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Methionine Oxidation Footprinting in Intact Proteins (MOFIP) Using Top-Down Proteomics.
Anju Teresa Sunny1, Kellye A Cupp-Sutton1, Zhitao Zhao2
1Department of Chemistry and Biochemistry, University of Alabama, Tuscaloosa, Alabama, USA.
Proteomics
|October 21, 2025
Summary
We developed Methionine Oxidation Footprinting in Intact Proteins (MOFIP), a novel top-down proteomics method. MOFIP reveals methionine solvent accessibility in intact proteins, offering structural insights missed by bottom-up techniques.
Area of Science:
- Proteomics
- Structural Biology
- Biochemistry
Background:
- Mass spectrometry (MS)-based proteomics, including hydrogen-deuterium exchange MS (HDX-MS) and hydroxyl radical footprinting (HRF), offers insights into protein structure and interactions.
- Bottom-up proteomics methods, commonly used in MS, digest proteins into peptides, potentially obscuring structural information of intact proteoforms.
- Existing footprinting techniques often require protein digestion, limiting the analysis of intact proteoform structures.
Purpose of the Study:
- To develop a novel top-down footprinting method, Methionine Oxidation Footprinting in Intact Proteins (MOFIP), for probing solvent accessibility in intact proteoforms.
- To evaluate the feasibility of MOFIP for analyzing complex biological samples, such as Escherichia coli (E. coli) lysate.
- To obtain structural information on intact proteins by characterizing methionine residue solvent accessibility.
Main Methods:
- Developed MOFIP, a top-down proteomics technique utilizing methionine oxidation by hydrogen peroxide (H₂O₂) to assess solvent accessibility.
- Incubated native protein lysates with and without H₂O₂ to differentiate between solvent-accessible and inaccessible methionine residues.
- Analyzed intact proteoforms using top-down MS to characterize the oxidation state of individual methionine residues.
Main Results:
- MOFIP successfully analyzed intact proteins within complex E. coli lysate, characterizing 69 proteoforms with suitable methionine residues.
- Solvent-accessible methionines were fully oxidized, while inaccessible ones remained unoxidized upon H₂O₂ treatment.
- Partially oxidized methionines suggested the presence of multiple conformations in native cell lysates, such as bound and free states.
Conclusions:
- The MOFIP approach effectively determines methionine solvent accessibility in intact proteins within complex biological samples.
- MOFIP provides valuable structural insights into proteoforms that are challenging to obtain with traditional bottom-up proteomics.
- This top-down method enables high-throughput analysis of methionine accessibility at the intact proteoform level, advancing structural proteomics.
