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tSIM IQ-X wavy Chromatogram

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tSIM IQ-X wavy Chromatogram gerrit hermann  2025-08-25 04:53
 

To reduce storage needs and speed up processing, we acquired data for seven small molecules in tSIM on an Orbitrap IQ-X.

Settings:

Multiplexing: Off

Isolation window: 1.2 m/z

Orbitrap resolution: 120,000

Data type: Profile

Polarity: Negative ionization

Two compounds are isomers, so we entered five precursor m/z values in the table.

When I load the data into Skyline (25.1.0.142), the extracted chromatograms look wavy compared with Thermo FreeStyle (see attached). I’ve spent several hours trying different filter options in Skyline and MSConvert but haven’t been able to fix it.

Has anyone run into this and found a solution?

Thanks in advance—cheers,
Gerrit Hermann
 
 
Nick Shulman responded:  2025-08-25 08:04
There is Skyline feature called "Spectrum Filters" which you can use to tell Skyline that a particular Molecule's chromatograms should only be extracted from a subset of the applicable spectra.
You can read about this feature here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=SpectrumFilters

It is likely that you will be able to find a spectrum property to filter on which will result in Skyline only using the appropriate spectra for each molecule.
If there does not seem to be a way to get this feature to work, then you could send us your Skyline document and one or more of your .raw files.

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.
The Share Document dialog gives you the option to include .raw files, or you can send those to us separately.

Files which are less than 50MB can be attached to these support requests.
You can always upload larger files here:
https://skyline.ms/files.url
-- Nick
 
gerrit hermann responded:  2025-08-26 03:07
Hello Nick,

Thanks for your quick reply.

I’ve spent several hours trying different spectrum filter settings but haven’t found a combination that works. I even considered evaluating the files in TraceFinder; however, as a committed Skyline user, I’d prefer to solve this within Skyline.

Could you take a look and advise? I’ve attached an export to this message.

Many thanks in advance,

Gerrit
 
Nick Shulman responded:  2025-08-26 06:40
Thank you for attaching that .sky.zip file.
It looks like there are spectra with the following SIM isolations:
-58.41 : -207.08
-86.45 : -207.08
-88.42 : 207.08
-100.46 : -207.08

The only property that you can use to distinguish those spectra in Skyline is "Scan Window Width".
If you wanted only the spectra -58.41 : -207.08 then you could have a filter "Scan Window Width > 148".
If you wanted only the spectra -86.45 : -207.08 then you could have a filter with two lines "Scan Window Width < 148" and "Scan Window Width > 118"

Is the upper bound of those isolation windows really supposed to be -207.08?
It sounds like you intended that the isolation width would be 1.2 m/z.

If those SIM isolations had been narrow windows around your target m/z's, then Skyline would have correctly only used spectra which contained the appropriate precursor m/z.
-- Nick
 
gerrit hermann responded:  2025-09-01 01:02
Hi Nick,

Thank you so much for your help—your suggestion did the trick. I set up compound-specific spectral filters, and the chromatograms look great now.

We also identified the original issue: Easy-IC was enabled while we were running in tSIM mode, and its spectra appear to have interfered with the target spectra.

Thanks again for the extremely fast and expert support.

Best regards,

Gerrit