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Integration problems? alejandro.cohen  2018-01-18 12:50
 
Hi Skyline people!

I'm running some metabolomics samples acquired on a Qtrap5500 by MRM. I downloaded the date, and for some particular targets, Skyline does not seem to pick the appropriate peak! Even when I select the likely peak manually, and then Apply Peak to All, it still doesn't seem to find the right 'obvious' peaks for integration.

Attached the file. For example, look at Target number 2 on the list: 1-Methyladenosine. A very prominent peak for most positive samples is at 14.7min in most 'Pos' labeled samples, yet Skyline finds other minor peaks all over the XICs.

Could you see if I'm missing something?
Thanks
Alex
 
 
Nick Shulman responded:  2018-01-18 15:02
Can you send us your .wiff files? I think we need to catch Skyline in the act of importing chromatograms in order to figure out what went wrong.

If you look at the chromatogram for the replicate "Pos_S01", Skyline did not even see a peak at 14.7 minutes where the largest peak is. There are retention time numbers are visible above the other smaller peaks that Skyline considered.

However, if you go to:
Edit > Manage Results > Rescore
(and then also reopen the Skyline document-- not sure why this is necessary) you will see that Skyline then does detect the obvious peak at 14.7.

Can you send me that file "120112_0738_114_Met-Pos_S01.wiff" (as well as the .wiff.scan file)? If it is less than 50MB, then you can attach it to this support request. Otherwise you can upload it here:
https://skyline.ms/files.url

It probably won't be necessary to see your other .wiff files, but if you feel like it, you can upload those too.
 
alejandro.cohen responded:  2018-01-22 13:23
Thanks Nick for your prompt response. I'm attaching the files now, just a few. Hope you can find the culprit!

Cheers

Alex
 
alejandro.cohen responded:  2018-01-22 13:34
I tried the rescoring trick... but it did not work for me :-( even after reopening). I'm working on the latest daily version.

Alex
 
alejandro.cohen responded:  2018-01-22 13:40
oops... it DOES work after I reintegrate one peak and 'Apply Peak To All'

Alex
 
Nick Shulman responded:  2018-01-22 16:07
I am stumped.

Is it possible that when you first imported chromatograms from these .wiff files your Skyline document looked completely different?
Specifically, did you maybe have all of your precursors under the same molecule, and only after importing chromatograms did you split them apart so you have one precursor per molecule?

I cannot think of any reason that Skyline would not have been able to detect the peak at 14.7. Certainly, when I do a "rescore" or I import chromatograms from the .wiff files you gave me, I cannot see Skyline make this mistake. The only explanation I can imagine at the moment would be that Skyline was looking at a whole bunch of different chromatograms at once.

Any ideas?
 
alejandro.cohen responded:  2018-01-23 10:21
Nick,

I have a template for this metabolomics workflow that I have used for other samples. It is possible that I have opened the .sky file from another set of samples, and then removed all the previous data and reimported the new dataset, I cant remember for sure. I dont think the template ever had the precursors under on same molecule.

I'll see if I get the same problem with other datasets. In any case, how do you recommend saving a Template file (with out any files/data) so I can open that file whenever I start a new dataset?

Cheers

Alex