TNFα, 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 mTNFα homotrimer, compaction of the mTNFα 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.

Covalent labeling and mass spectrometry reveal subtle higher order structural changes for antibody therapeutics.

Monoclonal antibodies are among the fastest growing therapeutics in the pharmaceutical industry. Detecting higher-order structure changes of antibodies upon storage or mishandling, however, is a challenging problem. In this study, we describe the use of diethylpyrocarbonate (DEPC)-based covalent labeling (CL) - mass spectrometry (MS) to detect conformational changes caused by heat stress, using rituximab as a model system. The structural resolution obtained from DEPC CL-MS is high enough to probe subtle conformation changes that are not detectable by common biophysical techniques. Results demonstrate that DEPC CL-MS can detect and identify sites of conformational changes at the temperatures below the antibody melting temperature (e.g., 55 á´¼C). The observed labeling changes at lower temperatures are validated by activity assays that indicate changes in the Fab region. At higher temperatures (e.g., 65 á´¼C), conformational changes and aggregation sites are identified from changes in CL levels, and these results are confirmed by complementary biophysical and activity measurements. Given the sensitivity and simplicity of DEPC CL-MS, this method should be amenable to the structural investigations of other antibody therapeutics.

Investigating Therapeutic Protein Structure with Diethylpyrocarbonate Labeling and Mass Spectrometry.

Protein therapeutics are rapidly transforming the pharmaceutical industry. Unlike for small molecule therapeutics, current technologies are challenged to provide the rapid, high-resolution analyses of protein higher order structures needed to ensure drug efficacy and safety. Consequently, significant attention has turned to developing new methods that can quickly, accurately, and reproducibly characterize the three-dimensional structure of protein therapeutics. In this work, we describe a method that uses diethylpyrocarbonate (DEPC) labeling and mass spectrometry to detect three-dimensional structural changes in therapeutic proteins that have been exposed to degrading conditions. Using ß2-microglobulin, immunoglobulin G1, and human growth hormone as model systems, we demonstrate that DEPC labeling can identify both specific protein regions that mediate aggregation and those regions that undergo more subtle structural changes upon mishandling of these proteins. Importantly, DEPC labeling is able to provide information for up to 30% of the surface residues in a given protein, thereby providing excellent structural resolution. Given the simplicity of the DEPC labeling chemistry and the relatively straightforward mass spectral analysis of DEPC-labeled proteins, we expect this method should be amenable to a wide range of protein therapeutics and their different formulations.