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.