The usual reason that a chromatogram might be missing for a transition that is in your document would be that the transition was added to the document after chromatograms were imported.

If you add transitions to the document you usually need to tell Skyline to extract chromatograms again so that the new transitions get chromatograms.

It sounds like you are saying that something else is going on, but I am not sure that I understand your question.
Could you send us your Skyline document and your raw file?
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms and spectral libraries.

If that .zip file and your raw file are less than 50MB you can attach them to this support request.
You can upload larger files here:
https://skyline.ms/files.url
-- Nick

Hi Nick,

I'm Nadine's colleague. What you describe is the issue we have. We use the settings in Skyline as a "mostly good" prefilter for the transitions we end up including in our document. However, after importing the data and curating it (adjusting boundaries/removing peaks; marking transitions non-quantitative etc.) we often enough want to adjust the selected fragments for targets that ended up with more usable fragments excluded than intended.

The way Skyline works (by design, I take from your explanation) makes it difficult to "rescue" targets that initially looked to be poorly supported. Namely, (1) there is no easy way of seeing what fragments we might be missing without opening up the full scans -- and for low abundant targets that don't have a known retention time, we wouldn't even know where to search for this full scan -- (2) and re-importing the data undoes the manual peak integration that's been performed already within the document, which turns this from being merely a 'clunky' experience to a real roadblock in our processing pipeline.

Of course, we could just be using the software suboptimally, so please do share if you know of a better way of dealing with this. But assuming we're working correctly it would be great if there could be an option to import every transition with a matching signal, even if they are not selected.

Kind regards,
Wouter

You should use the "Reimport" button on the "Edit > Manage Results" dialog to reimport the results.
When results are reimported using the Reimport button, Skyline is supposed to preserve any manual peak integrations that you may have done.
If you have a case where manually adjusted peak boundaries are being lost even though you used the "Reimport" button, that is probably a bug that we need to fix.

If you are reimporting results in a different way such as by first going to "Edit > Manage Results" and removing a replicate, saving, and then using "File > Import > Results", then you will lose any manually adjusted peak boundaries.

We have tried very hard to make sure that the "Reimport" button at "Edit > Manage Results" preserves manually adjusted peak boundaries. There have been some very unfortunate bugs in the past where manually adjusted peak boundaries were getting lost, so we try very hard to investigate any scenario where it sounds like someone might be saying that is happening. (Please send us your files if you think this might be happening).

I wonder if you might have an easier time in Skyline if your document began with all of the transitions selected, and then you refined the document by deleting transitions.

If you want Skyline to add all of the possible transitions to the document, you would first go to:
Settings > Transition Settings > Filter
and change the options in the "Product ion selection" dropdowns so that the "From:" box says "ion 1" and the "To:" box says "last ion".
Then, at:
Settings > Transition Settings > Library
you would choose the option "From filtered ion charges and types plus filtered product ions" so that all of the transitions matching the filter criteria will be added to the document.

Then you can go to:
Refine > Advanced
and check the checkbox next to "Auto-select all ... Transitions"

After you have the complete set of transitions in your document, you can tell Skyline to extract chromatograms.

I imagine that what you are trying to do is a very common scenario. Someone else on the Skyline support board might have a much better idea about how to deal with this.
-- Nick

Hi Nick,

Thank you for your detailed answers. Trying again under controlled conditions it appears that re-importing *as per the intended way* does not reset peak boundaries. Turns out we were sometimes re-importing the incorrect way, which confused our understanding. And it seems that your other steps do lead to a complete import, which will definitely help with a specific "in the weeds" analysis I am currently working on.

There was one missing step to solve our issue, but we managed to figure this one out ourselves. Namely, we want all of the data imported, but then to apply the document-wide "good enough" filter settings (as we don't want to manually re-disable everything). It turns out you guys made a useful little button on the transition selection menu to apply the (current) document settings.

So overall, we are adjusting our workflow as follows:

- Import raw files with all possible transitions enabled
- Then configure the document with our desired filter settings
- Apply the filter per target as we curate the data; this will hide but otherwise leave available all transitions to quickly toggle it on/off as we need to

Thanks for your advice!

Kind regards,
Wouter