Hi Skyline team

I am new to using Skyline for data analysis and am trying to generate PRM calibration curves, only using heavily-labelled versions of my peptides of interest. I have been following the data processing and analysis outlined in Skyline Webinar 17 in the first instance.

I generated a spectral library of my peptides of interest pooled together, and with data acquired using DDA mode on a QE-HF. I then spiked in mixtures of my 22 peptides of interest into a complex background at different concentrations and acquired PRM data using the appropriate inclusion list with the QE-HF, with the same LC parameters as the DDA run.

I imported the raw data, and filtered for my peptides of interest using Edit>Insert>Peptides. There are transitions showing up for the majority of the peptides I am interested in, apart from two, for which I get a message saying "Chromatogram information unavailable". These two peptides have the same primary sequence aside from the fact that one contains deamidated asparagine and the other contains asparagine linked to N-acetylglucosamine. These two modifications are included as variable modifications in within the appropriate settings.

I can see that both of these peptides are present within the spectral library, and when I make XICs of my RAW files using FreeStyle, they also seem to be present at the correct m/z.

I wondered whether you might be able to help me figure out why I am unable to see the peaks using Skyline, please. I have had a look on your forum, but I can't work it out.

Is there a way I can send screenshots privately to you, if you require them?

Thanks,
DanCam