Hi Morgan,

We're always looking to expand that list of supported search tools, if you have some that you work with we'd be interested to know about them.

One common way to get such data into Skyline is to read a NIST-style .msp file, if your search tools generate that.

Have a look at this tutorial for information on method refinement:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_method_refine

it's in terms of proteins but the principles apply to small molecules too.

Thanks for using the Skyline support board!

Brian Pratt

The group I'm working with uses SciEx's software, which to my knowledge does not support exporting libraries. To clarify, I was wondering if there were any small-molecule search tools that you already supported, or other streamlined approaches to creating small molecule libraries that you were aware of.

Regarding method refinement: I don't see any options in that tutorial or in the software that would address the issue I'm mentioning. The precursor peaks that I am referring to are within the fragmentation spectra, and sometimes quite abundant, so ranking by intensity, etc. would not work. Am I mistaken that the 'Precursor m/z exclusion window' is supposed to address this?

Hi Morgan,

We don't currently have any small molecule specific tools supported, but as I say we're very interested in that. Does the Sciex software produce any kind of report that might form a basis for library creation?

The 'Precursor m/z exclusion window' value controls how Skyline selects which fragments to use from an existing spectral library, so that's probably not quite what we want here.

But I'm not sure I'm completely understanding your situation - can you provide a Skyline document (use File|Share with include results files) for context?

• Brian

Hi Brian,

Thanks for the response, and I appreciate you for clarifyig that Skyline do not currently support any small molecule search engines; for now I'm happy to learn that I'm not creating the sll library inefficiently. Regarding SciEx output, I'm not sure as I'm relatively new to working with this instrument/software/group. I'll have to ask others in the group and possibly some of the SciEx reps themselves. I have a feeling the answer is no, but I'd love to be proven wrong.

Regarding the 'Precursor m/z exclusion window', what you just described sounds to me like what I am trying to do. I created ssl files from an mzml and selected spectra to generate a small spectral library to test the small molecule features. I'm trying trying to keep skyline from selecting the unfragmented precursor as a transition, despite the fact that it is relatively high abundance in the spectral library.

For example: I have a molecule with a precursor m/z of 177.1022. In the corresponding library entry, the unfragmented precursor (again, m/z=177.1) is the second highest ranked ion. I set the 'Precursor m/z exclusion window' to 10 mz and import the library entry into the document using the "Spectral Libraries -> Add All...." command. Based on my understanding of that window, I would expect that Skyline would not automatically select the 177.1 m/z peak, but it is nevertheless selected as a transition. Maybe this is a bug? I've attached my skyline document with the library included to help illustrate.

I appreciate your help

Thanks for the files.

I think you're right, 'Precursor m/z exclusion window' is not working properly for non-peptide molecules. I'll get that fixed ASAP.

Thanks,

Brian

Thank you for looking into that bug; fixing it will be extremely helpful.

Regarding SciEx output, I've been looking into the software, and it seems that I can export spectral libraries, but only in sciex's proprietary data format. Would that be useful to you folks?

Please do send an example, perhaps we can make some sense of it.

And if there's any kind of human readable report it generates that makes you think "I could probably figure out what goes into a small library based on what's written here", we'd like to see that too.

Thanks,

Brian

I've attached a few outputs from SciEx OS. I think the .lbp is going to be the most useful file for you; it should contain 400-some compounds with spectra acquired on a SciEx 5600 instrument.

Thanks for the help/interest.

Thanks for those. The lbp file does look promising, unfortunately it's in a binary format that doesn't seem to follow any standard database structure.

This link
https://sciex.com/support/knowledge-base-articles/how-to-backup-mass-spectrum-databases-with-libraryview-when-it-is-impossible-to-export-as-an-lbp-file-due-to-a-software-issue_en_us
suggests that the information might be available in a more standard format, though.

Good catch. I dug ours up and uploaded it to this support forum. (File too large to post directly here). The zip file is titled: 2024.09.18_MWMann_LibraryView_log.zip .

Hope this is easier to work with.

It does look like the .mdf file has sufficient information to associate a compound with a spectrum. Do you have anyone on staff that can work with this kind of thing?

We don't have much computational expertise I'm afraid, and I think I'm the only one here who is particularly invested in using Skyline for our purposes.

What kind of work would need to be done with these files? If it's not too complex, I might be able to invest some time on it.

I've been communicating more with SciEx on this, and they've shared that in their new (unreleased) library software, they have an option to export to plain text. They sent me some example files based on one of my libraries, and were fine with sharing with you guys, so I'm uploading it here, in-case this is something you folks would be able to support in the future.

For now, I think I can use these to generate .mzML files to recreate the spectra contained within, and then add those to a skyline library via a .ssl file.

Otherwise, any news on the precursor window exclusion? I've been scanning new release notes, but haven't seen anything on it.

That format does look useful.

re precursor window exclusion - my apologies, I did work on that then got sidetracked. I'm bumping it to the head of my work queue and will make sure it appears in a Daily ASAP. Thanks for the followup.

An update/question:

I was working with the output SciEx sent, and managed to convert it into an mzml file and associated ssl for library import. However, I get an error when I try to import into Skyline (attached as a text file). I'm guessing that something about the way I built the mzml doesn't agree with skyline's expectations, so I was hoping someone could look at it and let me know if there's a field/etc. that I could add.

Files attached.

Looking at the mzML in SeeMS (which uses the same reader code as Skyline) I see that the scan times are all set to zero. Is that intentional? That's not the kind of data Skyline is designed for.

Perhaps try converting with msconvert (instead of what appears to be "mzR", from looking at the mzML text)?

Regarding scan times:
Since I'm not starting with a real spectra file, (but rather with the text-based spectral library), there's not a true scan time associated with each spectrum. I can set an arbitrary, unique scan time for each, but that doesn't seem to fix the error. In any case, I wouldn't expect the scan time to be especially valuable for library building?

Regarding msconvert:
Since I'm not starting with a traditional spectral file, I don't think msconvert supports this? In this case, I created the mzml file using the R package "Spectra", which could export an mzml after I manipulated the text file in R. I did try to a few things on this end though:
1)I ran the generated mzml through msconvert using a trivial ms2-only subset filter, which adds a proteowizard step to the processing methods embedded in the mzml file.
2) I also tried to replace the processing method in the mzml file manually (i.e. by find and replace) with the sections from a more traditionally generated file.
Unfortunately, both approaches generate the same error as before, so I don't think that ms-convert alone is the magic bullet here.

If you have alternative suggestions than the "Spectra" package I used, which you think might work better with Skyline, I'd be happy to hear them. In this case, it was the only method I knew would work with the text file that SciEx provided.

Otherwise, I'd welcome other ideas on what might be missing/different about my mzml files to cause this issue. E.g. If skyline needs a chromatogram, or instrument information, or something similar, maybe I can add those fields before export.

Ah, right - this is a BiblioSpec issue, not Skyline per se. Let me investigate a bit more...

OK, a couple of issues here:
In the SSL file, get rid of "scan=", just need a bare number in that column. Easy fix.
In the mzML, there's no precursor information for those MS2 scans. Can you add that? Not strictly necessary for our purposes, but the mzML parser is tripping over it.

Adding precursor information (and the ssl change to scan number) fixed it! Everything imports into skyline smoothly now!

This is immensely helpful to me and my projects; thank you!

Excellent! Glad we could help.