Exploris PRM data import into skyline Fynn  2020-01-08 08:14
 

Dear Skyline-Team,

I am using a PRM method with multiplexing (MSX) for targeted measurements on a "Q Exactive HF-X" mass spectrometer. Here everything works well and I can observe precursors as well as transitions. After transferring the method onto the "Orbitrap Exploris 480" platform and loading of the raw files into skyline (and Skyline daily) I still see the precursors, but no transitions. When I open the raw files with "SeeMS" I can see both, the MS1 and MS2 scans. Since the skyline method worked for the HF-X runs I am a bit lost why it is not working for the Exploris runs. Do you have any suggestions how to solve the data import problem?

Best,
Fynn

 
 
Nick Shulman responded:  2020-01-08 11:06
Fynn,

Can you send us one of your .raw files (e.g. "20200106_EXPL2_FyHa_SA_PRM_BSA_01.raw")?

You can upload that file here:
https://skyline.ms/files.url

Usually bugs like this have been caused by Skyline being confused about what m/z was isolated in each MS2 scan and so the scans never get properly associated with the peptides in your document.
-- Nick
 
Matt Chambers responded:  2020-01-08 11:52
Even better would be one RAW file from each instrument so we can see the difference.
 
Fynn responded:  2020-01-09 00:48
Hi all,

I figured out why the import was not working. It was a problem with the Exploris settings after all. The new Xcalibur version has an option called "Reported mass" when you define an isolation offset. This option was set by default to "offset mass", which leads to the recording of an offset mass and not the original mass (see picture). After changing this point to "original mass" the import of the file into skyline works. I am very sorry for the confusion.

Best,
Fynn
 
Brendan MacLean responded:  2020-01-09 07:47
We should still get your data - both types. (please) From my read of the description you provided, I believe we would prefer the Offset mass, i.e. the central mass at which the quadrupole was set, and not the Original mass which I read as an instrument detected mass pulled from an MS1 spectrum that is likely the monoisotopic mass of the most abundant precursor in the isolated range. That is not really what we are expecting. So, it is a bit strange that this works better than the Offset mass.

We really need to take a closer look at how Thermo is representing these two masses. Thanks for bringing this to our attention.

--Brendan
 
Matt Chambers responded:  2020-01-13 10:25
The offset mass may be the central mass at which the quadrupole was set, but the offset is a constant 0.4 m/z. So if using a narrow extraction range around each isotope then we really need the original mass because the m/z after offset won't actually correspond with a peak. Like this: If using a wide window instead of multiple narrow windows, then the offset mass would be better.