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..

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Methionine oxidation plays a critical role in many processes of biologic and biomedical importance, including cellular redox responses and stability of protein pharmaceuticals. Bottom-up methods for analysis of methionine oxidation can suffer from incomplete sequence coverage, as well as an inability to readily detect correlated oxidation between 2 or more methionines. However, the methodology for quantifying protein oxidation in topdown analyses is lacking. Previous work has shown that electron transfer dissociation (ETD)-based tandem mass spectrometry (MS/MS) fragmentation offers accurate and precise quantification of amino acid oxidation in peptides, even in complex samples. However, the ability of ETD-based MS/MS fragmentation to accurately quantify amino acid oxidation of proteins in a top-down manner has not been reported. Using apomyoglobin and calmodulin as model proteins, we partially converted methionines into methionine sulfoxide by incubation in H 2 O 2. Using top-down ETD-based fragmentation, we quantified the amount of oxidation of various ETD product ions and compared the quantified values with those from traditional bottom-up analysis. We find that overall quantification of methionine oxidation by top-down MS/MS ranges from good agreement with traditional bottom-up methods to vast differences between the 2 techniques, including missing oxidized product ions and large differences in measured oxidation quantities. Care must be taken in transitioning ETD-based quantitation of oxidation from the peptide level to the intact protein level.

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.