No precursor ion chromatograms found

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No precursor ion chromatograms found bwidner  2019-04-30
 

Hi,

I am new to Skyline and trying to do something very simple, but I cannot get it to work. When I import the transition list with just the precursor information, everything works. I see the peaks as expected. However, when I add the product information, I get the "No Precursor ion chromatogram found" message. I double checked the product m/z and looked through the tutorials, and still I am quite befuddled!

The data is from a Thermo Fisher Fusion Lumos orbitrap. I have attached the skyline files and the raw file I am working with.

Thanks,
Brittany

 
 
Brian Pratt responded:  2019-04-30
Hi Brittany,

I don't see any attachments, perhaps you can upload to http://skyline.ms/files.url ?

Best Regards,

Brian Pratt
 
Nick Shulman responded:  2019-04-30
Brittany,

You should right-click on the chromatogram window and choose:
"Transitions > All".

I believe the problem is that at some earlier point you told Skyline to only show you the precursor chromatograms using the menu item "Transitions > Precursors".
-- Nick
 
bwidner responded:  2019-04-30
Hi Brian, I uploaded the files as you suggested.

Nick, If I do that I see "chromatogram information unavailable". I can only see peaks for Base Peak or TIC.

Thanks!
Brittany
 
Brian Pratt responded:  2019-04-30
You need to adjust your Full Scan settings to let Skyline know that you want to analyze MS2 data as well as MS1. You had MS/MS Filtering set to "none".

Thanks for using the Skyline support board!

Brian
 
bwidner responded:  2019-05-06
Thanks, Brian!
It worked. I am having another issue now though- sort of the opposite problem. I made a transition list for a bunch of compounds and imported some results files. It is showing me MS2s now, and it does show data for the precursor, but that data is actually the MS2 data. I know this because you can see that the scans are identical to the MS2 and the instrument is set to collect fewer scans for MS2s than for MS1s. The MS1 peaks look much smoother. Is it a setting that is incorrect? I have "f,p" in the Transition> Filter>Small Molecule settings. I uploaded the files to the file sharing drop box.

Thanks in advance!
Brittany
 
Brendan MacLean responded:  2019-05-06
Do you have MS1 filtering enabled in the Transition Settings - Full-Scan tab? This is really the only reason Skyline would extract your precursor from the MS2 scans.
 
Brian Pratt responded:  2019-05-06
Also, you click on any part of a chromatogram and Skyline will show you the raw data it's based on, which will tell you whether it's MS1 or MS2. (If you have the raw data, that is - you didn't provide that in your upload so I can't be sure).

Brian
 
bwidner responded:  2019-05-06
These are the settings I have. yes, MS1 filtering is enabled. Should it not be?
 
bwidner responded:  2019-05-06
Brian, When I click on the precursor chromatogram it says MS/MS. I do have the raw data.
 
Brendan MacLean responded:  2019-05-06
Your Full-Scan settings look fine. Though, higher resolution on the MS1 than I am used to seeing, and we now most frequently use Centroided with Orbitrap data.

But, the fact that you are getting your precursor ions extracted from MS/MS sounds like a bug in the small molecule code, if your settings are as you have shown.

Once, Brian has your Skyline document, I am sure he will be able to figure it out.
 
bwidner responded:  2019-05-06
Thanks!

I uploaded a raw file, in case that is helpful.
 
Brian Pratt responded:  2019-05-06
May I also see the transition list you imported into Skyline? There are some strange things here, like "5'deoxyadenosine 1" which has a fragment of the same mass as the precursor, which is likely what's causing the confusion.

Thanks

Brian
 
bwidner responded:  2019-05-06
Hi Brian,

Here is the transition list (columns B-H). Yes, the list does have the fragment with the same m/z as the precursor. I did that because of something I read in response to a question another user had on the skyline support... Sorry, I can't find that other conversation now. But when I did that for a single transition and some simple data, it seemed to work. Then I tried to make a big transition list and import different data ('real' data, not a standard)... and it is not working.

Thanks,
Brittany
 
Brian Pratt responded:  2019-05-06
It looks like Skyline is having trouble reading that transition list. For the lines that are precursor declarations you should either leave the fragment information blank, or be sure that the mz and charge declarations match.

What you have there for precursors is actually reasonable - precursor adduct [M-H] and fragment charge -1 are clearly describing the same ion for the precursor "fragment", but -1 is ambiguous enough (is that mz due to deprotonation or is the fragment inherently charged?) that Skyline gets confused and doesn't recognize that the fragment mass is actually the same as the precursor mass. If you change that "-1" to "[M-H]", or just leave it blank, things are fine.

As it doesn't work as you would expect, I consider that to be a bug and will get it fixed. But for now there's at least a clear workaround.

Thanks,

Brian Pratt
 
bwidner responded:  2019-05-06
Aha, ok. I did not realize that the charge could be [M-H] (and not just a number). It works now! Thanks a bunch!
Brittany