Points Per Peak sa825  2020-03-11 04:36
 

Good morning,

I am conducting PRM on the Thermo Q Exacitive and I wanted to ask if there was a setting in Skyline that allowed you to select the number of points per peak for the peptides of interest?

Thanks,

Shimon
 
 
Nick Shulman responded:  2020-03-11 07:41
I am not sure I understand your question.

Are you saying that you want to export a PRM method (i.e. isolation list) and you would like to control the number of MS2 spectra that are collected for a given precursor? I do not believe there is any way to do that. You can control the length of time in minutes that precursors are collected with the "Time Window" setting at:
Settings > Peptide Settings > Prediction > Retention Time Predictor
but there is no way to control that from Skyline in terms of number of scans.
Maybe someone who is more of an expert on Thermo software could help you.

Do you want to control how wide of peaks Skyline chooses when Skyline detects peaks in chromatograms? There is no way to control that.

Do you want to see the number of points across the peak for the chosen peaks? You can see that value in the Document Grid. The column is called "Points Across Peak" and it's on the Transition Result.

-- Nick
 
Brendan MacLean responded:  2020-03-11 11:03

Hi Shimon,
True, there is no way to control ahead of time the number of points per peak in PRM. The place you want to be looking, however, is View > Retention Times > Scheduling. When you have your settings for PRM all set up for scheduled PRM, then this graph will show you the number of concurrent precursors you will get exporting your method. This is covered for SRM in the Targeted Method Refinement tutorial:

https://skyline.ms/_webdav/home/software/Skyline/%40files/tutorials/MethodRefine-3_7.pdf#page=20

It is, however, left to you to know your dwell-time (plus switching time) and to do the mental calculations to figure out your expected points per peak. i.e.

points per peak = peak width time / concurrent precursor count * time per precursor

For example, a 30-second peak width and a 20 ms dwell time for a desired 10 points across a peak will come out to:

30 / 10 = 3 second cycle time / 0.02 seconds dwell time = 150 concurrent precursors

Want 50 ms dwell time?

3 second cycle time / 0.05 second dwell time = 60 concurrent precursors

15 points across the peak just to be sure?

30 / 15 = 2 second cycle time / 0.05 second dwell time = 40 concurrent precursors

This is currently how you would use the tools Skyline provides to answer the question you are posing.

Hope it helps. Thanks for posting to the Skyline support board.

--Brendan
 
sa825 responded:  2020-03-13 11:24

Thank you both for your answers. They were very helpful!

Shimon