With a TSV assay library, you should be using File > Import > Assay Library. It sounds like you are trying to use a method to build a spectral library, but you already built a spectral library with SpectaST and went further by converting it to the TSV assay library. If you now use the File > Import > Assay Library function this should:
- Create a new spectral library with the intensities from your assay library TSV.
- Give you the opportunity to create an iRT library as part of your spectral library.
- Add all of the targets in your assay library to the current Skyline document.
If you just want a spectral library that Skyline can understand, you can forget the step to convert your SpectraST sptxt file to the tsv and just add the sptxt file as a library to Skyline directly without any build step (Peptide Settings - Libraries - Edit List > Add...) Then you can build your iRT library by going to Peptide Settings - Prediction, click the button with the calculator icon, Add, Choose the Biognosys standards and click Add > From Library selecting the SpectraST library.
But starting with the TSV, you may want to use File > Import > Assay Library.
Hope this helps. Thanks for posting to the Skyline support board.