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Nick Shulman responded: |
2025-05-29 07:24 |
Can you 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.
Files which are less than 50MB can be attached to these support requests.
If that .zip file and/or the .raw files are larger than that, you can upload them here:
https://skyline.ms/files.url
The usual reason that you would not be seeing any chromatograms in Skyline would be that the precursor and product m/z's of the transitions in the Skyline document do not match the Q1 and Q3 values of the SRM chromatograms.
After we see your Skyline document and your .raw file we will probably be able to tell you what is going wrong.
-- Nick |
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guo xue responded: |
2025-05-29 19:10 |
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Nick Shulman responded: |
2025-05-29 21:49 |
There is a program called "SeeMS.exe" which comes with ProteoWizard which you can use to see the chromatograms that are in your .raw file.
I see that you have one SRM chromatogram where the Q1 value is -146.9 and the Q3 value is -100.
In order for Skyline to see that chromatogram in your Skyline document, you would need to add a molecule where the precursor m/z is -146.9 and the product m/z is -100.
I have attached a Skyline document like that.
When I convert your .raw file to .mzML using ProteoWizard MSConvert, I can see that it says that the Q3 isolation was actually a 100m/z wide window around -100. That is, everything from -50 to -150 was allowed through by the third quadrupole. However, for SRM chromatograms, Skyline does not pay attention to the Q3 isolation window. Skyline just sees that the isolation is centered on -100 m/z.
The setting which controls how close the Q1 and Q3 values need to be to the precursor and product m/z's in the Skyline document is "Method match tolerance m/z" on the "Instrument" tab at "Settings > Transition Settings".
However, the maximum value that you can set that to is 0.6, so there is no way that Skyline would ever understand that your SRM chromatogram had that wide of a Q3 isolation, so you really do need to make sure that the product m/z matches the center of the Q3 isolation window.
By the way, I see that you are using Skyline version 22.2.
We just released Skyline 25.1 last week.
We always recommend that you use the latest released version of Skyline unless you are trying to reproduce results from older experiments.
-- Nick |
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Nick Shulman responded: |
2025-05-29 22:18 |
Actually, when I look more closely at your data, I see that there are actually spectra in the .raw file, but Skyline really wants to treat them as chromatograms.
You can see the spectra in SeeMS.exe if you check the option "SRM as Spectra".
However, getting Skyline to recognize those as spectra would be much more tricky.
There might exist a way to use MSConvert to create a file where Skyline would recognize those things as spectra. That would probably involve making sure that the "SRM as Spectra" checkbox in MSConvert is checked, and then converting the .raw file first to something like ".ms2" so that the information which is causing Skyline to think those SRM spectra are chromatograms is removed. Then, you could convert the .ms2 file to .mzML.
My guess is that there is something you could change about your Thermo instrument method which would result in .raw files that Skyline was better able to understand.
I do not know much about Thermo instrument methods, but, if you tell us more about your Thermo instrument method, and what you were trying to acquire, someone else might be able to give you a better answer.
-- Nick |
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guo xue responded: |
2025-05-30 01:11 |
Hi Nick, thanks for your explanation. For this data, I only input parent mass to LTQ method and give a scan range of 50-150 for MS2. It is like a PRM but I think LTQ doesn't have the real PRM funtion. When I opened the raw files in Freestyle, which is the thermo software. I can see a peak with SRM MS 2 at 128.8. I think that should be the correct transition. Following your suggestion, I re-acquired another data, with scan range center at 128.8 with an isolation window at 2 m/z. So now I have two data, one is acquired with a PRM-like method, one is a MRM like method. And I can see the peak and MS2 in freestyle. but now when I import them into skyline, I still couldn't see any chromatogram, I am thinking to be processed with PRM, but LTQ is a low resolution instrument, so I change the instrument tolerance to 0.5 m/z. So now I can see the peak. but the precursor and product are the same. is that correct? and how can I export the intensity of each molecule?
https://drive.google.com/drive/folders/1NRmTNOcRyyxFuVHK4bx7RJHqJgLiLgKi?usp=sharing
thank you very much. |
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