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Multiplex PRM data

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Multiplex PRM data Andrea  2019-02-11 13:35
 

I plan to process PRM data in skyline produced by multiplexing the endogenous and heavy labeled peptide targets. The MS2 spectra will be a mix of heavy labeled and non labeled fragment ions. Is this possible to do?, if yes, are there any special processing parameters I would need to change
 
 
Tobi responded:  2019-02-12 02:27

Dear Andrea,

you can process both multiplexed and non-multiplexed PRM data.

In case of non-multiplexing skyline seems to be able to even differentiate ions from light and heavy if they have the same mass. It means that potentially all ion types can be used as long as each precursor is in just 1 isolation window. (This is from a personal quick test, i did not explore this throughout)

In case of multiplexing you need to know which fragment ion types still contain the label mass shift and you should only use those for quantification. In principle you can still use all ion types for visualization and potentially for identification, but light heavy fragment ions of the same mass will be added up and will be of the same intensity for light and heavy.

Please be aware that PRM can result in very slightly different light-heavy ratios than MS1 since it tends to very slightly overestimate the amount of heavy.

For transition settings you might want to allow all ion types and charges you want to build chromatograms from:
Precursor Charge: 2, maybe also 3
Ion Charge: 1,2
Ion Types: p, y (others might be used too but mostly just y-ions are quantitative)

From: Ion 3 (y1 and y2 are not very specific and can have isobaric interferences), fragment ions with a m/z >precursor are more specific so this might be a more stringent option

To: 6 ions or last ion

In Full Scan Settings, MS/MS Filtering set to targeted and input the settings of the acquisition, profile data recommended.

Other settings are (more or less) optional.

If you have a testrun of the heavy labled peptides you can use respective database searches to build a spectral library (or from the experiment results). This allows you to build chromatograms automatically just for the X most intense (most relevant) fragment ions for each peptide.

Hope it helps,
tobi
 
Andrea responded:  2019-02-12 07:30

Thanks, very informative and very helpful
 
Andrea responded:  2019-02-14 11:33

I have tried running two PRM experiments producing mixed MS2 spectra, 1 using multiplexing, and 1 with a wide isolation window to included heavy and light-target on light and use wide isolation to get heavy. In the raw spectra i see both heavy and light fragments in the MS2 spectra.

When i look at data in Skyline the multiplex data shows transitions(y-ions) for both heavy and light. But for the wide isolation data i only see light transitions(PRM)

Any other advice?

Thanks
 
Nick Shulman responded:  2019-02-14 11:58
I am not sure I understand your question. Can you send us your files?

In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request. Otherwise, you can upload it here:
https://skyline.ms/files.url

It sounds like you should also send us at least one of your raw files.
-- Nick
 
Andrea responded:  2019-02-14 13:18
Let me try again. I want to use a wide isolation width for a PRM method to isolate both the unlabeled (Pep_u) and the heavy labeled forms (Pep_h) of the target peptide. This produces a single MS2 spectra containing both sets of fragments-unlabeled and heavy labelled,e.g. y6Pep_u and y6Pep_h.

In my PRM method I target the mz for Pep_u and widen isolation width to include Pep_h. This produces a unlabeled/heavy mixed MS2 spectra. In skyline I can see the Pep_u : y6Pep_u transitions but not the y6Pep_h transitions, as an example.

I guess this is because in my PRM method i only targeted the Pep_u (in inclusion list) and not the Pep_h and Skyline has no way to link the Pep_h precursor to the y6Pep_h fragment?

I have attached a spectra showing fragments pairs

I will submit data if still needed

Thanks
 
Nick Shulman responded:  2019-02-14 13:40
You should tell Skyline that your MS/MS Filtering Acquisition Method is "DIA" instead of "Targeted".

When it is "Targeted", Skyline will only associate each MS2 scan with whatever target has an m/z that is the absolute closest.
If your MS2 spectra have wide enough isolation windows that they are supposed to do double duty, then you should tell Skyline that it's "DIA".

This will work, so long as your isolation windows do not overlap with each other. If they overlap, then you will have chromatogram points from the spectra that you intend, but, also, either your light or heavy precursor might get chromatogram points from the other less than ideal overlapping spectra too.

-- Nick
 
Tobi responded:  2019-02-14 15:08
Dear Andrea,

the issue with wide isolation windows is nicely described by Nick and depending on the number of targets and the scheduling it can occur quite frequently. It requires a check of every made inclusion list and potential deletion of otherwise suitable targets before the measurement. Measuring with overlapping windows can cause problems for quantification and for automatic peak picking in skyline.

If wide isolation windows fit your purpose go for it, but especially when your instrument is having a not too old quadrupole it might be best going for 2 small isolation windows 1-2 m/z each in the targeted fashion with optional multiplexing if doubling the resolution is required (in that case isochronous injection times are needed).

Best,
tobi