Export Isolation List

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Export Isolation List benjamin.thomas  2013-07-30
 
Hi Brendan,
I'm trying to setup a scheduled PRM method on our QExactive and use Skyline to generate the scheduled inclusion/isolation list for the Qexactive PRM method file.

I've made a spectral library and imported a .raw file containing the target peptides and I've used the MS1 Full Scan method tutorial to identify the retention times and elution profiles of the peptides I'm interested in.

Then I'm using File -> Export -> Isolation List
to generate a scheduled list for the QExactive Inclusion list/Isolation List in the PRM method

I've got a couple of questions though (I've attached a screengrab of the isolation list and Skyline)
1) Where is the retention time window tolerance set for the Isolation List export? - currently it is using 2 min windows
2) Where is the NCE collision energy profile information coming from - I guess this is supplied by Thermo and I don't need to calibrate it? Any feeling how good the NCE estimates in Skyline are for QExactives?


Many thanks for your help
Ben
 
 
Brendan MacLean responded:  2013-07-30
Hi Ben,
1. Set in Peptide Settings - Prediction tab under Use measured retention times when present, Time window field.

2. I am told this is supposed to be for Normalized Collision Energy, and that our lab may be using 25 for this value across the board. It looks like the developer on this feature chose to use the function in Skyline that calculates an actual CE for each precursor based on linear equations assigned in the Transition Settings - Prediction tab, used for SRM transition lists. This is not correct for this column on the Q Exactive, however, and we will fix this in the future. You should be able to use Excel to fill this column with whatever normalized CE works best for you.

Thanks for your feedback.

--Brendan
 
benjamin.thomas responded:  2013-07-31
Thanks Brendan,
I'll stick with our default NCE value of 28 for now but if I try the Skyline derived NCE values I'll let you know how they compare.
many thanks
Ben