All-ions fragmentation data workflow

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All-ions fragmentation data workflow dmesasan  2021-02-18
 

Hello!

I'm having some issues with importing results and exporting chromatograms of Agilent data that has been acquired using an all ions fragmentations mode. Maybe I need to adjust my settings somehow to accommodate this type of acquisition? I've attached an example data file. Thanks for the help!

 
 
Brendan MacLean responded:  2021-02-18

It seems you forgot the attachment.

 
Brian Pratt responded:  2021-02-18

It would also be helpful to have a Skyline document (use File > Share > Complete), so we can see what you have tried so far.

You can upload both to http://skyline.ms/files.url

Best Regards,

Brian Pratt

 
dmesasan responded:  2021-02-18

Oops! seems it was too big of a file! I uploaded at the link (allionsfrag.sky.zip and CTRLBrain_Slide9_Sample1_line18.d.zip). I am getting this error when trying to import:

At 5:13 PM:
No scans in J:\data\QTOF\Daniela\All Ions Fragmentation Imaging\02132021\CTRLBrain_Slide9_Sample1_line11.d match the current filter settings.

Inner exceptions:
Exception type: pwiz.Skyline.Model.Results.NoFullScanDataException
Error message: No scans in J:\data\QTOF\Daniela\All Ions Fragmentation Imaging\02132021\CTRLBrain_Slide9_Sample1_line11.d match the current filter settings.
No scans in J:\data\QTOF\Daniela\All Ions Fragmentation Imaging\02132021\CTRLBrain_Slide9_Sample1_line11.d match the current filter settings.
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 362
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 215
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.SetRequestOrder(IList`1 chromatogramRequestOrder) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 548
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 403
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 263

 
Brian Pratt responded:  2021-02-18

I have the data, thanks.

Looking at it in SeeMS I can see that it's all MS2 data, which explains why your MS1-only Skyline document isn't matching anything.

I'm not sure where to go from here. What are your thoughts?

Best.

Brian

 
dmesasan responded:  2021-02-19

Oh that's interesting. Agilent software shows it as MS1. I suspected something about that might be the issue. I took a look with SeeMS and it's showing some made up precursor of 899.79096 for everything. I adjusted my transition list and made my targets into products with this fictitious precursor and this seems to have worked. Will need to play around with it a bit more, and figure out if the made up precursor is consistent. Thanks for pointing me the right way!

 
Brian Pratt responded:  2021-02-19

Normally with all ions fragmentation we'd expect to see alternating collision energies (we look for precursors in the low energy scans, fragments in the high energy scans), but I'm not seeing that here.

I adjusted my transition list and made my targets into products

You shouldn't have to do that, unless that's actually what they are. But then, yes, there needs to be an associated precursor.

Perhaps you could explain a bit about your experiment?

 
dmesasan responded:  2021-02-19

Sure! We use Skyline in a bit of an odd way. We use it to extract chromatogram data from ion mobility mass spec imaging experiments, and then turn that into our images. We are using an Agilent 6560, and I was hoping rather than a traditional imaging experiment where everything is in MS1 and then later IDs have to confirmed by separate MS2 experiments I could just fragment everything at once, generate images for all fragments and then using drift alignment I'd be able to cluster all fragments from the original molecule. At this point I'm applying a set CE to everything that makes it past the drift tube and into the TOF component, but this can be modified in the future to drift dependent CEs. So basically yes they are products, but there isn't really a real precursor that I've pointed to.

 
Brian Pratt responded:  2021-02-19

That is interesting!

Internally Skyline notes that the MS1 data has nonzero CE and treats it as MS2 fragment data. Normally we'd also expect to see MS1 data with zero CE, which we treat as we would any other MS1 data. Can you set up the machine to do that - to alternate zero and nonzero CE values? Then set up your Skyline document to declare which precursors and fragments you're interested in, and all should be well.