That's certainly a new variant of MGF - of which there are many, of course. What software produced this?

I am not sure just how much work it will be to support this, I will investigate that. Thanks for providing the example data.

Best regards,

Brian Pratt

Thank you for the response.

One tool we use is Pyteomics (https://pyteomics.readthedocs.io/en/latest/index.html) to investigate the mgf files and associate charges to product ions.

Do you have a way where I can properly annotate product ions with the correct charge programmatically? Right now I am working with ~600-1000 transitions in the spectral library...

Additionally, will improper charge annotation of transitions affect the usage of the spectral library when importing DIA results?

Thanks

Do you have a way where I can properly annotate product ions with the correct charge programmatically? Right now I am working with ~600-1000 transitions in the spectral library...

If you're up for some SQLite programming, you could look at populating the RefSpectraPeakAnnotations table the the .blib file that gets produced. That's where we store fragment charge information for the (very few) import formats that provide it.

Additionally, will improper charge annotation of transitions affect the usage of the spectral library when importing DIA results?

No, but for now at least you'll have to live with the fragment charge information being ignored. So you'd lose the sense of the actual mass of the fragments, and only know the m/z. But that's always been true with the MGF format. While this does seem like a useful extension of the format I doubt you'll find a lot of MGF readers that recognize it (yet?).

I'll put this (i.e. getting that information into .blib's RefSpectraPeakAnnotations table) into our development queue.

Best regards,

Brian Pratt

Thank you for the response. I have investigated the .blib database file and see that I can fill in the RefSpectraPeakAnnotations table. I will try that.

Thank you for putting this in the queue.

1 minor question for filling out the RefSpectraPeakAnnotations table, which columns are required and is there a place I can go for information on each:

id RefSpectraID peakIndex name formula inchiKey otherKeys charge adduct comment mzTheoretical mzObserved

Thanks

This should be useful:

https://raw.githubusercontent.com/ProteoWizard/pwiz/master/pwiz_tools/BiblioSpec/tests/reference/tables.check

You'll need id, RefSpectraID, peakIndex, charge, mzTheoretical, mzObserved at a minimum.

Don't hesitate to ask if you have other questions.

Brian

I have filled in the id, RefSpectraID, peakIndex, charge, mzTheoretical, mzObserved information for the RefSpectraPeakAnnotations table but when I reload the spectral library to my skyline-daily session I am met with this error (attached).

Failed loading library 'automation_test'.
Specified cast is not valid.

I have taken the peakIndex from the SpecIDinFile column in the RefSpectra table (not sure if that is the proper index into the mz/intensity list for the RefSpectra)

Thanks

Perhaps I can see your modified.blind file?

.blib file, that is (autocorrect got in the way)

.blib file, that is (autocorrect got in the way)

Thanks for looking into this. Here is the modified.blib

Thanks. It looks like a misunderstanding of the "peakIndex" field. That's an index into the fragment [m/z, intensity] pairs for the RefSpectra noted in the RefSpectraId field. So instead of those all being 5 for RefSpectraId=6, I'd expect to see them increasing monotonically from 0 (assuming you're annotating every fragment).

... and then starting at 0 again when you move on to RefSpectraId=23

Ah I see. It looks like that worked. I will investigate my library and make sure it all checks out.

Thank you for all of the help! I will ask any other questions as they come.