Bibliospec library building from XL data searched on PD

Bibliospec library building from XL data searched on PD af1234  2023-03-29

I have a 4 proteins XL sample which I searched using PD (Xlink + Sequest + Xcorr CSM scoring). Now I want to build a library from the .pd result file. I am having issues because there is no qvalue (i.e no percolator qvalue) and even setting it to 0 raises an error.

I have seen and I wonder what would be the easiest way to get all the XL peptides imported into skyline.

Ideally I would prefer to not research my raw files with one of the proXL supported search engines. Is there any way to directly import XL peptides from PD?



Nick Shulman responded:  2023-03-29
Can you send us your .pdresult files? Files which are less than 50MB can be attached to this support request. You can upload larger files here:
(I am not sure whether Skyline needs any other files (.msf?) in addition to the .pdresult files, but, please include them if you think Skyline or BiblioSpec uses them as well.)

I am not sure, but it might not be possible to use the "set cutoff score to zero" workaround in Skyline. I could imagine that Skyline might be failing to figure out what score types are in the .pdresult file so you are never able to get to the page where you can specify the cutoff score.
It might be that the only way to specify a cutoff score of zero would be to run BlibBuild.exe from the commandline (and you would then have to run BlibFilter.exe after that in order to produce the non-redundant library that Skyline needs).
However, even if you did do the zero cutoff workaround, the spectral library would probably not be useful in Skyline because the crosslinks will not have been understood.

It might be possible for us to write a proxl converter for your crosslinked pdresult files. After I get your .pdresult files I will ask our proxl converter expert how feasible that would be.
-- Nick
af1234 responded:  2023-03-29
Hi Nick,

Thanks for the quick answer. I uploaded both the msf and the .pd result file. the prefix for both is default-ms2_DSSO_wDecoy
Nick Shulman responded:  2023-03-29
Thank you for sending that .pdresult file and .msf file.
It looks like that .pdresult file is completely empty and does not contain anything that looks like a peptide spectrum match.
You cannot build a spectral library from that .pdresult because even if you specify "1" for the score threshold, BiblioSpec will complain that the there is no such table "MSnSpectrumInfo".

It is possible to build a spectral library from the .msf file. You need to specify "1" for the "Score Threshold" value in the Import Peptide Search wizard.

I will ask around and find out if it would be possible to make a proxl converter for that .msf file.
-- Nick
af1234 responded:  2023-03-30
Hi Nick,

Thanks, now I can import my file, however even after defining cross linker (DSSO) and quenched (NH2/Tris/H20) it seems that Skyline does not recognize any peptide in the library as XL. Is there a working skyline template with XL that I can take a look at to copy the peptide settings? My guess is that Skyline matches the fasta file and disregard sequences which are not in the fasta (i.e my pep1-pep2 XL sequences).
Nick Shulman responded:  2023-03-30
Right. The spectral library that you create from that .msf file is not going to have anything in it that Skyline recognizes as a crosslinked peptide.
We are going to need a msf to proxl converter in order to be able to create a real crosslinked spectral library.
I have asked our proxl expert how difficult it would be to write such a converter but I have not heard back.
There might be some sort of workaround that we could do in the meantime.

Could you send me your Skyline document? I would like to understand what sort of modifications you have on your crosslinked peptides.
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:
-- Nick
af1234 responded:  2023-03-30
Here is the zip file. Hope there is an easy workaround!
Nick Shulman responded:  2023-03-30
Thank you for sending that Skyline document.

Can you give me an example of a spectrum where your search engine found a crosslinked peptide? I do not see anything in the .msf file which looks like what I would consider to be a crosslinked peptide, which would be two peptide sequences joined by a crosslinking molecule. It might be that I do not know what I am supposed to be looking for, so if you could tell me a raw file name and spectrum number (or retention time) where a crosslinked peptide was found, I could figure out what they look like in the .msf file.
It would probably also be helpful if you could send me the .raw file where the crosslinked peptide was found.
-- Nick
af1234 responded:  2023-03-31
Sure, find attached the search result and I uploaded the correspondent raw file lu0104186
Nick Shulman responded:  2023-03-31
Thank you for sending me that text file with the list of crosslink peptide identifications.
Is there a name for that type of file?

I was able to make a spectral library from your text file and the raw file you sent me. We are going to need to make some changes to BiblioSpec before you would be able to make a spectral library like this yourself. Specifically, BiblioSpec can read a simple text file format called "spectrum sequence list" (".ssl"). Unfortunately, it is not currently possible to have a crosslinked peptide sequence in a .ssl file, but I think we will be able to fix that soon.

I hope the attached .blib file will be useful to you.
You can add it to your document using the "Edit List" button at "Settings > Peptide Settings > Library" and after you have the library in your document you can use the "Add All" button on the "View > Spectral Libraries" window.
-- Nick
af1234 responded:  2023-04-03
Thanks for the fix!

This is just a test dataset and we plan to use Skyline for XL-MS more systematically in the future so I we are keen on getting a working solution.

Can you briefly explain (either here or via mail) what was done/how was the blib file generated?