Deuterium Exchange Mass Spectrometry

Peptide maps were database searched in ProteinLynX Global server 3.0 (Waters corporation, Milford, MA) with the following processing and workflow parameters: low and elevated intensity thresholds set to 100.0 and 50.0 counts; intensity threshold sets to 750.0 counts; variable modification: N-terminal methylation; non-specific primary digest reagent; false discovery rate set to 4%. Each fragmentation spectrum was manually inspected for assignment confirmation. The peptide map was refined in DynamX 3.0 (Waters corporation, Milford, MA) with a minimum product per amino-acid value of 0.4. DynamX 3.0 was used to extract the centroid masses; only one unique charge state was considered per peptide and no back-exchange correction was performed. HDX results are reported as relative deuterium exchange level expressed in either mass unit or fractional exchange. Fractional exchange data were calculated by dividing the experimental uptake value by the theoretically maximum number of exchangeable backbone amide hydrogens that could be replaced into each peptide in 78.6% excess deuterium. MEMHDX (Hourdel et al. 2016 (link)) was used to visualize and statistically validate the HDX data (Wald test, p-value < 0.01).