Spectrum missing

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Spectrum missing cunain1gx  2020-08-31
 

I am using Mascot DAT file to build a library in skyline. I can find a MS/MS spectrum for one peptide in mascot search result, but cant find this peptide spectrum in the skyline library. As I am trying to extract transition for this peptide from skyline, now it just not showed in the library. Should I change any parameters or anything? Because the spectrum can be observed with mascot search result. Thank you

 
 
Nick Shulman responded:  2020-08-31
When you are building the spectral library there are two settings that might affect whether you see a particular peptide.

One setting is the "cutoff score" and it controls allowable false discovery rate for peptide spectrum matches.
The default value for that setting is 0.95 (which represents a 5% false discovery rate).
If you change the cutoff score to zero, then you will see all of the peptide spectrum matches.

The other setting which might be affecting things is the "Include ambiguous matches". If that is unchecked, then any peptide spectrum match which was matched to more than one peptide will be excluded from the library. You will get more peptides in your spectral library if you change that checkbox to checked.

If neither of these ideas answer your question you can send us your DAT file and we can take a look. (Make sure you tell us which peptide you are looking for).

If that DAT file is less than 50MB you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url

-- Nick
 
cunain1gx responded:  2020-09-01
Hi Nick,
Thanks a lot for your reply, I did find more peptides when I set cut-off to 0 and checked the "Include ambiguous matches". however I still cant find the peptide I need. the Peptide is Y(phospho)S(phospho)PTSPK, Y1 and S2 got phsophrylation. Please help me with this issue. Thank you very much.
 
Nick Shulman responded:  2020-09-01
You need to also send us your .dat file in order for us to figure out what is going wrong.

If that .dat file is less than 50MB you can attach it to this supoort request.
Otherwise, you can upload it here:
https://skyline.ms/files.url
-- Nick
 
cunain1gx responded:  2020-09-01
I uploaded the dat file in the link https://skyline.ms/files.url. Description is Spectrum missing. Please help me. thanks a lot.
 
Nick Shulman responded:  2020-09-01
When BiblioSpec builds a library from Mascot search results, BiblioSpec only includes the top match for any particular spectrum.

The peptide YSPTSPK was theoretically one of the possible matches for spectrum #3196 (RT 7.87), but Mascot decided that the best match for that spectrum was YNHINAK.

Is there a reason that you expect to be able to find YSPTSPK in these search results? Is some other software telling you that that peptide should be there?
-- Nick
 
cunain1gx responded:  2020-09-01
Hi Nick, the phosphorylation of this peptide YSPTSPK is the experiment result after we knock down some genes. And mascot give me this spectrum with YSPTSPK rather than YNHINAK. How can I load the spectrum I need in skyline library? Can I change some parameter? Actually there are some other peptides, but I am not able to find them too. It will be quite troublesome if I can only observe in mascot but not in skyline as I need to do PRM. I attached this spectrum info here. Thank you for your help.
 
Nick Shulman responded:  2020-09-01
I modified my own copy of BiblioSpec so that it would include all of the peptide spectrum matches from the Mascot DAT file.

I uploaded the .blib file that I made here:
https://skyline.ms/_webdav/home/support/file%20sharing/%40files/full_filtered.blib
I am not sure whether this .blib file would be useful to you.

In your .pdf file, it looks like it is saying that YNHINAK got a score of 14.4, which is better than
YSPTSPK which got a score of 13.0

One thing that you can use this "full_filtered.blib" file for is to see which ions in the spectrum Skyline thinks match up with the theoretical ions from the peptide. With the default settings (0.5 m/z tolerance, and only showing b and y ions), only two ions from the observed spectrum match theoretical ions from your peptide (see "YSPTSPK_default.png").

In Skyline, you can adjust the tolerance with which Skyline matches theoretical ions to ions in a spectrum, using the "m/z match tolerance" setting at "Settings > Transition Settings > Library".
If you do that, and you turn on "A" ions, you get the picture I have attached "YSPTSPK_A_Ions_1mzTol.png"

My understanding is that your mass spectrometer (orbitrap?) is unlikely to produce A ions, and also should have a mass accuracy which is much better than 1 unit.

I am pretty sure that YSPTSPK is not the correct match for this spectrum.

Let us know if you think Skyline is doing something wrong with the way it is interpreting these Mascot search results.

Is there a reason that it is important to you that this peptide be in your spectral library.
You can always add peptides to your Skyline document using the menu item:
Edit > Insert > Peptides
You can specify the modified sequence of the peptide in the Insert Peptides dialog as:
Y[+80]S[+80]PTSPK
(You can also right-click on a modified peptide in the Targets tree and choose "Modify" to apply modifications to it).

One thing that spectral libraries are helpful for in Skyline is for choosing which transitions to monitor. However, in this case, you would probably do better choosing the transitions yourself. You can choose precursors and transitions by right-clicking on things in the Targets tree and choosing "Pick Children".

Hope this helps.
-- Nick