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

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has evolved as an essential technique in structural proteomics. The use of ion mobility separation (IMS) coupled to HDX-MS has increased the applicability of the technique to more complex systems and has been shown to improve data quality and robustness. The first step when running any HDX-MS workflow is to confirm the sequence and retention time of the peptides resulting from the proteolytic digestion of the nondeuterated protein. Here, we optimized the collision energy ramp of HDMS E experiments for membrane proteins using a Waters SELECT SERIES cIMS-QTOF system following an HDX workflow using Phosphorylase B, XylE transporter, and Smoothened receptor (SMO) as model systems. Although collision energy (CE) ramp 10-50 eV gave the highest amount of positive identified peptides when using Phosphorylase B, XylE, and SMO, results suggest optimal CE ramps are protein specific, and different ramps can produce a unique set of peptides. We recommend cIMS users use different CE ramps in their HDMS E experiments and pool the results to ensure maximum peptide identifications. The results show how selecting an appropriate CE ramp can change the sequence coverage of proteins ranging from 4 to 94%.