| Brian Pratt responded: |
2025-03-24 05:47 |
If you can you provide your Skyline document (use File > Share to create a .sky.zip file), and the .d folder, we can have a look. You can upload to http://skyline.ms/files.url or by any other means if you prefer something more private.
Thanks for using the Skyline support board,
Brian Pratt
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| NBL responded: |
2025-03-24 06:14 |
Hello Brian
Thanks for your reply.
How can we send it to you more privately? We will send the .d folder. We were not able to generate a skyline document. Eventually we want to do a peptide search.
Waiting for your reply.
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| Brian Pratt responded: |
2025-03-24 06:34 |
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| NBL responded: |
2025-03-25 02:25 |
Hello Brian
I just sent an e mail, thank you
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| Brian Pratt responded: |
2025-03-25 09:15 |
I received the file, thanks. It seems to be perfectly fine - I can look at it in SeeMS, which uses the same underlying data reader code as Skyline.
But Skyline itself isn't intended for viewing raw mass spec data, it's intended to find chromatograms in mass spec data using targets that you supply as a first step. There are several ways of providing those targets, including running a peptide search, or providing peptide search results, or just a FASTA file, or a simple transition list.
So when you say "load these results on Skyline" what do you mean exactly? It sounds like what you probably want to do is to run a peptide search on that file using the File > Search > Peptide Search menu item. File > Open will only show you Skyline document files (.sky).
Let us know how this works out for you!
Brian
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| NBL responded: |
2025-03-26 02:34 |
Hello Brian
We indeed did a run peptide search, added our .d (we dont know if skyline is reading a .d file or .d folder).
And here are the screenshots of the processing. At the end of the peptide search we get an error message.
Thank you
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| Brian Pratt responded: |
2025-03-26 14:16 |
Skyline (and msconvert, SeeMS etc) sees the .d folder as the mass spec data "file".
Unfortunately I don't have enough experience with that peptide search tool to say anything useful, hopefully another Skyline developer will weigh in here.
Brian
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| NBL responded: |
2025-03-27 05:42 |
Hello Brian
Thanks for your reply. Do we need to repost our issue for another skyline developer to see it, especially since we sent the .d folder to your email?
Thank you
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| Nick Shulman responded: |
2025-03-27 06:18 |
When you get the "No matches passed score filter" message I believe that means that none of the peptide spectrum matches were better than the "Max q-value setting".
One of the pages of the Peptide Search wizard has a setting "Max q-value" which defaults to 0.01.
If you set that to a larger number such as 0.1 you would probably get some peptide spectrum matches, but they would likely be very low quality.
The Max q-value is the false discovery rate, and the peptide spectrum matches which do not pass the default 1% filter are very likely to be misidentifications.
How big is the FASTA file that you are using?
One common mistake is to use a FASTA file which is too small. If the FASTA only has a couple of proteins in it then there are not enough peptides for the target/decoy method of controlling the false discovery rate to mathematically work.
Another mistake is to use a FASTA file which is too large or a FASTA file where too small a percentage of the proteins could possibly found in the sample. In these cases, there is not enough difference between the target peptides and the decoy peptides in terms of probability of being found in the sample.
-- Nick |
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| NBL responded: |
2025-03-27 07:35 |
Hello Nick
Thank you for your reply
We increased the Max-q value to 0.1. We used a human proteome fasta (in the link below). With the 0.1 Max-q value, we got only 11 proteins, and only 2 peptides for our protein of interest. Do you think this fasta file is too large and is therfore causing this problem?
thanks |
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| Nick Shulman responded: |
2025-03-27 08:12 |
The human FASTA is probably the right size.
Which search engine did you choose on the "Search Settings" page of the Peptide Search wizard?
In my screenshot I have MS Amanda selected but that is probably not a good idea for timstof data because MS Amanda does not know anything about ion mobility.
For ion mobility data ChatGPT recommends choosing MS Fragger.
Make sure that the MS1 tolerance and MS2 tolerance are appropriate values for your instrument. I have had cases where no peptides were found because I was telling the search engine that my numbers were ten times more accurate than they actually were.
-- Nick |
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| NBL responded: |
2025-03-31 08:37 |
Hello Nick
We have tried MS fragger with 2 PTMs (1 fixed and 1 variable) and we entered the MS1 and MS2 tolerance.
We got an error message saying "no spectra found" (please find attached the corresponding file).
Do you have any idea of why we are getting this error?
Thank you |
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| Nick Shulman responded: |
2025-03-31 12:35 |
Usually I believe that "Mismatch between spectrum id format of the spectrum file..." error means that you are using a .mzML file which is different from what MSFragger searched.
However, given that you are running the MS Fragger search inside of Skyline, I do not see how it could be possible that the wrong file is being used.
I'll send you an email so you can send me your files and I can try to figure out what is going on.
-- Nick |
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| Nick Shulman responded: |
2025-04-01 13:01 |
Thank you for sending me your .d folder.
One thing I noticed is that when you do an MS Fragger search on that .d folder, a file whose name ends in "_uncalibrated.mzML" gets created. I believe that is the actual file that MS Fragger ends up searching because MS Fragger does not know how to read .d folders.
The peptide search will ultimately fail in Skyline because of that message about "Mismatch between spectrum id format of the spectrum file...".
However, if you then do another peptide search where you tell Skyline to search the file whose name ends in "_uncalibrated.mzML" then everything does seem to work.
For some reason, if you tell Skyline to search the "_uncalibrated.mzML" files then the spectrum IDs in the .pepXML file will have things that like this:
spectrumNativeID="controllerType=0 controllerNumber=1 scan=36056"
However, if you tell Skyline to search the .d folder, then the things will look like this and not work:
spectrumNativeID="1699"
I will have to ask my coworkers why this is behaving like this.
-- Nick |
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| Nick Shulman responded: |
2025-04-02 16:56 |
I was mistaken about MS Fragger not being able to read .d folders.
MS Fragger does indeed search the .d folder, and the "_uncalibrated.mzML" file is created for other reasons.
Apparently, this problem with the spectrum IDs not being interpretable by Skyline when MS Fragger searches the .d folder is a known problem.
It is believed that this might be fixed in MS Fragger 4.2, but Skyline only knows how to run MS Fragger 4.1 so you would have to run MS Fragger yourself outside of Skyline.
When I did a peptide search on the "_uncalibrated.mzML" in Skyline using MS Fragger with a 0.1 false discovery rate, I did find 13 peptides.
I had to use the File > Import > Results menu item to tell Skyline to extract chromatograms from the .d folder because the .mzML file does not contain MS1 spectra.
13 is probably not a good number of peptides to be found especially with a 0.1 false discovery rate.
You might be able to lower the false discovery rate if you use a FASTA file more specific to the type of tissue in your sample.
-- Nick |
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test .d (run peptide search).pdf
SearchSettings.png
uniprotkb_UP000005640_AND_reviewed_true_2025_03_05.fasta
test msfragger skyline.PNG