Problems with transition list

Problems with transition list fdalessa  2023-07-12 08:54

we are measuring porphyrins via HPLC-MS in our lab. We've been using an eatablished protocol for a few months now. However, we had to adapt the HPLC-MS method in order to measure different kinds of porphyrins within one run. Scince that adaption we can't evaluate our Chromatograms with our established transition list as it falsly detects our MS°2 measurements as MS^3. Although we can clearly see our added target molecules and their fragments in the chromatogramms, skyline doesn't detect them at all.

Do you have any idea to solve our problem?

Thank you in advance!!

Nick Shulman responded:  2023-07-12 09:00
Can you send us your Skyline document and some 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.

If that .zip file and/or your raw files are less than 50MB you can attach them to this support request.
You can upload larger files here:

I cannot tell from your screenshots what might be going wrong.
Skyline is suppose to extract chromatograms from all spectra whose MS level is greater than or equal to 2, so long as the MS1 precursor matches the precursor in the Skyline document.

We will probably be able to figure out what is going on when we see your Skyline document and raw files.
-- Nick
fdalessa responded:  2023-07-13 00:02
Hi Nick,
thank you for you help!
Here is the Skyline File and two raw data files. The file 3071101 contains the data from the new protocol with 4 different porphyrins, whereas the file 3071001 contains the data using the old protocol with only thwo porphyrins.
Nick Shulman responded:  2023-07-13 10:51
I am not sure I understand what you were hoping to see in your mzML files.
I used ProteoWizard SeeMS.exe to look at the mzML files.
You can install ProteoWizard from here:

In 30711018, all of your MS2 spectra had two different MS1 precursors. The m/z values of those precursor pairs were:

In 30710018, there were some spectra where what was isolated was "563.29999", and there were other spectra where two precursors were isolated:

In your Skyline document, the things with missing chromatograms have a different precursor m/z from what could be found in your .mzML files.

Can you tell me what you were hoping you would see with this data?
-- Nick
fdalessa responded:  2023-07-14 03:03
Hi Nick,

basically our problem is that our new transition list fails to detect the ions from our raw data. For example for Protoporphyrin we identified the precursor ion at 563.3. The fragment ions of Protoporphyrin have m/z values of 504.2; 489.3 and 445.4 respectively. However, using our transition list, SkyLine falsly labels the ion 508.3 as the precursor ion.
Regarding Coproporphyrin, we detect the precursor ion at an m/z of 655.4. It’s fragment ions have an m/z value of 596.2; 537.3 and 523.3 respectively. For Coproporphyrin SkyLine doesn't automatically detect any signal at all, although we can clearly see the ions in our chromatogramm.

I have attached a file from our MS-data where you can see the corresponding MS-pattern.

Thank you for your help!
Nick Shulman responded:  2023-07-14 14:36
It sounds like you are saying that when you imported a transition list into Skyline, the m/z of a Precursor in the Targets tree was incorrect.
That is, the transition list that you imported said that the precursor m/z was 563.3, but what actually appeared in the Targets tree was a precursor with the m/z 508.3.

When something like this happens, you should have been able to see immediately that the Targets tree did not have the correct precursor and product m/z values in it.
You would not need to try to extract chromatograms in order to see that the precursor m/z of a molecule was incorrect.

In the transition list that you attached to the first post in this support request, I definitely see some rows where the value in the "Q1 First Mass" column is 508.2.

I do not think that I understand what transition list you are trying to import into Skyline and how that is different from what you are actually getting.
-- Nick
fdalessa responded:  2023-07-17 00:09
Hi Nick,

in your first sentence you summed up my problem pretty well. We used our transition list for different measurements including both precursor ions. However, I think my problem is easier to understand in the Skyline Document I have attached here, where only the 563.3 precursor ion is measured.

For protoporphyrin both raw data files that I have already sent you include the same Protoporphyrin at m/z = 563.3. As you can see in the screenshot, skyline only identifys the precoursor and target ions for protporphyrin of the file 30710018. However, in the file 30711018 the same molecule was measured so should be a signal in skyline too.

I don't know why i can't analyse my file 30711018 with the same transition list. To me it seems as skyline doesn't detect the precursor ion correctly.
Nick Shulman responded:  2023-07-17 04:22
Can you tell me the precursor m/z and product m/z of one of the transitions that you would like to extract a chromatogram for?
-- Nick
fdalessa responded:  2023-07-17 05:02
The precursor m/z is 563.3 and the fragment ions are m/z= 445.4; 489.4 and 504.3
Nick Shulman responded:  2023-07-17 05:31

When I look at scan number 626 in "30711018.mzML", I see the following:

                <cvParam cvRef="MS" accession="MS:1000827" name="isolation window target m/z" value="504.299987792969" unitCvRef="MS" unitAccession="MS:1000040" unitName="m/z"/>
              <selectedIonList count="2">
                  <cvParam cvRef="MS" accession="MS:1000744" name="selected ion m/z" value="563.299987792969" unitCvRef="MS" unitAccession="MS:1000040" unitName="m/z"/>
                  <cvParam cvRef="MS" accession="MS:1000744" name="selected ion m/z" value="504.299987792969" unitCvRef="MS" unitAccession="MS:1000040" unitName="m/z"/>

It sounds like you were hoping that Skyline would include that spectrum when Skyline was extracting the chromatogram for your precursor whose m/z was 563.299987792969 since that was one of the values in the "selectedIon" list there.

However, when Skyline is deciding whether to include a spectrum in a chromatogram, the only thing that Skyline actually looks at is the isolation window, which this spectrum says is 504.299987792969.

Usually the selected ion values are things that are inside of the isolation window. I do not know what it means when the numbers are the way that they are in your file.

Do you have any idea why things look like this in the mzML file? If you think something might have gone wrong when MSConvert converted the file to .mzML you could zip up and send us the original Bruker .d folder "30711018.D".
-- Nick

fdalessa responded:  2023-07-17 05:46

Ok that sounds reasonable to me. Well I don't know why my mzML file looks like this to be honest. But that must be the problem then.

fdalessa responded:  2023-07-19 00:21

Do you have an idea how to solve this problem with MCConvert?

Brian Pratt responded:  2023-07-19 17:08

Not yet, but we're working on it!

Is this really MS3 data? That's how msconvert understands it, if not that's an important clue.

fdalessa responded:  2023-07-19 23:43

So for the measurements of the file 30711018, Mesoporphyrin was measured in MS^3 and Protoporphyrin and Coproporphyrin were measured in MS^2.

Matt Chambers responded:  2023-07-20 10:53

I think this is the first MS3 data I've seen in the Bruker YEP format. So I'm not surprised we're misinterpreting the isolation/fragmentation data. For the MS3 above, is 504.29 the MS1 precursor and 563.29 the MS2 precursor?

fdalessa responded:  2023-07-20 23:51

Yes that's right. 563.29 is the Ms^2 precursor and 504.29 is the MS^3 precursor.

Matt Chambers responded:  2023-07-21 07:22

There are no MS2s recorded in that 30711018 file as far as I can tell, only MS1s and MS3s. That means there aren't any product ion scans for the 563.29 (MS1->MS2) precursor. Only for the 504.29 (MS2->MS3) precursor. If you have some evidence from the vendor viewing software showing MS2s in the 30711018 file I'd like to see it, because it doesn't seem to be available in their CompassXtract API.

The multiple selected ions thing is a bug but like Nick said that shouldn't affect Skyline. It does appear that the current code is correctly interpreting which m/z is the most immediate precursor (that's the one it puts in the isolation m/z).