Direct infusion Analysis?

Direct infusion Analysis? halwaseem  2023-05-02


Is there a way to compare/export the intensities of precursors when importing raw data from a direct infusion experiment. I have been trying to do this in
the molecules interface mode. I can see and export the TIC for all my raw files but cannot figure out how to look at the intensities of individual precursor masses. When I click through my transition list, I only see the TIC. If I check "precursor" under transitions, the error says no precursor ion chromatograms found.

I am trying to compare signal intensity of specific precursor ions as I am tuning different mass spec parameters (not CE optimization).


Nick Shulman responded:  2023-05-02
Skyline is not a good tool for looking at direct infusion data.
Probably the biggest problem is that Skyline assumes that the "background" level can be determined by taking the lower of either the amount of signal at the start or end of the peak:

When you have direct infusion data, the "background" level ends up being very close to the total signal.

It is possible to tell Skyline not to do background subtraction. If you go to "Settings > Transition Settings > Instrument" and check the "Triggered chromatogram acquisition" checkbox, then Skyline will no longer do background subtraction. That checkbox was put there to support Thermo SureQuant datasets, and, while it probably will also help for direct infusion data, there are probably other reasons that Skyline will not be able to do a good job on your data.

Even though the numbers that you get out of Skyline will probably not be useful, you should still be able to get chromatograms. I do not know why Skyline would be telling you that no chromatograms were found.

If you send us your Skyline document and your raw files, I could take a look.

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.
If that .zip file and your raw files are less than 50MB you can attach them to this support request.
You can upload larger files here:
-- Nick
halwaseem responded:  2023-05-02
Hi Nick,

Data is attached.

Nick Shulman responded:  2023-05-02
Thank you for sending those files.
The problem is that all of your molecules have an "Explicit Retention Time" specified and that time is always outside of the time range over which data was collected.
The Explicit Retention Time values that you can set in the Document Grid are supposed to be in minutes, but it looks like you might have been treating them as seconds.
I see that the lowest specified Explicit Retention Time is 2.79 minutes, but your runs are about a quarter minute long (15 seconds).

You can use the "Molecules" report in the Document Grid to set all of your Explicit Retention Time values to smaller numbers, or make them blank.
After you do that, you could tell Skyline to extract chromatograms again (by going to Edit > Manage Results > Reimport) and you should see some chromatograms.
-- Nick
halwaseem responded:  2023-05-02
Thank you so much!