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missing a detected dominant fragment in our PRM data

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missing a detected dominant fragment in our PRM data ru wei  2017-08-11 08:55
 
Dear Skyline team,

We have been using Skyline for years now, thanks for great work!! Also appreciate it very much for addding quantitation module. We recently start PRM assay on our QE HF platform and use Skyline for quantifying proteins using external calibration curves.

One issue we encountered when processing our PRM data is missing a detected and dominant fragment ion (y15++) for one of our assayed peptides. Please see details in the attached pptx file.

Thanks in advance,

Ru
 
 
Brendan MacLean responded:  2017-08-13 13:01
Hi Ru,

It seems that you didn't originally include the missing ion y15++ when you first imported your data, which would certainly cause what you are seeing. In fact, what you are seeing would be expected in that case. Skyline only extracts the chromatograms in your targets list when you import a file.

So, the only surprising point is the comment that you tried to reload the file but it did not help. Everything up to that point is completely expected.

Your effort should be directed toward understanding why the "reload" did not have the expected impact of causing Skyline to include the y15++ ion in its extraction. I would probably try the following:

  1. Just close and re-open the file to see if it was an update problem where the re-import actually worked, but Skyline failed to notice the new chromatogram.
  2. Use Edit > Manage Results to Remove All replicates. Save the empty document. Reimport on file to see if the problem persists, which would be really surprising, but also very informative.
  3. Assuming the above did not reproduce the problem. Remove the one replicate again. Remove the y15++ ion and repeat your original steps to see if you can create a simplified set of steps that reproduce the problem with a single document.
Once you have narrowed down what exactly you did and where Skyline is failing to meet expectations, report back and we will want the minimal set of files you used to reproduce the problem.

Also, can you give more detail now on what method you used to "reload" the replicate you are showing? Did you fully remove it using Manage Results and then use File > Import > Results to add it back, or did you use the "Re-import" button in the Manage Results form? And what version of Skyline are you using? (see Help > About)

Thanks for the screenshots with your report and your appreciation of our work.

--Brendan
 
ru wei responded:  2017-08-14 14:11
Dear Brendan,

Thank you very much for your quick reply and clear troubleshooting suggestions. Although it's not straight forward, it worked now. One time, after removing and re-load the file, I had to click y15 transition to bring it on. In other cases, it requires close/re-open skyline files; or remove-reload the 2nd times to have y15 fragment. The most importantly, we can have y15 fragment included. Great thanks!!

Ru
 
Brendan MacLean responded:  2017-08-14 14:16
Hi Ru, That is too bad. I guess we need to try more of that here ourselves to retest this functionality. We have a lot of automated tests, and I am pretty sure this has worked better in the past. Sometimes things do slip.

Thanks for letting us know of your experience, and glad to hear this is not blocking you now.

--Brendan