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Adding the PepCalMix to your SWATH ion Library as a Retention Time Calibration Protein
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The most important consideration in adding a new RT calibration protein to your library is making sure you are getting the retention time for both into the same retention time or chromatographic frame. 

Below is a list of steps that walks you through how to do this.

  1. Select a single SWATH acquisition run to act as your RT time frame, this SWATH file must have both the matrix from the main ion library and the PepCalMix dosed in. It doesn’t matter if this isn’t the same gradient you will always use, you just need to set the RT frame to a single frame.
  2. Load your ion library (*.group file) into SWATH 2.0 microapp in PeakView® Software and associate it with the above SWATH datafile.
  3. Perform retention time calibration if needed to adjust the RTs of the ion library to that of the SWATH file.
  4. Save the library using the Saved Ion library with Updated RT option.
  5. Next, load the PepCalMix ion library and associate it with the same SWATH file
  6. Perform retention time calibration, and save the library using the Saved Ion library with Updated RT option.
  7. Open the exported PepCalMix ion library in Excel and first delete the RT-Cal protein that was exported. Next for the remaining peptides in the PepCalMix protein, change the Accession number to [ RT-Cal protein ], the protein name to Retention Time Calibration Protein and N to -1. Save. The reason you have to do this is the RTs that are correct for the SWATH file are in the RT_Detected column of the PepCalMix rows.
  8. Open the main ion library in Excel. Delete the [ RT-Cal protein ] from the main library and paste the new [ RT-Cal protein ] for PepCalMix into the top of the main ion library.
  9. Finally, copy the RT-detected column (column C) over into the iRT column (column M), then rename the column back to iRT. Save the ion library. This now means that all the peptides in the library are in the same RT frame and that new frame is saved in the iRT column for use in future SWATH processing.

 Note: if your main library is too large to open in Excel you will need to use a tool to combine libraries such as SWATHXtend ( .

This topic was modified 5 months ago by Trish Reed
Posted : November 23, 2016 11:19 am
Ali Azimi

Hi Christie,

At point No 3 and 6, when you say "perform retention time calibration", how do we actually do it? At this stage, we don't have the PepCalMix peptides coming up in the list to be used as RT-Cal peptide. Should I select any other peptides for RT-Cal or there is any other way for doing this. Sorry for keep asking quiestions. 🙂 I will also really appreciate if you have detailed method for generating ion library and processing SWATH files and if you could send them to me. 

Thank you,


Posted : September 18, 2017 12:58 am

Hi everyone,

Is it possible to have a faster way including the analysis of the PepCalMix peptides with Protein Pilot at the same time the DDA analysis with the database we choose? say an automatically way when you have several SWATH analysis to do?

Thank you,


Posted : March 3, 2021 1:20 pm