ERROR on imported spectral library generated by Spectronaut software

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ERROR on imported spectral library generated by Spectronaut software Winnie  2020-02-17
 

Skyline team,

Hi! I have problems in importing the spectral library (.csv) generated by Spectronaut software (file >import> assay library) int skyline. The detailed error information attached. According to the error information, the Modification tab in the Peptide Settings, the m/z types on the Prediction tab and the m/z match tolerance on the Instrument tab of the Transition Settings were re-checked. See the attachment for a screenshot of the parameter settings. I did not find any errors.

Is there any wrong in importing the spectral library? The attachment for a screenshot of the spectral library was also upload.

Thanks in advance,
Winnie

 
 
Nick Shulman responded:  2020-02-17
Winnie,

Can you send us that .csv file that you are trying to import?
Also, you should send us your Skyline document.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files.

If those files are less than 50MB you can attach them to this support request. Otherwise, you can upload them here:
https://skyline.ms/files.url

It sounds like Skyline disagrees with that .csv file about what the m/z values for the peptides should be. It looks like all of the errors have something to do with the Carbamidomethyl modification, since the amount of the disagreement is always either 57.02, or a fraction thereof corresponding to different charge states.

Your Cabamidomethyl modification settings in Skyline correct (it looks like the way that we have them in all of our experiments). Does Spectronaut think that your Cysteines are supposed to have that modification on them?

I am not sure from your error messages whether the problem is that the modification is being incorrectly applied to Cysteines that are supposed to be unmodified, or the other way around. I am sure we will be able to figure it out once we see your Skyline document and .csv file.

-- Nick
 
Winnie responded:  2020-02-18
Nick,

  Hello, it seems to something error in modification settings.

   I reassembled a small library that contains error peptides reported during import and normal peptides during import. The detailed spectral library and
   Skyline document attached.

   Thanks for taking the time to post your feedback.
 
Nick Shulman responded:  2020-02-18

It looks like this new .tsv file has different errors than what you were originally asking about. I don't think these errors have anything to do with cysteine modifications.

Here is the first error:
Product m/z value 756.3709 in peptide YWPTMGPQK has no matching product ion.

And here is the corresponding line from the .tsv:

PrecursorMzProductMzPrecursorChargeProductChangeLibraryIntensityNormalizedRetentionTimePeptideSequenceModifiedPeptideSequencePeptideGroupLabelProteinIdUniprotIdFragmentTypeFragmentSeriesNumberCollisionEnergyPrecursorIonMobilityTransitionGroupIdDecoy
562.2656845756.3708863217.8441487.285915YWPTMGPQKYWPTM(UniMod:35)GPQKYWPTM(UniMod:35)GPQK_2P23780BGAL_MOUSEP23780BGAL_MOUSEy7-1-1

Your .tsv file wants the mass of the y7+ ion of the peptide YWPTMGPQK to be 756.37 but Skyline thinks it should be 758.3865.

One way to tell what m/z Skyline is going to assign to a fragment ion of a peptide is to add the peptide to your Skyline document with the menu item "Edit > Insert > Peptides". Then, once you have the peptide in your document you can right-click on the precursor under the peptide and choose "pick children". You might need to press the filter button in the child picker window if the transition that you want to see is not there.

This website also agrees with Skyline that the y7 ion of this peptide should be 758:
http://db.systemsbiology.net:8080/proteomicsToolkit/FragIonServlet?sequence=YWPTMGPQK &massType=monoRB&charge=1&bCB=1&yCB=1&nterm=0.0&cterm=0.0&addModifType=&addModifVal=

By the way, I noticed that you have a column called "ProductChange" in your .tsv which should probably be "ProductCharge".
-- Nick