• Sign In
  1. Requests

MRM windows are smaller than in the measurements

support

View Request

view list print
MRM windows are smaller than in the measurements schweitzer theresa  2023-07-28 04:39
 

Hello,
we are currently working with a 7500 QTrap and are measuring scheduled MRM data of cell lysates. We also use spiked in SIS peptides of our peptides of interest and saw that heavy and light peptides show some retention time shifts. Everything fine so far, we increased our MRM window to 60s instead of 30s and can see the data being collected in Sciex OS (attached file: Troubleshooting Skyline.jpg) at 9.1 to 11.1 min, however when importing the data to Skyline we only see the signal from 9.6 min to 10.6 min which would fit our old 30s windows (Troubleshooting Skyline_RT.jpg).

We already checked every skyline setting, but nothing seemed to fix the problem and now we are running out of ideas, so if you have some idea how we could get all the collected data shown in skyline that would be awesome.

Thanks in advance.

Kind regards,
Theresa
 
 
Nick Shulman responded:  2023-07-28 12:02
Can you send us the files "230727_Inf10_Trypsin.wiff" and "230727_Inf10_Trypsin.wiff.scan"?
If those files are less than 50MB you can attach them to this support request.
You can upload larger files here:
https://skyline.ms/files.url

One thing I noticed is that at "Settings > Transition Settings > Full Scan", you have "Retention time filtering" set to "Use only scans within 5 minutes of MS/MS IDs".
With PRM data, you would normally want that set to "Include all matching scans" because you have already told the mass spectrometer what time range to collect data for each precursor.

The "Retention time filtering" setting is definitely not the cause of your problems (if it were, you would probably see chromatograms that were 10 minutes long).

After I see your .wiff and .wiff.scan files I will probably be able to tell you why Skyline is doing what it is doing.
-- Nick
 
schweitzer theresa responded:  2023-07-30 23:55
Dear Nick,
here are the files. Thank you for your fast response :)

Kind regards,
Theresa
 
Nick Shulman responded:  2023-07-31 00:27
Thank you for sending that wiff file.
I used ProteoWizard SeeMS.exe to look at the chromatograms in that file.
For Q1=765.353, Q3=1053.5, you can see in the graph that SeeMS.exe thinks that the chromatogram only goes from 9.6 minutes to 10.6 minutes.
However, in the "Id" for that chromatogram, it does say "start=9.1 end=11.1" which is a little suspicious.

I will ask my coworkers and find out if ProteoWizard is doing something incorrect.
-- Nick
 
schweitzer theresa responded:  2023-08-03 00:50
Thanks a lot, Nick.
Did you stumble across any cause for the mismatch of the ID information and the SeeMS.exe output?

Best regards,
Theresa