Inline Liquid Chromatography-Fast Photochemical Oxidation of Proteins Allows for Targeted Structural Analysis of Conformationally Heterogeneous Mixtures

Abstract

Structural analysis of proteins in a conformationally heterogeneous mixture has long been a difficult problem in structural biology. In structural analysis by covalent labeling mass spectrometry, conformational heterogeneity results in data reflecting a weighted average of all conformers, complicating data analysis and potentially causing misinterpretation of results. Here, we describe a method coupling size exclusion chromatography (SEC) with hydroxyl radical protein footprinting using inline fast photochemical oxidation of proteins (FPOP). Using a controlled synthetic mixture of holomyoglobin and apomyoglobin, we demonstrate that we can achieve accurate footprints of each conformer using LC-FPOP when compared to off-line FPOP of each pure conformer. We then applied LC-FPOP to analyze the adalimumab heat-shock aggregation process. We found that the LC-FPOP footprint of unaggregated adalimumab was consistent with a previously published footprint of the native IgG. The LC-FPOP footprint of the aggregation product indicated that heat shock aggregation primarily protected the hinge region, suggesting this region is involved with the heat shock aggregation process of this molecule. LC-FPOP offers a new method to probe dynamic conformationally heterogeneous mixtures such as biopharmaceutical aggregates, and obtain accurate information on the topography of each conformer.