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MS2 signal missing in SureQuant experiment

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MS2 signal missing in SureQuant experiment michele puglia  2024-07-03 07:55
 

Dear Skyline team,

I am having a problem when importing raw files acquired on an Ascend instrument using the SureQuant method (user designed peptides).
I am following the "SureQuant tutorial 2" instructions: https://skyline.ms/wiki/home/software/Skyline/download.view?entityId=14124b80-46d4-1038-b053-e465a393788a&name=Tutorial-2-tuesday-surequant.pdf

My library (build in MQ) works very well and all my heavy peptides are in the library. I can see a very good MS1 signal for them as well as a Library match and the Peptide ID times (see picture attached). Unfortunately NO MS2/product signals, even though I can see them in the Library and listed in the "rank".

I tried importing several times the raw as well as mzML (after conversion with Proteowizard), no success.
I tried several parameters in the MS/MS filtering option (SureQuant, DDA, PRM, centroid, etc...) but NO success, after re-importing the files each time.

I am sharing a very partial version of the Skyline document with just 2 peptides and 3 mzML files, hoping you can help me.
I used "profile" for MS/MS spectra as specified in the Thermo SureQuant template, can this be the issue? should I have acquired the samples in "centroid" mode? this is the only option left that comes in my mind...

I really hope you will be able to help me with this issue since I was really looking forward to set several SureQuant experiments.

Best
Michele
 
 
Nick Shulman responded:  2024-07-03 22:41
Thank you for uploading that Skyline document and those .mzML files.

The problem is that the MS2 spectra have the "Scan Description" set to values which begin with "SQ_" but which are not exactly what Skyline is expecting and so, Skyline thinks that none of the MS2 spectra are intended for the molecules that you were hoping they would be used with.
You can find out more about this here:
https://skyline.ms/announcements/home/support/thread.view?rowId=49364

One thing that you could do is use a text editor to remove all of the "Scan description" values from the .mzML files.
I have done that with your three .mzML files and I uploaded them here:
https://skyline.ms/_webdav/home/support/file%20sharing/%40files/MichelePugliaNoScanDescriptions.zip

A different thing that you could do is change your instrument method so that it does not set the "Scan Description" values on anything and acquire your data again.
-- Nick
 
michele puglia responded:  2024-07-05 05:32
Dear Nick,

thank you for reply so quickly. Your suggestion worked and Skyline is now able to extract the MS2 signals.
From now on, I will leave the "Scan Description" field empty when I am measuring using SureQuant.

Thanks again
Best Regards
Michele