Get hands-on experience with iRT, a technique for storing calibrated, empirically measured peptide retention times in a library for future use in retention time prediction for scheduled acquisition and peak identity validation. In this tutorial, you will calibrate your own iRT calculator and also learn more about the iRT-C18 calibration proposed by Biognosys for the peptide standards in their iRT-Kit. You will calibrate new iRT values from SRM data, a spectral library and chromatogram peaks filtered from MS1 scans in a discovery experiment. And, you will learn how to recalibrate these iRT values to schedule SRM acquisition on a new column with a new gradient. You will see how more accurate retention time prediction based on iRT can give you higher confidence in your chromatogram peak identity validation. (36 pages)
* - written on v1.2, updated for v1.4, updated for v20.1
On May 12, 2015, the Skyline Team produced Webinar #7: iRT Retention Time Prediction with Skyline, another great resource to learn more about iRT retention time normalization and library building concepts in Skyline.
On January 25, 2017, the Skyline Team produced Webinar #15: Large Scale DIA with Skyline with updated, advanced DIA workflows and using larger data sets. Webinar #14 also touches on iRT integration into method development.
Also, see our paper in Proteomics (please cite)
Using iRT, a normalized retention time for more targeted measurement of peptides