How to analyze and quantify multiplexed peptides (Q Exactive PRM) with Skyline?

How to analyze and quantify multiplexed peptides (Q Exactive PRM) with Skyline? gillespiekevinp  2020-04-09

I'm having a lot of trouble with analyzing my RAW files generated by a Q Exactive HF.

I am only trying to analyze one protein. In my "Inclusion List" I constructed with the Thermo software, for each peptide, I have Multiplexed multiple precursor charges. But I have the heavy internal standard peptides and light peptides Multiplexed separately.

Using Xcalibur Qual Browser, I check for the precursor ions of these peptides (Full Scan) or the product y and b ions (DIA), and my peaks are present and reasonably intense.

However, I can't figure out the correct Transition Settings in Skyline to reflect any of this Multiplexed PRM data when I import it. And I can't figure out how I would generate and export the ratios of either Light/Heavy precursor ions or Light/Heavy MS2s from Skyline.

Any tips? I can attach files as needed to clarify. I've gone through tons of tutorials by this point, and any additional help would be appreciated.

Nick Shulman responded:  2020-04-09
Can you send us your Skyline document and at least one 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.

You can upload that .zip file and your .raw file here:

It sounds like you have a MSX method where the MS2 scans have multiple non-overlapping isolation windows. Skyline is supposed to be able to handle this. In the settings at:
Settings > Transition Settings > Full Scan
I would expect that this would work if you either
1: Set the MS/MS filtering Acquisition Method do "Targeted"
or 2: if you set that to "DIA" and set the Isolation Scheme to the one called "Results Only"

When we see your data files we will probably be able to figure out what is going wrong.
-- Nick
gillespiekevinp responded:  2020-04-10
Hi Nick,

I was able to solve my issue. I will explain my mistake and solution here for future reference.

You were correct and the issue was with the MS/MS filtering settings in Transition Settings. I had first tried to create a new Isolation Scheme to reflect the Multiplexed Data. However, I found that with Acquistion Method > DIA and Isolation Scheme > All Ions, Skyline was able to handle all of the RAW files. Since the only things I multiplexed were multiple charges of the same parent ion, it automatically separated the precursors and detected transitions for each.

In regards to exporting Light/Heavy normalized data, under Settings > Peptide Settings > Quantification Tab, set “Normalization method: Ratio to Heavy". Once RAW files are imported and peaks are selected and integrated, under File > Export > Report…, choose “Transition Results” (or create a Custom Report) and click Edit List > Edit...

Under “Columns” in the left panel, .expand Peptides > Precursors > Transitions > Transition Results and click "Area Ratio". When the CSV file ix exported, the Area Ratio column represents the peak area for the Light / peak area for the Heavy transition for each Peptide from each Replicate.

Got exactly what I wanted. Thank you so much.

Tobi responded:  2020-04-15
Dear Kevin,

thank you for posting all the information here. Do I get it right that you multiplex multiple charges of one peptide together but heavy and light are in different isolation batches? It is an interesting approach for me, but do you know if the quantification by light/heavy ratio is still accurate?

Its just because with multiplexed PRM its possible to screw up quantification without noticing it, the data can look fine but light/heavy ration can be off by a factor of 2 or sth. Did you by chance check for that?