PRM search xhuang  2018-10-08
 

Hi, I'm a new user for skyline. I have a more general question as I browse previous post and can't find the answer. I'm running targeted msms using Q-exactive. When I try to set up those transitions, for example, I can't find the instrument listing as QE. Shall I choose orbitrap instead? Also how do you define those values (such as library-ion matching tolerance, instrument-method matching tolerance)? Where to define the dot color (red/yellow/green) next to the peptide fragment? Thanks!

 
 
Nick Shulman responded:  2018-10-08
I believe we currently recommend that you choose "Centroided" on the "Settings > Transition Settings > Full Scan". This causes Thermo software to be used in terms of turning profile scans into centroided scans. This vendor-provided centroiding usually works better than Skyline's method of summing all of the profile data across a m/z channel.

If you did not choose "Centroided", then you should choose "Orbitrap". The purpose of this setting is to tell Skyline how wide of a m/z channel to sum across when extracting chromatograms. The different types of mass analyzers all have slightly different behavior in terms of how the width of a peak depends on the m/z.

The library match tolerance is used when Skyline is looking at a spectral library, and needs to figure out which observed peak in the spectrum is the y7+ ion, etc.
The method match tolerance is used when Skyline is looking at a SRM or PRM file and needs to figure out which peptide a particular chromatogram or scan is for.

The dot color means:
green: all transitions had a nice looking peak
orange: more than half of the transitions had a nice looking peak
red: less than half of the transitions had a nice looking peak

The definition of "nice looking peak" depends on whether you have the menu item "Settings > Integrate All" checked. If "Integrate All" is checked, then anything that has an observable bump slightly above background is good enough. If "Integrate All" in unchecked, then the peak apexes all of have to be close to each other, and each transition's peak needs to be of similar shape.
-- Nick
 
chen qian responded:  2022-09-20
Hi Nick,

Thank you for your reply. I got it now regarding to the red dot. But MS2 problem is still not clear. I double checked, my current setting is already "Transitions > All". When I chose "Transitions > Products", it displayed " No product ion chromatograms found". Since the list has those fragment ions listed below each precursor ion, it should have the product ion information, right?

Thanks,
Chen
 
Nick Shulman responded:  2022-09-20
I am definitely seeing product chromatograms.
Is there a particular peptide and replicate for which you are seeing the "No product ion chromatograms found" message?
With DDA data, sometimes there really might be no MS2 spectra which matched your precursor. In those cases, Skyline would show you the "No product ion chromatograms found" message, because when Skyline extracted chromatograms the product transition chromatograms ended up having zero extracted points in them.
I do not see any peptides like that in the Skyline document that you sent me.
-- Nick
 
chen qian responded:  2022-09-20
Hi Nick,

I can see fragment ions now. I had the Transition>Only quantitative checked before. I just unchecked this one, and I can see the fragment ions now.

Thanks,
Chen