Potential mismatch peptide library and results

Potential mismatch peptide library and results t j f m voermans  2019-02-25

Dear Skyliners,

When analyzing my QTOF results and having them imported in Skyline, I asked Skyline to match the spectra with the peptide library. Multiple matches were found (seeing the green dots), but after analyzing the spectra myself via various software, I came to the conclusion that the m/z values found by Skyline do match, but the number of charges doesn't. Therefore, the fragments from the library probably are probably not the fragments found by the QTOF.

Are there some settings that I need to change, or am I making mistakes with my reasoning?

Than you!

Nick Shulman responded:  2019-02-25
I am not sure that I understand your question.
Can you send us your Skyline document?
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file is less than 50MB, you can attach it to this support request.
Otherwise, you can upload it here:

It might also help if you post a screenshot of what you are seeing in Skyline and in your other software.
-- Nick
t j f m voermans responded:  2019-02-28
Hi Nick,

Sorry for the delayed answer. I have made some screenshots and created the zip file.

To clarify my previous post, simply said I find different results when I manually check the precursor masses of my peak with a program like Magtran than Skyline. I do find a peak in my spectrum with around the same number as a 2+ precursor (sequence: K.IVIGMDVAASEFYR in skyline, precursor mass: 1570,7934) predicted by Skyline, however the peak found in my spectrum is not 2+ (See PDF: '5,2 789,09 peak' and '5,2 789,09 peak zoomed in'). In other words, I am not sure to what extent I can trust Skyline matching the fragments with the chromatogram I created.

The other PDF's show the chromatogram, spectrum and Magtran results of the spectrum. Because Skyline says that it found K.IVIGMDVAASEFYR at 5,2 but I couldn't find that exact number I choose a range starting at 5,15 till 5,30. The range in Magtran was set at 1500 till 1600 because that is the mass of the precursor: K.IVIGMDVAASEFYR.
Nick Shulman responded:  2019-02-28
It looks like you are only extracting MS1 chromatograms for all of your peptides.
In that case, Skyline ends up choosing peaks based on whether the M, M+1 and M+2 chromatograms coelute with each other. This is not enough information for Skyline to get the right answer in most cases.

Was this a DDA experiment? Can you run a peptide search on these results and give those results to Skyline so Skyline can do a better job?
The MS1 full scan filtering tutorial shows how to use peptide search results from another program to then tell Skyline to extract chromatograms for the peptides that were identified in those search results.

If you cannot give Skyline peptide search results, there are ways to tell Skyline to look at more features other than the coelution and intensity when choosing peaks. This involves training an mProphet model and is covered in the Advanced Peak Picking tutorial. You probably do not want to do that because with only MS1 data to work with, the false discovery rate will probably still be too high, but here's a link to that tutorial:

Hopefully you can do a peptide search on your data. Skyline does not know how to do a peptide search itself, but here is a list of all of the peptide search formats that Skyline understands:
-- Nick