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Peptide data will not import

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Peptide data will not import perzanowska an  2016-06-13 08:33
 
Hello,

When importing my MRM data from an Agilent 6495 triplequad to Skyline (ver. 3.5.0.9319) one of the peptides in my list of 25 peptides will no longer import/show data and only the text “Chromatogram information unavailable” appears in all samples. This was never a problem before, and just noticed this around three months ago.

The MRM method used for data acquisition on the instrument does not appear to be the problem as the m/z of the precursor and fragment ions are correct and this particular peptide data is present and visible when extracted by the Agilent MassHunter Qualitative viewer.

The peptide, using identical transitions, was also measured and viewed previously in older versions of Skyline. When I open these old Skyline files in the new version of Skyline the old chromatograms for that peptide appear as before but when I re-import the data the results in the chromatograms disappear for only that one peptide.

Changing (increasing) the match tolerance settings does not help, and the settings are as always (library: ion match tolerance: 0.5 m/z. Instrument: method match tolerance: 0.055 m/z.)

Peptide sequence and transitions:
LEGLTDEINFLR
710.3775++
    G [y10] - 1177.6212+
    T [y8] - 1007.5156+
    D [y7] - 906.4680+
    E [y6] - 791.4410+
    G [b3] - 300.1554+
715.3817++ (heavy)
    G [y10] - 1187.6294+
    T [y8] - 1017.5239+
    D [y7] - 916.4762+
    E [y6] - 801.4493+
    G [b3] - 300.1554+

Thank you for any suggestion on how to solve this issue,
Anna
 
 
Brian Pratt responded:  2016-06-13 10:23
Hi Anna,

I'd like to take a look at that data.

Can you please send me the raw file, and the Skyline document that shows it properly loaded? Use File|Share|Complete to create a zip file which contains the Skyline document and all supporting documents.

I'll contact you privately about arranging the transfer.

Thanks

Brian Pratt
 
perzanowska an responded:  2016-07-27 07:49
Hi Brian,

Did you get my email with requested files?

Thank you,
Anna
 
Brendan MacLean responded:  2016-07-27 09:09
Hi Anna,
Brian is currently away on vacation. He will be back in the lab next week. Sorry to leave you hanging.

--Brendan
 
Brian Pratt responded:  2016-08-02 12:58
Hi Anna,

Apologies for dropping the ball on this. I'm not sure what happened there.

Looking at the data, it seems that there just isn't much intensity for that ion in this replicate (I used the SeeMS program from ProteoWizard to inspect the data outside of Skyline.)

I'm not sure there are actually peaks to be found there?

Thanks

Brian
 
perzanowska an responded:  2016-08-10 08:49
Hi Brian,

Recently, I’ve sent you two Skyline files that indicate that when I open the old Skyline files (saved before ver. 3.5.0.9319) the chromatograms for the peptide appear correctly however after re-import of the data the chromatograms disappear – I hope you have received this answer?

Now, as one of our computers has still the old version of Skyline (2.6.07176) I imported the data that in Skyline version 3.5.0.9319 caused the described issues and I found out that the data was imported correctly and chromatograms for that peptide appeared normally.

Thank you for your help.
Anna
 
Brendan MacLean responded:  2016-08-10 22:20
Hi Anna,
Brian is out this week, but I am sure he will be back in touch early next week. Sorry for the delay.

--Brendan
 
Brian Pratt responded:  2016-09-02 17:30
Just leaving a note to say that we worked this out off-list, and the issue should be resolved in the next Skyline-Daily.