Multiple precursors per peptide sa825  2019-08-27 02:41
 

Good morning,

I was wondering why there are 3 precursors of diff m/z’s for the peptide? And is it alright remove the ones with irank 2 & 3?

The reason I am asking about this is because when I do a DDA run using the same peptides on the Thermo Q Exacitive, my peptide is detected at the expected retention time but when I do a targeted run (PRM), the Thermo is unable to detect my peptide.

I fear its because the Thermo is looking for 3 precursors at the same retention time when it should only be looking for one?

I have attached an image to show what I mean.

Thanks,

Shimon
 
 
Nick Shulman responded:  2019-08-27 15:32
Those three precursors ("M", "M+1", and "M+2") represent the first three masses in the isotope distribution of the precursor ion.
You can control how many of those Skyline gives you with the "Isotope peaks included" setting at "Settings > Transition Settings > Full Scan".
The chromatograms for these precursor transitions are extracted from MS1 scans.

In a PRM method, you should always target the monoisotopic mass of the precursor. Skyline will only extract MS2 chromatograms from MS2 spectra whose precursor isolation window contains the monoisotopic mass of the precursor.

It sounds like you are saying that your peptide search engine is unable to detect the peptide in your PRM raw files. Which peptide search engine are you using?

If you send us your .raw file, we could certainly take a look at it and see if it looks right. Files which are less than 50MB can be attached to this support request. You can upload larger files here:
https://skyline.ms/files.url

It would also be helpful if 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 including extracted chromatograms.

-- Nick