Data Processing.
Protein Metrics Inc. (CA) Byos HDX 4.6-37 searched the 6 data files from equilibration buffer samples (0 sec labeling time, control and heat denatured, 3 technical replicates each) to identify (glyco)peptides using MS/MS spectra. The search database included spike and both proteases. Both HCD and EThcD tandem mass spectra contributed to peptide spectral matching. Putative (glyco)peptides were then searched for in data files from all samples at the MS level (and appropriate retention times) to identify both unlabeled and deuterated peptides and visualize their isotopic envelopes. Initial spike results (heat-treatment vs. control, 1271 peptides) were narrowed by 1) default software filters (MS2 score > 15, minimum alt_rank_score/primary_rank_score > 0.99, maximum precursor m/z error ± 40 ppm, maximum retention time deviation ± 5 min) leaving 1056 peptides, and 2) removing peptides with MS/MS score < 150 leaving 106 of 205 glycopeptides, removing peptides with more than ± 10% average, maximum, or minimum “deuteration” in 0 sec samples, and removing peptides causing standard deviations >10% at any labeled time-point, leaving 561 peptides. Additional manual curation involved adjustment of the extracted ion chromatogram (EIC) window used to integrate MS data and generate an isotopic envelope, optimizing the intensity and specificity of that envelope. Peptides with inadequate intensity EICs to estimate deuteration were discarded.