small molecule sMRM - chromatogram information unavailable

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small molecule sMRM - chromatogram information unavailable paul leonhardt  2021-06-09
 

Hi Skyline team,

i currently use skyline as open-source tool to analyse data that has been acquired on a Sciex 5500 qqq instrument. My research focuses on nucleotides and their modifications, that's why i often need to detect the same [M+H]+ mass during different retention times. Currently I only want to get a quick overview of the data, so i need the chromatogram, peak area and retention times. The problem i have is that skyline most of the time only recognizes one of my isobaric sMRM transitions.

I have acquired my data as full Range MRM scan and also as scheduled MRM scan. When creating the transition list for the full range MRM data i did not have so many problems - apart from adjusting the integration manually. But now when i want to use scheduled MRM data most of the time skyline does not find the corresponding chromatogram. Is there a way to assign this manually?

Attached you find a file for demonstration which includes 4 results, two of them are full range and two scheduled - you will see that some transitions are only detected within the full range files even though the peaks are present.

I'd really appreciate if you take a look into it

Kind regards,
Paul

 
 
Nick Shulman responded:  2021-06-09
Can you send us some of your raw files? (Actually, I guess in your case they are .mzML files-- that works too).

If those files are less than 50MB you can attach them to this support request. You can upload larger files here:
https://skyline.ms/files.url

When Skyline is deciding which SRM chromatogram belongs with which transition in your document, Skyline looks at the Q1 and Q3 m/z's. Also, if you have specified an Explicit Retention Time for your molecule, then Skyline also looks at the time range over which the chromatogram was collected.

A common reason that Skyline will not find chromatogram for a particular molecule is that there are no chromatograms in the raw file whose time range overlaps with the Explicit Retention Time that was specified.

We will be able to tell you what is going wrong after we see your mzML files.

By the way, if you want to look at all of the chromatograms in a raw (or mzML) file, you can use "SeeMS.exe" which gets installed if you install ProteoWizard.
-- Nick
 
paul leonhardt responded:  2021-06-10
Hi Nick,
thanks for having a look a it.
sure i can share the files. Files from 07/05/2021 were acquired using MRM scan and files from 07/06/2021 are scheduled MRM + full range MRM for some analytes that have not been clearly identified yet.

I have confirmed the presence of the peaks in the mzML files. For example the transition 282,072 -> 150,10 detects methyl adenosine at RT 4,0 and 8,4 min. Skyline can not find the peak for RT 4,0 even though it's in the mzML files

Cheers,
Paul
 
Nick Shulman responded:  2021-06-10
Thanks for sending those files.

When Skyline is reading chromatograms from your raw file, Skyline cannot tell the difference between your two molecules which both have the precursor m/z 282.072. Those two molecules in your document belong to different molecule lists, and they have different explicit retention times, but, the only thing that Skyline's chromatogram extraction code ends up looking at is the precursor m/z. Because of this, the only chromatograms that make it into the .skyd file are the ones for the first of those two molecules, which means in Skyline you only see the chromatogram which contains the 4 minute retention time.

You can work around this problem by specifying a "Precursor Name" when you insert your Transition List. I have attached a picture of what I mean. If your precursors have names then their chromatograms in the .skyd file will be able to be distinguished from each other.

I am going to ask around and see whether we can fix this behavior, or, at least, provide a warning that two molecules in the document are trying to store their chromatograms in the same place in the .skyd file, and only one is going to be able to do so.
-- Nick
 
paul leonhardt responded:  2021-06-11
Thank you Nick! This has been really helpful, i will also share this with my colleagues.

Cheers,
Paul