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Unable to import Thermo Stellar SRM .raw: “chromatogram information unavailable” error

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Unable to import Thermo Stellar SRM .raw: “chromatogram information unavailable” error busra demirtas  2025-09-29 08:43
 
Dear Skyline support team,

We recently equiped with a Thermo Stellar triple-quadrupole LC-MS instrument and ran our first batch (SRM) this week. The ".raw" files open normally in Thermo FreeStyle and chromatograms are visible there, but when importing the same .raw files into Skyline (latest version) we get “chromatogram information unavailable” and no XICs/SRM traces appear.

What we have already tried is conversion of .raww to mzML with MSConvert, but MS convert noted that input contains only chromatogram data. Also, tried to convert it to .mzML with ThermoRawFileParser v1.5.0-alpha.2 (with .NET8); conversion completed but import to Skyline failed with "invalid cvParam accession (1003409)" and conversion log contains many WARN Spectrum #... has no m/z data lines.

Verified transitions/polarity/RT settings, method is a standard SRM and worked previously on Waters TQ Absolute (with same transition list on Skyline).

The mzML contains a cvParam line MS:1003409 (name:"Stellar") that appears to be a new PSI-MS cv entry. It seems the Stellar vendor chromatogram stream/new cvParam is not being interpreted by our conversion tools or by Skyline’s reader, so, Skyline ends up with no usable chromatogram traces.

Does Skyline currently support Thermo Stellar SRM vendor chromatogram stream (MS:1003409) ? If yes, which Skyline build and which Thermo RawFileReader / RawFileReader SDK / ThermoRawFileParser version do you recommend for correct import?

We can provide a small example .raw, the mzML produced by ThermoRawFileParser, or the Skyline audit log.
Please advise which one would be useful.

Thanks in advance, any guidance will be very helpful.

My email address:
busra.demirtas@istinye.edu.tr

Best regards,
Büşra
 
 
Nick Shulman responded:  2025-09-29 08:58
We are going to try to fix the way that Skyline handles Stellar SRM data soon.

In the meantime, the workaround that I have heard works is to use MSConvert to convert the .raw files to .mzML, and, make sure to check the checkbox "SRM as spectra" in the MSConvert graphical user interface, or specify the command-line argument "--srmAsSpectra".

I believe that the "SRM as spectra" checkbox ends up producing a .mzML file which Skyline will be able to read from as if it were PRM data. (I am not 100% sure about that-- if PRM does not work then maybe try treating it as SRM data which would mean making sure that "None" is selected for both MS1 and MS2 on the "Full Scan" tab at "Settings > Transition Settings".)

Yes, it would be helpful if you could send us some of your .raw files.
You can upload them here:
https://skyline.ms/files.url

I will also send you an email directly in case you would prefer to send us your files some other way.
-- Nick
 
Mike MacCoss responded:  2025-09-29 09:16
Hi Büşra,
One thing to keep in mind is that the Stellar isn't a triple quadrupole, it is a hybrid quadrupole-ion trap. https://pmc.ncbi.nlm.nih.gov/articles/PMC11956834/

The data should be collected as parallel reaction monitoring (PRM) and not SRM. One of the main advantages of the Stellar is that you collect the full spectrum and not just individual SRM transitions. This enables you to refine the product ions post analysis. You shouldn't need to do any file conversion.

If you are looking for some example Skyline documents to determine your settings you can check out these example datasets.
https://panoramaweb.org/stellar_ms_platform.url
https://panoramaweb.org/prm_conductor.url
https://panoramaweb.org/stellar-biofluid-prm.url

Let us know if you have any problems with Skyline collecting the data as PRM.

Cheers,
Mike
 
Nick Shulman responded:  2025-09-29 10:31
Büşra,

Thank you for uploading that .zip with the .raw file in it.
Skyline does not seem to have any trouble reading chromatograms from your .raw file.
This looks to Skyline like ordinary SRM data, which means that you should have "None" chosen on the "Full Scan" tab at "Settings > Transition Settings".

You can use a program such as "SeeMS.exe" which comes with ProteoWizard to see what chromatograms are available in the .raw file.
One of the chromatograms in the list is:
SRM SIC 118,44.25
which means that you should have something with a precursor m/z of 118 and a product ion m/z 44.25 like I have in my attached "test.sky.zip".

The exact closeness that the m/z's in the Skyline document need to be to the Q1 and Q3 values of the SRM chromatogram in the raw file is controlled by the "Method match tolerance m/z" value on the "Instrument" tab at "Settings > Transition Settings".

The method match tolerance defaults to 0.055. It might be that if you change that to a larger number the missing chromatograms will immediately appear.

If you are still having trouble you can send us your Skyline document.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including whatever chromatograms might have read out of the .raw file.

We were having some discussions last week with Thermo about Skyline not handling Stellar SRM data correctly. However, Skyline does not seem to have any trouble with your data so what I said about converting to mzML and telling it to treat SRM as spectra is not correct.
-- Nick