For top down analysis experiments involving MS2 data, the ProSightBP Fragment Map pane on the Process and Review page displays the proteoform-specific fragment map generated by the ProSight Lite application after processing the MS2 spectra. A fragment map shows the location of the PTMs and matching fragment ions within a protein (proteoform) sequence.

Procedure

  1. Open the results of your experiment from the Queue page or from the Load Results page.
  2. See Open results.
  3. The Process and Review page opens by default and displays the results of your top down analysis experiment, including the ProSightBP Fragment Map (if applicable).
  4. On the Process and Review page, select the Intact Fragmentation Results tab at the bottom left (if necessary).
  5. Select the ProSightBP Fragment Map subtab (next to the ProSightBP Output subtab) at the bottom right (if necessary).
  6. Select the row of a peak in the Intact Fragmentation Results table.
  7. The ProSightBP Fragment Map pane displays the fragment maps containing the identified fragment ion results for all of the proteoforms processed under the selected peak.
  8. The name of each proteoform appears above each map, followed by the name of the loaded raw data file (for a batch experiment or there an experiment with only one loaded raw data file) or "Combined Results" (for a multiconsensus experiment with multiple loaded files), and finally the Residue Cleavages (%) value.
  9. image/svg+xml Colored barsrepresentingidentifiedfragement ions C- terminal Green letterrepresentinga modifiedamino acid N-terminal
    ProSightBP Fragment Map pane
  10. If there is only one proteoform generated for the experiment, only one map appears showing the identified fragment ions for that proteoform.
  11. If there are multiple proteoforms generated for the experiment, multiple maps appear. The first map contains the results from the unmodified sequence (if you select to include it as a searched proteoform), followed by one map for each of the generated proteoforms, stacked one on top of the other.
  12. If you load only one raw data file for the experiment or you are running a batch experiment, the results for each map are from one file.
  13. If you load multiple files for a multiconsensus experiment, the results for each map are from the combined results. To generate the combined results, the application sends the MS2 deconvolution mass results from each individual raw data file to the ProSight Lite application for searching. After receiving the individual search results, the application then combines them for display.
  14. Interpret the ProSight fragment map results for your top down analysis experiment according to the following information:
  15. •The map lists the amino acid letters in the protein sequences from left to right and from top to bottom.
  16. •The gray "N" at the top left corner of the map represents the N-terminal of the proteoform.
  17. •The gray "C" at the bottom of the map represents the C-terminal.
  18. •All cysteine (C) residues appear in yellow.
  19. •All modified amino acids appear with green backgrounds.
  20. When you hover your cursor over an individual amino acid letter, a tool-tip box appears showing the corresponding residue number.
  21. The map also contains different vertical bars, depending on the identified ion type:
  22. •A red bar with a serif at the top pointing left represents the termination of a c ion.
  23. •A red bar with a serif at the bottom pointing right represents the start of a z ion.
  24. •A blue bar with a serif at the top pointing left represents the termination of a b ion.
  25. •A blue bar with a serif at the bottom pointing right represents the start of a y ion.
  26. •A green bar with a serif at the top pointing left represents the termination of an a ion.
  27. •A green bar with serif at the bottom pointing right represents the start of an x ion.
  28. These bars can overlap if combinations of identified ions exist.
  29. When you hover your cursor over a colored bar, a tool-tip box appears showing the identified fragment ion type, mass (measured in Da), and number of identifications.
  30. Double-click a colored bar or a set of overlapping colored bars on a fragment map.
  31. The Matching Fragment Detail dialog box appears, displaying the masses and mass differences for the selected bars.
  32. image/svg+xml
    Matching Fragment Detail dialog box
  33. Each row in the table represents one identified fragment ion for the selected location in the sequence.
  34. See Matching Fragment Detail table parameters.
  35. You can save an image (as a .png file) of the ProSightBP Fragment Map by right-clicking in the pane and selecting "Save as (.png)" from the shortcut menu.
  36. See ProSightBP Fragment Map pane commands.