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Issue with Bruker bbCID (AIF) analysis for small molecule

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Issue with Bruker bbCID (AIF) analysis for small molecule christophef  2018-08-27 12:54
 

Dear support,

I am reporting an issue I have with analyzing small molecule data acquired on Bruker Impact II in bbCID mode.
The idea was to evaluate Skyline for small molecule quantification using MS1, AIF or SWATH. MS1 and SWATH data from Bruker works just fine as expected, but I have a curious issue when importing AIF data. It looks to me like the full scan data with low CE and high CE are just inverted in Skyline, meaning the precursor and the product ion are extracted from the high CE and low CE, respectively. I am expecting the other way around.

Could it be an issue with how Skyline is handling Bruker AIF data file or I am just not doing it right?
I am using Skyline 4.1 and tried Skyline Daily as well. Bruker Compass 1.9 is used to control the mass spectrometer acquistion

Please find attached some slides on the issue I have ( example with 1 small molecule transition)
Slide 1: general view of Bruker data analysis bbCID data
Slide 2: Bruker DataAnalysis vs Skyline, wrong scenario
Slide 3: Bruker DataAnalysis vs Skyline, correct scenario in Bruker DataAnalysis
Slide 4: Skyline mass spectra and transitions setting applied.

Do you have an idea where the issue is and if this has something to do with the Bruker bbCID file structure?

Thanks for your help.

Christophe
 
 
Brian Pratt responded:  2018-09-05 06:48
Hi Christophe,

I would be very happy to look into this. Can you provide the Bruker file, and the Skyline document?

For the Skyline document, please use Skyline's File>Share>Complete menu item to create a .sky.zip file.

You can upload everything to http://skyline.ms/files.url and we will sort this out. If you prefer to upload someplace more private, we can arrange something else.

Thanks,

Brian Pratt
 
christophef responded:  2018-09-05 12:58

Hi Brian,

Thanks for your answer and to look into this.
Please, find the Skyline document attached and the Bruker Raw file in files.url.

Name of the file is:
180824_IROA-Matrix-MSe-20eV_i1_1-A,2_01_3939.d.zip

Thanks again for your help.

Best regards,

Christophe Folly
 
Brian Pratt responded:  2018-09-10 17:24

Hi Christophe,

I've been looking at this, I can't see what we're doing wrong. I set up a little Skyline document based on your example that has a second molecule with the precursor and fragment mz values reversed, with the idea that if we really are swapping the high and low CE scans, the fake molecule should work properly. But it doesn't - the fragment intensity is much greater than the precursor intensity, which seems wrong.

I've attached the file, can you play with it a bit and offer your thoughts?

Brian
 
christophef responded:  2018-09-11 04:04

Hi Brian,

Thanks for your answer and document. I have looked at it and do think you have noticed with the "fake molecule" approach, the same issue, meaning inversion of Low CE and High CE in Skyline document.

I have attached some slides to explain what I see.

  • Slide 1 and 2 are showing that for "compoundA" (175 ->116 m/z) and "fake_molecule" (116->175 m/z), precursors ions are extracted from high CE scans (even scan number), when products ions are extracted from low CE scans (odd scan number).
    This is further confirmed by precursors ions intensity you expect to see higher in MS1 vs MS/MS and products ions intensity you expect to see higher in MS/MS vs MS1.

-Slide 3 should convince us that Low/High CE scans are inverted in Skyline vs Bruker DataAnalysis. Notice the scan numbers, the corresponding MS level and our target precursor/product ions intensity in the mass spectra.

-Slide 4 is looking at the Bruker Raw file with SeeMS and confirming that odd scan numbers are Low CE and even scan numbers are High CE, because of the fragmentation spectra profile that correspond to what we have seen in DataAnalysis.

Please, have a look and tell me if my interpretation seems correct to you?
If yes, I would invert MS level information given to odd/even scan during data import. On the other hand, I do not know if this is a constant data structure in Bruker bbCID file? My knowledge stops here...and I rely on your help.

Thanks again for looking at it.

Best regards,

Christophe