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Trouble viewing MS2 data from Bruker maXis for systyem suitability applications

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Trouble viewing MS2 data from Bruker maXis for systyem suitability applications paul mathews  2023-03-08 13:57
 

Hi Skyline team,

I am in the process of building SS protocols across our LC systems using Skyline and Panorama AutoQC function. In this request I am investigating DDA MS2 data acquired on a bruker maXis 2 for a single compound, resperine, ran on with a c18, 4 min uplc method with bruker autoMSMS detection.

I have followed the tutorials for small molecule quantitation and generated two skyline documents, one from scratch and one using the "Auto MSMS_MS and MSMS filtering" template. In the scratch generated document, I am unable to see MS2 information. In the modified template, I can see my MS2 transition of interest, but the chromatogram appears wonky with a straight line. It seems selecting "DIA" or "none" I see in another post this may just be a bug. Is there something I can do within my transition settings or transition list to get the transition ion (which appears in the MS2 scan of m/z 609) to appear and be useful for SS? We may ultimately just quantify on the MS1 TIC, but for further use (including using GCMS data) I'd like to find a way to use a fragment ions in quantitation, without a MRM method.

Attached are screenshots of the skyline documents. For the auto MSMS template file there are two shots, one with DIA and one with the targeted(obsolete) method (which strangely disappears once you modify the template)
 
 
Nick Shulman responded:  2023-03-08 14:14
When MS2 chromatograms have long straight lines in them it is because there was a long period of time between MS2 spectra which matched the precursor. This often happens if you try to extract chromatograms from DDA data.

If your Transition Full Scan settings specify that the acquisition method is "Targeted", we recommend that you change it to "PRM". We added the PRM acquisition method at the end of 2021. Skyline will allow you to continue to use the "Targeted" setting, but makes it very hard for you to choose that setting if it is currently set to something else.

I do not understand what your data looks like, but if you send us your Skyline document and one or more of your raw files we will probably be able to tell you what is going on.
In Skyline you can use the menu item:
File > Share
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. You can upload larger files here:
https://skyline.ms/files.url

It would probably also be helpful if you could zip up your Bruker .d folder and send it to us as well.
-- Nick
 
paul mathews responded:  2023-03-08 14:53
Thank you. Attached is the skyline zip file and the 3 data acquisitions.
I noticed the PRM setting did not show chromatograms.

Thank you for your help on this.
 
Nick Shulman responded:  2023-03-08 15:20
Thank you for sending those files.
I see that your precursor m/z is 609.2807.
There is one MS2 spectrum in "20230303_SS_resperine_r002_1-F,2_1_4126.d" whose isolation window contains that m/z.
That is a spectrum at 2.26 minutes which isolated "606.67654-611.89511".

When Skyline tries to draw a chromatogram with only one point in it, it ends up looking the way that it does in your screenshot.

What were you hoping that Skyline would do with this data?
Skyline is great for extracting chromatograms but is not very good for looking at individual spectra.
-- Nick
 
paul mathews responded:  2023-03-14 08:44
Hi NIck,

Thanks for your response.
What we are trying to do is establish a system suitability procedure. Currently we are figuring out which compounds to use, with reserpine being a candidate. I am mostly just trying to figure out skyline at this point using our DDA data from the maXis qtof. I will also be trying to use skyline for full scan MS1 GC-MS data.

In the MS2 spectrum file, I 'd like to add quantitative information for the other main fragment at m/z 195.0653. basically treating it like MS1 data but using these peak area ratios to track performance...if that makes sense!