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

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 validate that we can achieve accurate footprints of each conformer using LC-FPOP when compared to offline 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 that 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 that can be separated by SEC such as biopharmaceutical aggregates and to obtain accurate information on the topography of each conformer.