Methionine Oxidation Footprinting in Intact Proteins (MOFIP) Using Top‐Down Proteomics

Mass spectrometry (MS)‐based proteomics methods, including protein footprinting methods such as hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) and hydroxyl radical footprinting (HRF), can give unique insight into protein structure and interactions. These methods primarily utilize bottom‐up proteomics techniques that require the digestion of intact proteins into small peptides before MS analysis. This digestion can obscure structural information relevant to the function of the intact proteoforms. Here, we have developed a novel top‐down footprinting method, Methionine Oxidation Footprinting in Intact Proteins (MOFIP), to probe solvent accessibility in intact proteoforms. For MOFIP, natively folded protein lysates are incubated with and without hydrogen peroxide (H 2 O 2 ) to evaluate solvent accessibility of methionine residues. Top‐down proteomics analysis allows the characterization of the solvent accessibility of each methionine residue within intact proteins to obtain structural information. Here, intact proteins in Escherichia coli ( E. coli ) lysate were used to evaluate the feasibility of complex biological sample analysis using the MOFIP platform. In total, we characterized 69 quantifiable proteoforms that contained at least one methionine residue suitable for methionine footprinting. We evaluated the oxidation state of individual methionine residues within each proteoform upon exposure to H 2 O 2 to determine accessibility to solvent. Upon incubation with H 2 O 2 , solvent‐accessible methionine residues were fully oxidized, and solvent‐inaccessible residues remained unoxidized. Moreover, partially accessible residues showed incomplete oxidation, which may suggest more than one conformation existing in the native cell lysate (e.g., binding and free states). Overall, our novel MOFIP approach successfully characterized the solvent accessibility of methionine residues in intact proteins within complex samples, providing insights into proteoform structure that is difficult to obtain using bottom‐up proteomics. Summary We introduce methionine oxidative footprinting for intact proteins (MOFIP), a top‐down proteomics technique that enables high‐throughput analysis of methionine solvent accessibility at the intact proteoform level. MOFIP has been benchmarked using an E. coli model system, and methionine accessibility was examined in 69 intact proteoforms. Determining methionine accessibility in intact proteins provides structural insights into proteoforms that are difficult to obtain using bottom‐up proteomics methods.