Large scale PRM data (msx count= 8) Wael  2019-03-20 09:01
 

Dear Skyline team,
I have a question please regarding PRM analysis. In short, I have an inclusion list consist of about 300-500 peptides and I ran this in multiplex PRM mode (msx count = 8) without RT optimization, as a fast exploratory experiment.
I am getting the MS2 ions, but skyline is integrating the peaks for all peptides over 15 min (LC method 20 min), please find attached pic as an example.
My normal RT window for a pepetide is about 0.2 min. I have the resolving power 140,000 at 200m/z.

Is skyline able to process such a workflow and if yes how can I improve the peak integration, pelase ?

Please let me know if you would like me to share skyline file with you.

Many thanks
Best
Wael
 
 
Nick Shulman responded:  2019-03-20 10:03
I do not understand your question, but it might help if you could send us your Skyline document and one of your raw files.

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

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 probably also send us at least one of your raw files.
-- Nick
 
Wael responded:  2019-03-21 03:14
Hi Nick,
I have attached the zip file which contains some peptides from the inclusion list (PRM_integration issue.sky).
sorry, I will try to explain again. I have ran a targeted prm method, but because I had too many peptides I have ran this in 8-plex mode, trying to compensate cycel time.
When I imported the raw files into skyline, peptides were intigrated over the whole gradient ie. skyline integrates for a peptide 10 min RT window (see atatched pic).
I hope that thettached file and pic. explain the issue.
Best
Wael
 
Nick Shulman responded:  2019-03-21 08:37
Can you send me one of your .raw files, for instance "NMI_QEI_01356.raw".

It looks like nearly all of your chromatograms have only one or two points that are not zero. For this reason, the chromatograms look like triangles, with just a single apex which is not on the x-axis.
In your screenshot, it looks like you have applied some sort of smoothing or other transformation, so your peaks look round and good. You should right-click on the chromatogram and choose "Transform > None" to see the actual data that was collected.

-- Nick
 
Wael responded:  2019-03-22 00:54
thanks, that was really helpfull.
I will need then to decrease the scan resolution or peptides number with some optimization and then it should be fine. As I understood, there should be no proplem for skylline to work with high multiplexed orbitrap data (msx count > 6) and the rest will be my task.

Many thanks again

please find the attached rawfile NMI_QEI_01360 and NMI_QEI_01356.

Best
Wae