Hi,

I am trying to build a spectral library based on the DDA data, for this I am using the comet and X!Tandem as sequence database to be searched into. Then I am using PeptideProphet for the statistical validation and Mayu for FDR estimation. I am using Biognosys iRT-C18 peptide kit for the RT normalization. But the problem I am facing is that the out of 11 iRT standard peptides, only 2 are present in the output pep.xml files of Comet, none in X!Tandem. So, due to which I am not able to normalize the RT properly. So is there any way out that I can perform RT normalization?

If no, then I want to use CiRTs for the RT normalization, but I am confused from where to start, do I need to follow the same protocol as explained for SiRT (Biognosys) explained in your tutorial or is there any way of doing this in Skyline?

Thanks in advance.