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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.

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|August 7, 2023
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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.

Keywords:
AnExSPGlu-C digestionLC-FAIMSPEPPI-MSproteoform

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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.