By default, the Hydrogen Deuterium Exchange subpage of the processing method editing wizard is inactive. To edit the parameters on this page, select the Enable HDX checkbox.
The Hydrogen Deuterium Exchange page includes six sections: Basic Parameters, Labeling Conditions, Quench/Digest Conditions, LC Conditions, Protection Factor Chi2 Modeling, and Back Exchange Chi2 Modeling.
Select the Show Advanced Parameters checkbox to edit advanced options that are hidden by default. Normally, these advanced parameters do not need to be edited.
| No. | Description | No. | Description |
|---|---|---|---|
1 | Parameters tab (processing method editing wizard) | 6 | Quench/Digest Conditions area |
2 | Subpage navigation bar | 7 | LC Conditions area |
3 | Enable HDX checkbox | 8 | Show Advanced Parameters checkbox |
4 | Basic Parameters area | 9 | Protection Factor Chi2 Modeling area |
5 | Labeling Conditions area | 10 | Back Exchange Chi2 Modeling area |
The table below describes the parameters on the Hydrogen Deuterium Exchange subpage.
Parameter | Description |
|---|---|
Basic Parameters | |
Peptide Detection | Select this checkbox to perform peptide detection. You can choose to perform peptide detection at the same time as HDX modeling. Or, you can select to perform peptide detection first, manually edit the peptide list, and then perform HDX data processing separately by selecting only the HDX Modeling option. If you select this checkbox, the application does not display the modifications in the Identification column of the Results table for HDX experiments. However, labels for the peptide ions do appear on the experimental MS/MS spectra. If you clear this checkbox, the application displays the modifications but not the labels. |
HDX Modeling | Select this checkbox to perform HDX modeling. |
Number of Simulations | Specifies the number of Monte Carlo simulations to perform. |
Number of Recorded Solutions | Specify the number of top solutions to record. The application discards the remaining solutions. |
Labeling Conditions | |
D Concentration | Specifies the concentration of deuterium in the labeling solution. For example, if you initiate the HDX by a 10-fold dilution of the sample into a D2O buffer, then the D Concentration is 0.9. |
Temperature (°C) | Specifies the temperature at which the application performs the HDX. The application uses this value to calculate the theoretical intrinsic rate of exchange to calculate the protection factor. |
pD (read) | Specifies the pH of the labeling solution as read from a pH meter. The application uses this value to calculate the theoretical intrinsic rate of exchange to calculate the protection factor. |
Quench/Digest Conditions | |
Temperature (°C) | Specifies the temperature during digestion. The application uses this value to calculate the theoretical back exchange rate of each backbone amide hydrogen during digestion. |
pH | Specifies the pH condition during digestion. The application uses this value to calculate the theoretical back exchange rate of each backbone amide hydrogen during digestion. |
LC Conditions | |
Temperature (°C) | Specifies the temperature during liquid chromatography. The application uses this value to calculate the theoretical back exchange rate of each backbone amide hydrogen during peptide separation. |
pH | Specifies the pH value during liquid chromatography. The application uses this value to calculate the theoretical back exchange rate of each backbone amide hydrogen during peptide separation. |
Protection Factor χ2 Modeling | |
Smooth Absolute | Specifies the absolute increase of the χ2 value for determining the weight of the smoothness function. |
Smooth Relative (%) | Specifies the relative percentage of increase of the χ2 value for determining the weight of the smoothness function. |
Differential Absolute | Specifies the absolute increase of the χ2 value for determining the weight of the differential function. |
Differential Relative (%) | Specifies the relative percentage of increase of the χ2 value for determining the weight of the differential function. |
Back Exchange χ2 Modeling | |
Absolute | Specifies the absolute increase of the χ2 value for determining the Lagrange multiplier. To minimize χ2 and maximize smoothness at the same time, the application applies the Lagrange multiplier, which is a weight, to the smoothness function. |
Relative (%) | Specifies the relative percentage of increase of the χ2 value for determining the Lagrange multiplier. |
Select Back-Exchange Internal Standard | (Optional) Select the checkbox for one or more chains to designate as internal back exchange standards (short peptides that exchange very quickly) to help reduce run-to-run variations. These chains come from the protein sequence that you selected for the experiment. If you use the tetrapeptide (PPPI) as an internal standard for the intrinsic rate, select it here. The processing method does not save your internal standard selections because these vary depending on the selected protein sequence or sequences. The next time you use the same method, you must make these selections again. |
Show Advanced Parameters (checkbox) | Select this checkbox to display advanced options that are hidden by default. |