MSe Collision Energy Optimization for the Analysis of Membrane Proteins Using HDX-cIMS

Area of Science:

• Structural proteomics
• Biophysical characterization
• Mass spectrometry techniques

Background:

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is vital for structural proteomics.
• Coupling ion mobility separation (IMS) with HDX-MS enhances applicability and data quality for complex systems.
• Initial step in HDX-MS workflows involves confirming peptide sequences and retention times from non-deuterated proteins.

Purpose of the Study:

• To optimize collision energy (CE) ramp settings for HDMS^E experiments in membrane protein analysis.
• To evaluate the impact of different CE ramps on peptide identification and sequence coverage.
• To provide recommendations for maximizing peptide identifications in HDX-MS workflows using cIMS.

Main Methods:

• Utilized a Waters SELECT SERIES cIMS-QTOF system for HDMS^E experiments.
• Employed Phosphorylase B, XylE transporter, and Smoothened receptor (SMO) as model membrane proteins.
• Optimized and compared various collision energy ramps (e.g., 10-50 eV) within the HDX workflow.

Main Results:

• A collision energy ramp of 10-50 eV identified the highest number of peptides for Phosphorylase B, XylE, and SMO.
• Optimal CE ramps were found to be protein-specific, with different ramps yielding distinct peptide sets.
• Selecting appropriate CE ramps significantly altered protein sequence coverage, ranging from 4% to 94%.

Conclusions:

• Pooling results from multiple CE ramps in HDMS^E experiments is recommended for comprehensive peptide identification.
• CE ramp optimization is critical for maximizing sequence coverage in HDX-MS studies of membrane proteins.
• The findings offer practical guidance for users of cIMS-coupled HDX-MS systems.