"Chromatogram information unavailable" message

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"Chromatogram information unavailable" message DanCam  2021-09-20
 

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

 
 
Nick Shulman responded:  2021-09-20
I will contact you by email so you can send us your data privately.

One common reason that you will see the "Chromatogram information unavailable" is that the raw file did not contain any MS2 spectra which matched the precursor. If your MS/MS Acquisition Method is "Targeted", then a particular MS2 spectrum will only be associated with the one precursor in the document whose m/z most closely matches what the raw file says was isolated.

After I see your Skyline document and raw file I will be able to figure out what is going on.
-- Nick