Custom made modification [Phospho-18O (STY)] is 'filtered out' during import of peptide search results

support
Custom made modification [Phospho-18O (STY)] is 'filtered out' during import of peptide search results jmvb885  2022-06-13
 

Hi!

I want to explore the MS1 signal of some samples in which proteins were phosphorylated with heavy ATP. I know that the gamma phosphate of my heavy ATP has two Oxygen-18 atoms. Hence when I type in the formula in both Maxquant and Skyline, I get a mass of 83.9748. I did my database search using Maxquant and now I am trying to import the results into Skyline to explore the MS1 signal.
For some reason, it seems that Skyline is not recognizing the Phospho-O18 (STY) modification I've created, since the wizard for importing the search results doesn't suggest its addition (in contrast to when I do the exact same thing using results from a sample with light ATP, meaning the regular Phospho STY (80) is added). But most importantly, after the wizard finishes importing the raw files, the 18O modified peptides are not detected.
To me it seems like Skyline is filtering these peptides out during the import of the database search results, hence they are not in the target list and the chromatograms are not extracted.
I have already confirmed that the 18O peptides are there (they are detected by Maxquant and I've seen their MS1 signal in Qualbrowser)
Any ideas on how to fix/solve this?
Thanks in advance for the help!
Best regards,

Juan M. Valverde

 
 
Nick Shulman responded:  2022-06-13
I do not understand your question.

Can you 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 including spectral libraries.

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

-- Nick
 
jmvb885 responded:  2022-06-13
Hi Nick,

I have database search result files from MaxQuant. Here I searched peptides containing a specific modification (Phospho-O18). Now, I am trying to upload these results to Skyline to assess the MS1 signal. When I load the database search results and .raw files I cannot see the modified peptides. I don't see them in the library or in the target list.
For this I've been following the 'MS1 Full-Scan Filtering' tutorial. I uploaded the file via https://skyline.ms/files.url. Name is MS1_Filtering_Heavy.sky
Best,

Juan M.
 
Nick Shulman responded:  2022-06-13
Thank you for sending that file.
When I go to:
View > Spectral Libraries
Skyline brings up a window where it says it could not determine the meaning of some of the modifications in the library.
Those modifications are on S, T, and Y and have the mass +81.97476.

A regular phospho modification has a mass +79.966331

Are you sure that the modifications are supposed to have two 18O atoms? The modifications in your spectral library seem closer to what you would get if you had only one 18O. If it had two heavy oxygens, the mass would be closer to 83.9.

I think what you need to do is go to:
Settings > Peptide Settings > Modifications
and use the "Edit List" button next to the structural modifications list, and add the definition for your modification which has a mass of 81.97476.

-- Nick
 
jmvb885 responded:  2022-06-14
Hi Nick,

Yes, I am aware of the presence of the +81.97476 modification on S, T, Y. We think this is due to presence of phosphate groups with just one 18O atom. Despite this, we know that >99% of the ATP we used for this reaction has two 18O atoms. MaxQuant successfully detects phosphopeptides with the +83.97 modification (two 18O atoms). You can confirm this in the msms.txt file I am using to generate the spectral library. That is why I think the problem is that Skyline is not 'detecting' peptides with this modification, or filtering them out for some unknown reason.
I can also confirm that these phosphopeptides with two 18O atoms are present: I have one sample in which I have equal parts of Light ATP, which would generate 'regular' phosphorylation (+79.966331), and heavy ATP (two O18 atoms, +83.97). I did another search in MaxQuant looking only for the regular phosphorylation, and when I load the results into Skyline and explore the MS1 signal I can see the presence of the 'light' phosphopeptide and the 'heavy' phosphopeptide. Check the snip attached. It belongs to a +2 charge precursor, so you will see a difference of roughly 2 m/z units between the two species. This is compatible with a difference of +4 Da (83.97-79.97=4).
Best regards,

Juan
 
Nick Shulman responded:  2022-06-14
I am not sure that I understand what you want Skyline to be doing.

How is Skyline supposed to know that it should be applying a +83.97 modification to your peptides?
Usually, if you wanted Skyline to use a modification like that you would go to:
Settings > Peptide Settings > Modifications
and add a definition for this modification.

Did the peptide search results which you used to build your spectral library look for this modification? There are no peptides with the +83.97 modification on them in the .blib file. If there are supposed to be some of those in the library, then you should send us your peptide search results and we can figure out why those particular modified peptides are not showing up.

If you would like to learn more about all the different types of modifications in Skyline, some people have found this webinar useful:
http://skyline.ms/webinar10.url
-- Nick
 
jmvb885 responded:  2022-06-14
That is the point I've been trying to make: Yes, the peptide search results I am using to build the library found the +83.97 modification. I created this modification on MaxQuant when I did the database search. I also created this modification in Skyline. Still, when I load the peptide search results into Skyline these peptides are not recognized.
Find attached the msms.txt file from MaxQuant, which is the same one I've been using to load the library results into Skyline.
Best,

Juan
 
Nick Shulman responded:  2022-06-14
Can you also send me your "mqpar.xml" file?
If you also have a "modifications.xml", "modifications.local.xml" and/or "evidence.txt" you should send those files.
-- Nick
 
jmvb885 responded:  2022-06-16
H Nick,

I just uploaded the full txt file form the maxquant database search. The name is 'Phospho_018_Maxquant_txt'.
There you can find all the tambles and mqpar.xml file.

Juan
 
Nick Shulman responded:  2022-06-16
Thank you for sending those files.

I think this will work correctly for you if you use Skyline-Daily instead of Skyline 21.2.

When I build the library using Skyline 21.2, it thinks that the mass of those modifications is 81.97476 but when I build the library using Skyline-Daily it correctly says that the mass of the modifications is 83.97482.
You can install Skyline-Daily from here:
https://skyline.ms/project/home/software/Skyline/daily/begin.view?

When reading the "modifications.local.xml" (or "modifications.xml") file BiblioSpec used to think that the mass of the element "Ox" was 17 Daltons, but at the end of last year it was fixed so that "Ox" has the mass of 18 and "Oy" has the mass of 17.
-- Nick