Abstract
Structural analysis of proteins in a conformationally heterogeneous mixture has long been a difficult problem in structural biology, resulting in complex challenges in data analysis or complete failure of the method. In structural analysis by covalent labeling mass spectrometry, conformational heterogeneity will result in data reflecting a weighted average of all conformers, greatly complicating data analysis and potentially causing misinterpretation of results. Here, we describe a method coupling size exclusion chromatography in an HPLC format with Hydroxyl Radical Protein Footprinting (HRPF) using online Fast Photochemical Oxidation of Proteins (FPOP). Using controlled mixtures of myoglobin and apomyoglobin as a model system to allow for controllable conformational heterogeneity, we demonstrate that we can obtain HRPF footprints of both holomyoglobin and apomyoglobin as they elute off of the SEC column. Comparison of online SEC-FPOP data of both mixture components with traditional FPOP data of each individual component shows that we can obtain the exact same footprinting pattern for each conformation in an online format with real-time FPOP. Using this method, conformations within conformationally heterogeneous mixtures can now be individually probed by SEC-FPOP, and the stability of the FPOP label allows this structural information to be retained.