RT Calibrators for SWATH

RT Calibrators for SWATH taperez  2021-09-21

One of my coworkers is working on SWATH data with skyline and says you're able to process data on skyline without needing to use retention time calibrators. Are retention time calibrators necessary, and if so why?



Nick Shulman responded:  2021-09-21
I am not sure I understand your question, but I think the answer is "No", retention time calibrators are not necessary for DIA (swath) data.

What is a retention time calibrator? Is that an iRT standard that you have added to your sample? iRT standards and an iRT database can improve peak picking but are not necessary.

If you would like to learn more about iRT in Skyline it might be helpful to look at the iRT tutorial:

-- Nick
Brendan MacLean responded:  2021-09-21
You definitely want to use iRT (retention time normalization) with DIA/SWATH data. Performance without it will be very poor, but we have recently (in 2020) done work to make it relatively easy to use endogenous peptides as the iRT anchor peptides. So, in that sense, your colleague is right that Skyline does not require you to inject an iRT standard mix. Though, Skyline also works very well with all of the industry-standard retention time calibration mixes that we know of.

Nick has pointed you to our existing tutorial on iRT. Though, the DIA/SWATH tutorial is also a good place to look. The tutorial uses the Biognosys iRT mix, but you can now ask Skyline to automatically calibrate iRT and it will use endogenous peptides, if it does not detect one of the known RT standard mixes.


Hope this helps. Thanks for posting your question to the Skyline support board.