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Integration chosen from light or heavy peptide

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Integration chosen from light or heavy peptide cilento  2018-08-22 15:41
 

Hello,

Greetings from the lab of Dr. Jing Zhang here at UW. I am new to SRM quantification and have a question regarding differences found between my analysis and another member of the lab. We have a panel of peptides in which heavy synthetic peptides have been spiked in to aid in the light detection (pretty standard). In the targets that worked well (peaks clearly detectable in both light and heavy, well above background, and all transitions are detected good), we get identical results, however, in the peptides which are close to detection limit in the light, we found that our values are very different if the integration is dragged from the heavy peptide or the light peptide to guide the other corresponding partner. If dragged from the light partner, peak area replicate comparison demonstrates all transitions detected quite readily (even if some are close to background noise), however if dragged from the heavy partner, often Skyline seems to use some kind of algorithm to determine that some transitions in the light may be background (as they don't show up in in the peak area replicate comparison).
Mainly, I am trying to determine which approach is best for generating a "real" signal in the light- forcing all lights to generate transition signals (which may perhaps be noise), or use the heavy to guide that light signal which may in turn not detect transitions at times (determined somehow by the software). Either way, signal to noise criteria will be applied to the peptides, but advice on this starting point would be very helpful.
Thank you!

Gene
 
 
Brendan MacLean responded:  2018-08-22 15:56

Hi Gene,
We are going to need at least screenshots of what you are describing, and if your Skyline file is small enough, you can post it to this support request, reduced as much as you like to illustrate just what you are talking about or, if your file is over 50 MB, you can post it to:

http://skyline.ms/files.url

Use File > Share - Minimal to create a .sky.zip file you can share with us.

Thanks for reporting the issue.

--Brendan
 
cilento responded:  2018-08-22 16:25

Hi Brendan,

Thank's for the quick response. I attached two screenshots to the original message and on this response. As you can see in one, dragging the cursor under the light peak created a much larger overall signal than dragging under the heavy peak. In the other, the overall intensity of the of the bar graph didn't change substantially, however the ratio of transitions are changed (blue transition appeared in the light once dragged under the light peak).

Thanks and let me know if I can provide more information!

Gene
 
Brendan MacLean responded:  2018-08-22 16:36

Hi Gene,
I am not sure why your attachments didn't show up in the original email from the support board, but maybe because one was named "Skyline.exe", which is an executable file, and I have no removed it from the website.

Those graphs are a little tricky to interpret, but I think there must be something wrong, as I would generally expect to see something in the Peak Areas graph in the cases where you are showing nothing. It may be useful to select the transition element in the Targets view for the transition that is not showing a peak area. Then, Skyline should highlight how it is interpreting Area and Background for that individual transition.

But, it will be quickest for us to understand, if we can have a look at the Skyline document you created the screenshot from, using File > Share - Minimal to create a .sky.zip file, as I suggested before.

Thanks again.

--Brendan
 
cilento responded:  2018-08-22 16:49

Hi Brendan,

Attached is the skyline file, with the known heavy peak at ~50.5mins. I did not do any manual integration on this file, so what you see is simply the results following import of the data.

Hopefully this will help!

Thanks, Gene