This code change shouldn't be necessary (and isn't really proper either - explicit RT is a different concept than library RT - but we appreciate the effort!).

Normally you'd just set a tight tolerance for "Transition Settings > Full-Scan > Retention Time Filtering > Use only scans within <tolerance> minutes of MS/MS IDs". But if that's not working for some reason, let's learn more about your situation.

Thanks for using the Skyline support board,

Brian Pratt

and isn't really proper either - explicit RT is a different concept than library RT - but we appreciate the effort!

Are these concepts documented somewhere? Then it would probably be clearer to me why this wasn't doing what I expected in the first place :)

Our issue:
In small molecule target quantification, we start from RT + accurate mass. Explicit RT and RT window are fundamental to this workflow. We use the MS2, if we have it, to further confirm what we already know from RT + accurate mass. We might not have an MS2 confirmation for our target in every specific file.

We are quite happy with importing CSV transition lists with formula and explicit RT, which can include MS2 fragments. However, in this case, the users don't get the MS2 dotp.

On the other hand, we can import spectra from our in-house curated spectral library with matching retention times. However, in this case, only formula and spectrum are imported; the users would have to add explicit RT by hand.

I just want to provide a way for our users to add compounds to their documents with

• formula
• explicit RT
• spectrum
  so they can do quantification based on RT + accurate mass and still get a dotp for their spectrum if there is one.

(Aside, it would be cool to have a head-to-tail plot for spectrum vs library spectrum somewhere. If this exists, I haven't found it yet...)

Yes, we should put together a support tip that explains the interactions of explicit RT, library RT, and iRT. It is somewhat complicated. In software there's always tension between flexibility and simplicity...

At any rate, you have a couple of options, both of which start with loading a spectral library.

Once you have that:
Set up your transitions by importing a transition list that contains explicit RT and explicit RT Window information
or
Add the contents of the spectral library to the document, then use the Document Grid to add Explicit RT information to the precursors. This can be done pretty easily as a columnwise cut and paste action (assuming, of course, that you have the order right).

Spectral libraries don't carry RT window information, so even if we added a "Use Library RT as Explicit RT" option when adding spectral library entries to the document, it wouldn't solve the problem (unless, I suppose, an additional option "Use this Explicit RT Window for all precursors" would suffice).

Aside, it would be cool to have a head-to-tail plot for spectrum vs library spectrum somewhere. If this exists, I haven't found it yet..

The Library Match window is meant to convey that kind of information - is it not quite what you need?

Best regards.

Brian

Hi,
thanks for getting back to me;

I tried setting up the method starting from an inclusion list with RT as you suggested. It works "partly" but not fully.
(Option with copy/pasting RT into document grid: I don’t want to suggest this to my users, as it’s quite error-prone. At that point I’d probably write a script that modifies the XML directly.)

Steps to reproduce
See the upload in the "file sharing folder": 20240214_Skyline_MS2.zip

• Start Skyline
• Import Transition List
• Type: Quantification
• Molecule Settings: Library -> add «Eawag_45». Pick mol matching Library, rank by Picked Intensity
• Transition settings:
  • Filter: precursor [M+H]+, Fragment [M+] ion types f, p;
  • Library: tolerance 0.5 m/z, [x] pick ions, 3 product ions
  • Full scan: ms1: count, centroided, 3 peaks, 5ppm; msms: dda, centroided, 5 ppm, include all
• Paste data from transitions.xlsx
• Enable auto-select
• Save document
• Import results: import the two raw files
• Adjust the view somewhat, then Save again

Results

• Library spectrum is correctly shown in “Library match”
• The MSMS spectrum is correctly extracted, and can be seen clicking on the trace / potentially selecting “MS/MS” if MS1 is selected
• However, no dotp value is shown in the “Targets”. Also, in the Results grid, “Library dot product” is #N/A
• The “Library match” only shows the library spectrum
• We hope to see a head-to-tail plot, or some other visualization of the measured vs reference spectrum, such as this example: https://www.mdpi.com/metabolites/metabolites-10-00158/article_deploy/html/images/metabolites-10-00158-g006.png , ideally with the library dot product shown somewhere
  • Interestingly, while I’m on a “Spectrum information unavailable” view, I can select “Mirror” in the context menu. This option doesn’t exist when I have a small molecule library match.
  • While I now have a “Spectrum” and a “Mirror” selector, I can only choose “Eawag_45”, not the measured spectrum there. The measured spectrum is only in the “Full scan” window.

Further potential bug

• If i now adapt the explicit retention time using right click -> modify and set venlafaxine to 14.1, the InChIkey is lost (this can be confirmed in the document grid, where the inchikey is now blank).
• It is also not possible to add the InChIKey back in, as this field is seemingly readonly in the document grid
• As expected, the “library match” screen then shows “Spectrum information unavailable”.
• (This doesn’t happen if I adapt the inchikey directly in the document grid)

I can have a look at this early next week.

Having those files in hand to reproduce your steps is a huge help, thanks for that.

It's not clear that it is a good idea to add m/z's from the library to the document, since it's difficult to know whether a particular m/z is characteristic of the molecule of interest

That can be certainly discussed in general, however for this it shouldn't matter whether the molecule is added from the library via "Add" or via matching, right? Doesn't the user choose this behaviour / agree to it by setting "If a library spectrum is available, pick its most intense ions" and "Pick X product ions"?

By the way, an interesting consequence of "If a library..." checkbox for small molecules; I don't know if this is intended or not:

• if the checkbox is activated, the viewer in Settings -> Spectral Libraries highlights the top-n ions in the spectrum in blue
• if the checkbox is deactivated, the viewer in Settings -> Spectral Libraries highlights all ions in blue, and "add" inserts all fragments to the document. See attached screenshot.