Maximizing Sequence Coverage in Top-Down Proteomics By Automated Multimodal Gas-Phase Protein Fragmentation

Area of Science:

• Proteomics
• Mass Spectrometry
• Bioinformatics

Background:

• Top-down protein sequencing using tandem mass spectrometry (MS/MS) requires efficient fragmentation under various conditions for comprehensive amino acid sequence coverage.
• Current methods necessitate optimized experimental parameters for analyzing intact proteins.

Purpose of the Study:

• To develop an automated R-package, "topdownr", for constructing multimodal MS/MS fragmentation methods.
• To systematically analyze spectra generated from diverse fragmentation techniques for intact protein sequencing.
• To derive guiding principles for efficient intact protein sequencing.

Main Methods:

• Development of the "topdownr" R-package for automated MS/MS method construction on an Orbitrap platform.
• Application of multimodal fragmentation (CID, HCD, ETD, ETciD, EThcD, UVPD).
• Analysis of thousands of MS/MS spectra for five intact proteins (10-30 kDa).

Main Results:

• Achieved 90-100% amino acid sequence coverage for tested intact proteins.
• Generated and analyzed extensive MS/MS spectral data.
• Established guiding principles for optimizing intact protein sequencing.

Conclusions:

• The "topdownr" R-package and multimodal MS/MS approach provide a framework for optimizing protein sequencing.
• The software facilitates comprehensive characterization of protein pharmaceuticals.
• Future applications include de novo sequencing and detailed characterization of intact proteins.