Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition...

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Hydrogen/deuterium exchange (HX) coupled with mass spectrometry (MS) is a powerful technique for studying protein structure and dynamics.
• Accurate deuterium incorporation calculations and peptide identification are crucial for reliable HX-MS data analysis.
• Current methods can be time-consuming and require manual validation, limiting throughput and application scope.

Purpose of the Study:

• To introduce a novel HX workflow coupled to tandem mass spectrometry (HX-MS2) for enhanced data acquisition and analysis.
• To enable true auto-curation of HX data through the simultaneous acquisition of peptide precursors and fragment ions.
• To validate the utility of HX-MS2 for high-throughput, proteomics-grade analysis of complex biological samples.

Main Methods:

• Development of a hydrogen/deuterium exchange workflow integrated with tandem mass spectrometry (HX-MS2).
• Utilized data-independent acquisition (DIA) methods compatible with most mass spectrometry platforms.
• Employed collisional-induced dissociation (CID) to generate deuterated fragments for peptide identification and deuterium calculation authentication.
• Applied a combinatorial strategy for confidence assessment of deuterium calculations based on fragment ion redundancy.

Main Results:

• The HX-MS2 workflow enables simultaneous acquisition of peptide precursors and fragment ions, facilitating true auto-curation.
• Deuterated fragments generated by CID confirm peptide identity and authenticate MS1-based deuterium calculations with high confidence.
• The method significantly reduces analysis time and increases throughput for complex samples.
• Demonstrated the application of HX-DIA in drug binding analysis of the protein kinase DNA-PKcs.

Conclusions:

• HX-MS2 provides a robust, proteomics-grade approach for hydrogen/deuterium exchange mass spectrometry.
• The auto-curation capability and high throughput of HX-MS2 streamline complex sample characterization.
• This method offers a straightforward switch for laboratories utilizing DIA, promoting widespread adoption and application in various biological studies.