There are several factors to consider to ensure that the BioPharma Finder application accurately assesses variance between sample runs and adequately estimates back-exchange. Important considerations for HDX sample preparation and data collection are described in the table below.
Sample | Sample preparation | Data collection |
|---|---|---|
Control samples | ||
0% deuteration | Prepare the 0% deuteration control sample by quenching the protein/H2O sample directly into a buffer of the same concentration used in the analytical samples, followed by digestion. If a 0% deuteration control sample is not available, use an unlabeled sample as a control. Note: Typically, there is negligible difference between a 0% deuteration sample and an unlabeled sample when an on-column digestion is performed. However, there is often a small difference between the two samples when an in-solution digestion is performed. |
|
100% deuteration | To ensure complete labeling all backbone amide groups, prepare the 100% deuteration control sample by running the labeling reaction as follows:
| |
Internal standards | Short peptides can be used as internal standards to correct variations between runs. Additionally, reagents such as tetrapeptide Pro-Pro-Pr-Ile (PPPI) can be used to account for the intrinsic exchange rate in the protein. | |
Precursors | When collecting MS data of precursors, segregate the precursors by mass range or charge state to maximize peptide identification. For example, collect MS/MS of doubly charged precursors in the first run, triply charged precursors in the second run, and all others in the third run, etc. | |
Analytical samples | ||
Sample data collected at various time points | In general, prepare the analytical samples in a way that minimizes the difference in digestion conditions between the control and analytical samples. Collect full-scan data of analytical samples for a minimum of six time points, each in duplicate to ensure the application adequately assesses variance across runs. Important: For HDX Peptide Mapping Analysis experiments, the application assumes that only one major protein exists in the sample; that is, the sample is assumed to be pure. Therefore, Thermo Scientific recommends that you do not use modified peptides in HDX experiments unless you are certain that your sample has been completely modified. Glycosylations are permitted and are an exception to this rule, due to their inherent variability. For example, if your protein contains a methionine residue susceptible to oxidation, you must ensure that your sample is either 100% oxidized (modified) or 100% unoxidized (unmodified) for best results. |
|