Applying manual integration windows when adding new transitions/fragments for an existing compound in Targets

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Applying manual integration windows when adding new transitions/fragments for an existing compound in Targets Emmanuel  2020-07-27
 

Dear Skyline team,

I've manually adjusted the integration of a targeted small molecule across a dozen of samples and I'd like to add few more transitions/fragments for that compound (additional fragments found in another database than the first ones).

Is there a simple way to apply the manual integration boundaries to the new fragments without using the workaround of exporting/re-importing the peak boundaries as a report (as suggested by Tobi some years ago)?

When re-importing the results, it seems that the new transitions are integrated according to the default integration settings without taking into account the manual integration from the previous transitions. Therefore, I need to manually re-integrate all the samples ....

Thanks in advance for your help.

Emmanuel

 
 
Nick Shulman responded:  2020-07-27
If you add new transitions to molecules that are already in your document, and then you reimport your results by going to:
Edit > Manage Results > Reimport
then your manually chosen peak boundaries are supposed to be preserved.

So long as you are using the "Reimport" button on the Manage Results dialog, we would expect the peak boundaries to be preserved.

It might be that something strange is going on. You can send us your Skyline document and we can take a look.

In Skyline, you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files.

If that .zip file is less than 50MB you can attach it to this support request. Otherwise, you can upload it here:
https://skyline.ms/files.url

You should also send us your raw file that you are reimporting results from.

-- Nick
 
Emmanuel responded:  2020-07-27
Dear Nick,

Please find attached a reduced dataset with 3 blank runs and 3 replicates of a pooled sample.

I did the manual integration of the first 4 transitions (prec + frags down to m/z 97). Then I've added 4 frags in the target list with the expected RT at 1.86 min without setting a RT tolerance window.
For the blank runs, the noise is integrated with the default settings, but not according to the manual integration boundaries previously defined. While for the pool the RT has shifted to 2 min and the 4 new transitions are not integrated at all (even after re-importing the results ...).

I'll send you the rawdata through the alternative large files url .

Thanks for your help.

Best,

Emmanuel
 
Nick Shulman responded:  2020-07-28
Emmanuel,

Thank you for sending that file.

I see what you mean. If you add new transitions to precursors in your document, and those precursors have manually chosen peaks, then, when you do a reimport, the new transitions end up with null peak areas, similar to (but not exactly) what you would get if you had used the menu item "Remove Peak" to remove those new transitions' peaks.

I think the workaround that you have of exporting peak boundaries and reimporting them might be the best way to deal with this.

As far as I can tell, this has always been the behavior in Skyline, and I have never noticed since it's a rarer scenario than reimporting after adding new precursors or other settings changes.

I am not sure when we will be able to fix this in Skyline. Have opened this issue to keep track of the work item:
https://skyline.ms/issues/home/issues/details.view?issueId=749

Thank you for reporting this issue.
-- Nick
 
Nick Shulman responded:  2020-08-10
Emmanuel,

I have been looking at this problem a little more, and it seems like you can work around this by making a tiny change to the settings.

For instance, if I go to:
Settings > Transition Settings > Instrument
and change, for instance, the "Method match tolerance m/z" (e.g, change it to "0.0551") then Skyline will go ahead and reintegrate all of your new transitions for you (and then you can change it back to "0.055")

Does this workaround work for you?

If this is not a helpful workaround, then let me know, because I might not be understanding your original question.
-- Nick
 
Emmanuel responded:  2020-08-15
Hi Nick,

Thanks for your suggestion but this workaround works partially ... Indeed the new transitions are integrated and an area can be calculated (which was not the case previously). However, the peak boundaries of the first set of transitions (which were previously manually integrated) are not propagated to the new transitions.

I've attached three sequential screenshots trying to explain what I mean (look at the three pool replicates - red circled):

a) after adding new transitions, those ones are not "integrated" for the reasons explained in your first email (July 28th)
b) when changing the "method match tolerance" according to your last email (Aug 10th), the new transitions are integrated, but the peak boundaries are different from the first integration
c) what I would like to have, i.e. all the transitions with the same peak boundaries (I could get those results by manually re-integrating the three pool replicates.

Hope this helps and thanks again for your support. Let me know if you need more explanations.

Have a great WE.

Best.

E.
 
Juan C. Rojas E. responded:  2020-08-21
Hi Nick and Emmanuel,

I have observed this problem as well and my guess has been that Skyline treats every transition separately and not linking them to the same molecule as you would expect from a truthful molecule. This could be useful for first pass checks to check the validity of the integration, but in your case I guess you have already defined this.

As good practice I export peak boundaries before doing any changes to the document (if I have already corrected integration boundaries) and reimport them. Here is where an alternative workaround could be applied that I have been thinking for some time (haven't applied it yet to other priorities):

   - modify the peak boundaries .csv file.

You would have to make sure the formatting is kept the same, but you could add rows for each of the new transitions you want to include for each of the molecules and files in the document. If these are few you can definitely do it in Excel, but if you have hundreds of molecules (and possibly new transitions for each) and many files then a iterative programming script would be the approach (my scenario and hence why I haven't tried yet to invest the time into it).

Hope the comment helps.
Sincerely,
JC