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how to detect peaks with the same MRM transition

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how to detect peaks with the same MRM transition a van dam  2017-01-24 01:30
 
For our metabolomics project we always made use of Multiquant.
My colleagues, working with proteomics, are very content about skyline for quantification.
I wanted to compare the multiquant output with the skyline output for my project, but there seems to be a problem.
The acquisition method contains some identical MRM transitions for different metabolites such as for ex D-ribose-5-phosphate and Ribulose-5-phosphate. We can distinguish because of the difference in RT.
In Skyline the results for Ribulose-5-P at RT 7.2 min seems to be okay, but there are no data for D-rib-5-P at RT 6.3 min.
Even that part of the chrom is not present, it starts at 6.6 min instead of 6.0 min
In Multiquant both of the components have been quantified.

We can probably overcome this problem to adjust the masses of the MRM transitions in the acq method, but then also Multiquant has to be adapted.

Another question is the way of integration. Sometimes it would be preferable to draw an angled baseline instead of straight basline at the x-axis. Is there a tool to do that?

With kind regards,
Annie van Dam
 
 
Brian Pratt responded:  2017-01-24 09:09
Hi Annie,

I would like to investigate - may I have a look at your Skyline document, and the raw data files? I will contact privately you by email.

Thanks,

Brian Pratt
 
david fuentes responded:  2024-11-15 05:48
Hi Annie and Brian:
I have the same problem. 3x targets on a scheduled MRM at 5, 5.4 and 12 min. I see them on SCIEX Analyst and the data has been correctly accuired. Skyline however, only shows me the strongest transition at 12 min, regardless of the scheduled window on the acquisition method.
Did you manage to find a solution for this???

Many thnaks
David
 
Nick Shulman responded:  2024-11-15 07:52
David,

The usual way to tell Skyline what time to expect your molecule to elute is by setting the "Explicit Retention Time" value on the molecule.
You can use the Document Grid to create a report which includes that column.
You can learn about the Document Grid in the Custom Reports tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

When you have set the Explicit Retention Time on a Molecule, Skyline will try to choose a peak which is close to the specified retention time.
-- Nick
 
david fuentes responded:  2024-11-18 05:10
Hi Nick:
Thanks for the info. However I have tried that and it doesn't work for me. I have tried on the Document Grid and the Modify Molecule option and the chromatograms dissapear. When I try to import data, it removes the chromatograms with explicit RTs.
I attach a Powerpoint
Am I doing something wrong?
Many thanks
David
 
Nick Shulman responded:  2024-11-18 06:01
David,

When you have specified an explicit retention time on a molecule, Skyline will not use any chromatogram which does not overlap with that explicit retention time value.
It sounds like the chromatograms in your scheduled MRM runs cover a shorter retention time than the chromatograms in your unscheduled MRM run, and that the retention time range does not include the value that you have set as the Explicit Retention Time.
I am not sure I understand what you were hoping Skyline would do in this case.

Can you send me your Skyline document and your .wiff files (or .wiff2) and the .wiff.scan 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 there is the option to include the mass spec data files or you could send them 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
 
david fuentes responded:  2024-11-19 04:45
Hi Nick
I am running a scheduled MRM experiment with 1 min detection windows. 2 windows are at 5,1 and 5,4 min and the last window is at 12 min. Skyline only shows the peak at 12 min for all of the targets. I attach a powerpoint which shows the right peaks in SCIEX Analyst and the wrong peaks on Skyline. The problem is the same for 2x groups of peaks with same transitions. A zip file of the data is attached

Thanks
David
 
Brian Pratt responded:  2024-11-19 12:02
Hi David,

Can we have a look at the transition list that was given to Skyline? It looks like many of the Q1 values have been rounded to whole numbers, somehow.

Thanks,

Brian Pratt
 
david fuentes responded:  2024-11-20 04:34
Hi Nick:
It looks like that is the case. I wouldnt expect this to be such a big issue (?).
Attached is the transition list used to build the Skyline method.
Regards
David
 
Nick Shulman responded:  2024-11-20 10:37
David,

Can you send us your .wiff and .wiff.scan files?
-- Nick
 
david fuentes responded:  2024-11-21 04:10
Hi Nick:
.wiff and .wiff.scan files are attached
Cheers
David
 
Brian Pratt responded:  2024-11-21 12:58
Is there any way you can generate a transition list that doesn't have those grossly rounded values?

Looking at your powerpoint, I took a subset of your transition list and restored the missing precision:

Precursor Name;Precursor m/z;precursor adduct;precursor charge;Product m/z;Product Charge;Explicit collision energy
3,5-Dihydroxybenzoic acid;152,962;[M-H];-1;108,901;-1;20
3,5-Dihydroxybenzoic acid;152,962;[M-H];-1;65,001;-1;26
Protocatechuic acid;152,962;[M-H];-1;108,9;-1;20
Protocatechuic acid;152,962;[M-H];-1;65,0;-1;26
2,6-Dihydroxybenzoic acid;152,963;[M-H];-1;108,9;-1;20
2,6-Dihydroxybenzoic acid;152,963;[M-H];-1;65,0;-1;26

With these details Skyline does come up with distinct peaks. The transition list you're using rounds away the information Skyline needs to make the choices you're expecting.

I hope this makes sense?

Brian
 
david fuentes responded:  2024-11-22 05:59
Hi Brian:
This seems to have done the trick. See the attached powerpoint. Can you explain why my symbols have changed even though I only changed the values for 5 targets (2nd slide on powerpoint)? Why is there a triangle istead of a tick?

Just for my understanding, if I'm using a triple quad to acquire my data, why does skyline need 3 decimal places to get the right chromatogram? I would not have expeceted such an effect. The rounding may have happened when the data was copied to Excel, is Skyline affected by the rounding of Q1 only or does the rounding of Q3 also have an effect on the skyline results?

Thanks
David
 
Brian Pratt responded:  2024-11-22 11:19
>> Why is there a triangle instead of a tick?

May I see the revised transition list, and the Skyline document as pictured?

>> why does skyline need 3 decimal places to get the right chromatogram?

It's not about number of decimal places per se - it's about resolving ambiguity. Ask yourself, why does the method take care to use unique combinations of Q1 and Q3? If the method Q1/Q3 values were all rounded, would you be able to manually determine which chromatogram corresponded to which precursor? Or, if the method values weren't rounded, but I only gave you rounded Q1/Q3 values for trying to assign each precursor to a chromatogram, how would you decide which is which?

I hope that helps.

Do send that transition list and your current Skyline document so we can see what's going on there.

Brian
 
david fuentes responded:  2024-11-25 05:41
Hi Brian:
The transition list with only some of the Q1 and Q3 values changed is attached. The 2 skline files are the same but the one named Test, was made using the attached transition list

Regards
David
 
Brian Pratt responded:  2024-11-25 09:59
Hi David,

You're getting that yellow caution triangle ("not everything was matched") because you've accidentally told Skyline to expect chromatograms for precursor ions as well as fragment ions. Change "Settings > Transition Settings > Filter > Ion types" from "f,p" to "f" and you're good to go.

Best regards,
Brian
 
david fuentes responded:  2024-11-26 09:27
Thanks Brian:
I don't even know how I did that. I simply used the updated transition list to to make a new template like I normally would and that is what I got. In any case, it has worked!!!!!
Thanks for your help!

David
 
Brian Pratt responded:  2024-11-26 11:52
Excellent! Glad I could help.