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Electron Transfer Dissociation Parameter Optimization Using Design of Experiments Increases Sequence Coverage of
Milos Cejkov1, Tyler Greer1, Reid O'Brien Johnson1
1Analytical Chemistry, Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591-6707, United States.
Journal of the American Society for Mass Spectrometry
|February 17, 2021
Summary
A design of experiments approach optimized mass spectrometry parameters for middle-down analysis of monoclonal antibodies (mAbs). This method significantly increased sequence coverage for antibody subunits, improving structural insights.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Proteomics
Background:
- Middle-down analysis of monoclonal antibodies (mAbs) using tandem mass spectrometry (MS2) offers detailed primary structure insights with minimal sample preparation.
- Enzymatic cleavage into Fc/2, LC, and Fd subunits followed by RPLC-MS2 is standard, but maximizing sequence coverage for structural information is challenging.
Purpose of the Study:
- To optimize MS2 parameters for middle-down analysis of mAbs using a design of experiments (DOE) approach.
- To enhance sequence coverage of antibody subunits by improving electron transfer dissociation (ETD) efficiency.
Main Methods:
- Implementation of a design of experiments (DOE) strategy to optimize MS2 parameters, focusing on electron transfer dissociation (ETD).
- Application of the DOE approach to the NIST monoclonal antibody standard (NISTmAb) using reversed-phase liquid chromatography tandem mass spectrometry (RPLC-MS2).
Main Results:
- Initial DOE application yielded high sequence coverages: 67% (Fc/2), 67% (LC), and 52% (Fd) for NISTmAb subunits.
- Further DOE modeling to maximize fragments across mass ranges resulted in even higher coverages: 75% (Fc/2), 78% (LC), and 64% (Fd).
- The DOE approach achieved high sequence coverage using only ETD fragmentation.
Conclusions:
- A DOE approach effectively optimizes MS2 parameters for middle-down mAb analysis, significantly increasing sequence coverage.
- This strategy enhances structural characterization of antibody subunits and can be adapted for other fragmentation techniques.
