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 acquisition file must have both the matrix from the main ion library and the PepCalMix mixed 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 acquisition 2.0 microapp in PeakView software and associate it with the above SWATH acquisition datafile.
3. Perform retention time calibration if needed to adjust the RTs of the ion library to that of the SWATH acquisition 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 acquisition file
   1. Download PepCalMix-Ion-Library
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 acquisition 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 from step 7 into 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 acquisition data 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 (http://www.proteome.org.au/Services/Bioinformatics/SWATHXtend).

RUO-MKT-18-13709-A