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GeLC-FAIMS-MS workflow for in-depth middle-down proteomics
Ayako Takemori1, Philipp T Kaulich2, Ryo Konno3
1Advanced Research Support Center, Institute for Promotion of Science and Technology, Ehime University, Ehime, Japan.
Proteomics
|August 7, 2023
Summary
Middle-down proteomics (MDP) using GeLC-FAIMS-MS enables deeper analysis of large proteins. This method enhances detection of protein groups and post-translational modifications beyond conventional top-down proteomics.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Middle-down proteomics (MDP) characterizes large proteins difficult for top-down proteomics (TDP).
- Existing MDP methods face limitations in detecting extensive protein information.
Purpose of the Study:
- To apply and validate the GeLC-FAIMS-MS workflow for deep middle-down proteomics.
- To demonstrate both global and targeted MDP applications for enhanced proteoform analysis.
Main Methods:
- Utilized optimized limited Glu-C digestion for generating large peptides (>3 kDa).
- Implemented a multidimensional separation: gel electrophoresis, C4 reversed-phase liquid chromatography, and FAIMS MS.
- Applied GeLC-FAIMS-MS for both global and targeted analysis of proteins, including integrins in exosomes.
Main Results:
- Achieved significant increases in detectable peptide length and sequence coverage.
- Enhanced the detection of protein groups and post-translational modifications (PTMs).
- Successfully applied targeted MDP for specific protein analysis in exosomes.
Conclusions:
- GeLC-FAIMS-MS provides an in-depth MDP approach, expanding proteoform characterization.
- This method complements TDP by increasing detectable protein information and PTMs.
- Supports broader exploration of the proteome beyond conventional methods.
