Nick Shulman responded: |
2017-03-13 04:43 |
Anna,
Can you send us your .raw files? If they are less than 50MB, you can attach them to this support request. I will also send you an email with instructions about other ways to send us files. |
|
perzanowska an responded: |
2017-03-13 04:56 |
Hi Nick,
Thank you for the quick response.
I attached the .raw file.
Anna |
|
|
Nick Shulman responded: |
2017-03-13 07:08 |
It looks like this can happen whenever you have two precursors that are within approximately 0.6 m/z of each other. In this case, Skyline gets confused about which chromatogram belongs to which precursor, and then, instead of you getting the 11 CE optimization chromatograms that you would expect, all you get is one chromatogram per transition.
Let me get back to you on this. |
|
perzanowska an responded: |
2017-03-14 07:46 |
Hi Nick,
If the small difference of m/z caused this error, why S100A9 LGHP and Galectin LPDG peptides (difference in m/z smaller than 0.6) show CE optimization data in one Skyline file and the error occurred only after importing the results for CEA5 INGI which m/z is much higher?
Furthermore, in my list of all analyzed peptides I have 6 more peptide pairs which m/z is smaller than 0.6 m/z and in spite of this fact the CE optimization results are shown properly.
Also, if Skyline gets confused because of m/z of precursor ion how it knows to show proper chromatogram with fragment ions (in View-> Transition->All) for these peptides and makes a mistake only for CE optimization data which are fragment specific?
Thank you for your help,
Anna |
|
perzanowska an responded: |
2017-03-31 01:45 |
Hello Nick,
I was wondering if you had time to look into this issue?
Thank you,
Anna |
|
alwest responded: |
2023-06-29 22:52 |
I believe I'm experiencing the same issue.
I've performed a CE optimization (1 protein, 53 precursors, 66 transitions; many different modifications/charge states of the peptides). After import of the data, Skyline shows only one chromatogram and one red peak area-bar for most of my transitions, and at most two, after specifying View->Transitions->Single (and Split Graph).
Opening an old project using the same Skyline version, those files work well.
I'd be happy to email data files if you have time to look into this.
(Aquired on Sciex QTRAP 6500+. Imported as One New Replicate, Optimize Coll. Energy. Skyline Daily v.22.2.1.501) |
|
Nick Shulman responded: |
2023-06-29 23:24 |
alwest,
Yes, it would be great to see your data.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
It would also be good if you could send us your .wiff and .wiff.scan (and maybe .wiff2 if you have it).
Files which are less than 50MB can be attached to this support request.
You can zip up larger files and upload them here:
https://skyline.ms/files.url
There have been some small changes to how Skyline stores CE optimization chromatograms in the .skyd file. These changes happened a few months ago as part of the work that we needed to do to support CE Optimization in PRM data. We would not expect that you would notice these changes with old CE optimization experiments, unless there was something wrong with the old data.
With most CE optimization data, Skyline expects to find a series of SRM chromatograms which have the same Q1 value and where the Q3 values are 0.01 units apart. Older versions of Skyline would sometimes group together chromatograms where the difference between the Q3 values was not actually 0.01, but the current version of Skyline-daily is more strict in this regard.
-- Nick |
|
alwest responded: |
2023-06-30 00:26 |
I realize I may have caused this issue - since Sciex Analyst has issues with multiple rows with identical Q1 and Q3 masses, I've slightly edited the third decimal, perhaps also second decimal, of the masses before inserting the transition list to Analyst (it's a triple quad instrument). Therefore, each row for CEopt will not have identical masses with the others. Perhaps this may have caused the issue this time around? Not been able to test this idea yet.
Due to privacy/security issues, id prefer to send the data directly and not on the server that\s open to everyone. |
|
Nick Shulman responded: |
2023-06-30 07:17 |
alwest,
I will send you an email directly so that you can send me your files.
-- Nick |
|