detection of too long peptide (34 residues)

detection of too long peptide (34 residues) wangrui2011pku  2024-06-03 18:34

I’m running a PRM of only one long peptide (~34 residues) with thermo orbitrap 480. Although pairs of ion fragment under charge+6 precusor, it's too difficult to get a high quality of PRM result as following attachment. Because there is almost no any signals about +2/+3/+4 precusors, I only choose the highest signals of +6 precursor which also have only some y ions detected. How could I improve my PRM method about this long peptide?

Nick Shulman responded:  2024-06-03 22:53
Can you send us your Skyline document?
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files.

If that .zip file is less than 50MB you can attach it to this support request.
You can upload larger files here:

In your screenshot it looks like you might be looking at the second derivative of your chromatogram instead of the actual chromatogram. That would only be appropriate if you were trying to troubleshoot Skyline peak detection. You should right-click on your chromatogram and choose:
Transform > None

wangrui2011pku responded:  2024-06-04 00:06
Dear Nick,
Thanks for your quick reply.
I have upload shared files with several supporting PRM files and one DDA file if possible.
The std peptide sequence is "PKIRYSDVKKLEMKPKYPHCEEKMVIVTTKSMSR" without any modification in our design.
In raw file "5pmol_20240530DDA", charge+8 (510.9017) seems nice matched signals? But I'm not sure whether "large charges & high concentration" is available for PRM monitoring? That's why I choose charge+6 (680.8665) from files "2500fmol_DDA".
If any necessary, pls contact me to provide.


Nick Shulman responded:  2024-06-04 08:51
Yes, you can use higher charges with Skyline.
In order to be able to add precursors and transitions with higher charges to your document you need to go to the "Filter" tab at "Settings > Transitions" and change the "Precursor charges" and "Ion charges" settings so that they include those higher numbers.
With an experiment like this where you are still trying to figure out which transitions will work the best, I would recommend changing the settings on the "Filter" tab so that Skyline gives you a very large number of transitions.
For "Product ion selection" I would recommend choosing "Ion 2" for the "From:" option and "last ion" for the "To:" option.
When you choose "last ion" for the "To:" option, for a 34 residue peptide, Skyline will give you all of the y ions up until y33.

I see that at "Settings > Transition Settings > Full Scan" you have "Isotope peaks included" set to "Count" and "Peaks" set to "1".
This means that Skyline will only extract a chromatogram for the monoisotopic precursor.

People usually would set that "Peaks" value to a higher number because having the whole isotope envelope for MS1 chromatograms is helpful.

I see that you are using Skyline version 22.2.
The latest released version of Skyline is 23.1 and we always recommend using the latest version of Skyline unless you are trying to reproduce results from old experiments.

I hope this helps.
I am not sure whether I understood your question.
-- Nick
wangrui2011pku responded:  2024-06-05 21:39
Thanks so much for your nice suggestions.
It seems working in a concentration-dependent way now. But still problem is that the long peptide is always providing ions so hard and less as I watched.
I will try new skyline 23.1.