Procedure

  1. In a text editor program (such as Notepad or Microsoft Word), create and save a text file containing all chains of the protein sequence and any internal standards (short peptides, PPPI, etc.) used in FASTA format.
  2. Use the Protein Sequence Editor to import the FASTA file, specify the appropriate modifications, and save the sequence.
  3. See Creating and managing protein sequences.
  4. On the BioPharma Finder Home page, in the Experiment Types pane, select the Peptide Mapping Analysis workflow link.
  5. The Peptide Mapping Analysis page opens.
  6. In the Peptide Mapping Analysis Definition area, type the name of the experiment in the Experiment Name box.

  7. TIP

    Experiment names can contain up to 50 characters, including alphanumeric, space, underscore (“_”), and period (“.”) characters.

  8. IMPORTANT

    You can overwrite a completed experiment by entering the same experiment name.

    You cannot overwrite an experiment that is currently submitted to the run queue or is actively processing or canceling in the run queue.

  9. In the Load Raw Data area, select the Select Raw Data (“...”) button.
  10. The Add Analysis File(s) dialog box opens.

  11. NOTE

    The default file location for raw data files is C:\Xcalibur\data.

  12. In the Add Analysis File(s) dialog box, locate and select the raw data files (.raw) you want to add to the experiment.

  13. IMPORTANT

    Load the reference raw data file as the first file for HDX Peptide Mapping Analysis experiments.

  14. TIP

    To add a set of raw data files to the experiment (for example, replicates of the same sample or samples with various experiment conditions), select contiguous files using the SHIFT key or noncontiguous files using the CTRL key.

  15. Select Open.
  16. The application adds the imported raw data files to the table in the Load Raw Data area.
  17. The order of the files in this table defines the order that they appear in the Results tables on the Process and Review page.
  18. In the Load Raw Data area, in the Condition box, type the experiment condition for each raw data file, as indicated in the table below.
HDX experiment condition assignments

Raw data file

Condition

Reference

Type "Ref" or "ref".

Important: Load the reference raw data file first.

Control: 0% deuteration

For one conformation condition, type "0%".

For multiple conformation conditions, type "A:0%", "B:0%", and so on.

Control: 100% deuteration

For one conformation condition, type "100%".

For multiple conformation conditions, use "A:100%", "B:100%", and so on.

Labeling time points

Use the format "Condition:HH:MM:SS" for each condition.

For example, for condition A with labeling time 2hr:15min:30sec, type "A:2h15m30s".

  1. In the Reference Condition box, select the reference condition for the experiment from the drop-down menu.

  2. NOTE

    The default reference condition is the first entered condition in the Condition box.

  3. The application uses the indicated reference condition to calculate various analytical ratios during processing. These ratios are reported in the Results table(s) on the Process and Review page.
  4. If you load multiple raw data files for an experiment, in the Result Format for Multiple Raw Files area, select either the Batch Processing or Multiconsensus option.

  5. TIP

    For more information on these results formats, see Result formats: Batch Processing and Multiconsensus.

  6. In the Sequence area, select the checkbox in the table row for the sequence you want to assign to the experiment.
  7. The application applies the selected sequence to the experiment during processing.
  8. In the Processing Method area, select the checkbox for the HDX Default Method processing method.
  9. (Optional) Select the Enable Automatic Parameter Values checkbox if you want the application to automatically determine several parameters for the experiment, such as the absolute signal threshold and retention time shift, using the raw data files.
  10. Select Edit Method to open the processing method editing wizard and edit the experiment parameters before processing.
  11. Select Start Processing to submit your experiment for processing.
  12. Once you submit the experiment for processing, the Queue page opens, displaying the jobs in the run queue table.

  13. NOTE

    Depending on the complexity of the data, processing an HDX experiment might take a substantial amount of time to complete.

  14. When the processing job is complete, you can view the protection factor plot on the Hydrogen Deuterium Exchange subpage of the Mapping page.

  15. TIP

    Once the processing of an HDX experiment is complete, the application generates a set series of .csv files containing the HDX experiment results and stores them within the original raw data file folder in a folder named "ExperimentNameHDXResults". You can open these .csv files in an Excel spreadsheet and plot the data as appropriate.

    For more information on the HDX experiment result files, see HDX experiment output files.