To set up and validate NTMS processing in Chromeleon CDS software, use an injection of your peptide that was spiked with the PRTC standard. After you have set up NTMS processing, the comparison of a PRTC-spiked sample injection against an unspiked sample injection yields a list of peaks that correspond to the PRTC peptides.

Later, during PQA monitoring, a NTMS processing comparison of your sample injection to a pure standard yields a list of unexpected peaks, should they be present. An injection is marked as Passed if NTMS processing lists no new peaks.

Prerequisites

  1. You have acquired data in a sequence with an unspiked injection and a PRTC-spiked injection.
  2. In the sequence Properties > Features, you have selected BioPharma Finder as the NTMS New Peak Detection Algorithm.

  3. NOTE

    In Chromeleon CDS software, you can choose from two algorithms for new peak detection. For MAM 2.0, the BioPharma Finder algorithm is recommended for compatibility with the entire workflow.

  4. You have opened the sequence in the Chromatography Studio
  5. In the Chromatography Studio, you have selected the NTMS Processing category.
  6. If the work area does not display the Processing Method pane already, use the ribbon to display it.

Procedure

  1. In the navigation pane, select the PRTC-spiked injection.
  2. In the Processing Method pane, select the unspiked injection as the Control Injection.
  3. Check that the Auto-compute basic parameters option is selected and select Preview.
  4. The Processing Method pane is populated with auto-computed values.
  5. Clear the Auto-compute basic parameters option, inspect the auto-computed parameters, and modify their values as needed. For example, consider the following points:
  6. Set the Typical Chromatographic Peak Width to 0.15 – 0.30 mins to allow for minor shifts in retention time between the processed injection and the control injection.
  7. To detect low-abundance peptides, do not change the MS Noise Level value, but decrease the S/N Threshold value.
  8. Crop the time window of the chromatogram, if you do not expect any significant peaks before a certain retention time.
  9. In the Processing group of controls in the ribbon, select Start, and wait for the processing to complete.
  10. The Component Results list in the navigation pane is now populated with a list of all detected components. If you have the Component Results Table displayed in the work area, it lists these components together with their details, like the retention time, the m/z value of the base peak, the charge state, and so on. Note that many of the columns of the results table are pairs, where one column reports the value for the processed injection while the other column reports the value for the control injection.