Must have MS1 scans in PRM methods?

Must have MS1 scans in PRM methods? ziyang zhang  2020-12-16

Hello fantastic people at skyline,

I am quite new to quantitative mass spec and have very limited sources to learn - hope you don't mind these very basic questions...

  1. Is it mandatory that MS1 scans be include in PRM methods for skyline to perform quantification? My current method only has an inclusion list for precursor masses to cycle through. When I analyze the data acquired this way, Skyline is able to extract the ion chromatograms but cannot quantify the results. Also giving red tags for all the transitions and dotp=0. Screenshot attached.
  2. I have gone through the tutorial ( without much issue. I do wonder if there is a way for plot the extracted chromatograms for a particular transition on the same plot (overlaid)?

Again, I know these questions may be due to simple lack of understanding of skyline features or mass spec fundamentals. If there are tutorials somewhere that can save you some frustration with my ignorance or time explaining simple concepts, please do point me to them!

Many thanks in advance!


Nick Shulman responded:  2020-12-16

No, it is not necessary for there to be MS1 scans.

It looks like for most of your precursors, Skyline did not find any matching MS2 scans, and that is why they did not get any chromatograms.

The way that Skyline matches MS2 scans to precursors depends on:
1. The method match tolerance that you have specified at "Settings > Transition Settings > Instrument"
2. The MS/MS filtering Acquisition Method and Isolation Scheme that you have specified at "Settings > Transition Settings > Full Scan"
3. If the Acquisition Method is "Targeted" (i.e. PRM) then MS2 scans will be matched to the one precursor in the document whose mz is closest to what was isolated in the scan. Sometimes this means that one precursor in your Skyline document might steal MS2 scans intended for a different precursor.

I cannot tell which of these things (or maybe something else) is going wrong in your screenshot.

Can you send us your Skyline document and 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.

If that .zip file is less than 50MB you can attach it to this support request.

Otherwise, you can upload it here:

Please also send me one of your raw files that you extracted chromatograms from.
-- Nick
ziyang zhang responded:  2020-12-16
Hi Nick,

Wow, thanks for the fast response! You were right that the MS2 scans were stolen by other precursors. I cleaned up my library (some of the peptides in the screenshot are from the contaminant list defined in MaxQuant) and now it's looking much better now!

Still attaching the file and appreciate your advice if I'm still not doing things in the right way. (P20201112-12.raw uploaded in folder "20201216_Ziyang")

Also wondering if it's possible to overlay chromatograms from different replicates?

Thanks again!

Nick Shulman responded:  2020-12-16
Everything looks good to me in your Skyline document.

In order for another precursor to have been stealing MS2 scans, its m/z would need to be different from, but still within the mz match tolerance of the other precursor, which is 0.055 (the default) in your document. It did not look like you had any other precursors like that in your first screenshot (it's conceivable that such a precursor was there, but scrolled off?).
Another thing to remember is that if you add more Targets to your document you need to tell Skyline to extract chromatograms again by going to:
Edit > Manage Results > Reimport

Skyline does not have a way to view chromatograms from different replicates on the same graph.
You can use the menu item:
View > Arrange Graphs > Tiled
to see the graphs next to each other.

-- Nick
ziyang zhang responded:  2020-12-17
Excellent, this is super helpful, thanks so much!