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.