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CE Optimization - Product Ion m/z is different in exported transition list

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CE Optimization - Product Ion m/z is different in exported transition list Peter Jackson  2022-01-21 09:43
 

Hello,

I'm using the molecule interface and performing some CE optimization experiments. When I export my transition list, my product ion values are being changed along with the CE value. I've attached an example to this post.

Thanks,

Peter
 
 
Brian Pratt responded:  2022-01-21 09:54

Hi Peter,

Can you provide a bit more context? Perhaps you could supply the Skyline document in use here, using Skyline's File | Share menu item to create a .sky.zip file.

Thanks for using the Skyline support board,

Brian Pratt
 
Peter Jackson responded:  2022-01-21 10:02

Thanks Brian.

Yes I'm optimizing CE for three compounds. I have attached the .sky.zip here.

Peter
 
Brian Pratt responded:  2022-01-21 10:13

Thanks for that.

What you're seeing is normal - Skyline introduces a slight variation in the exported fragment m/z value so that the different CE values can be tried and distinguished in a single run. There's no way to tell the mass spec "try these different CE values on this specific transition" so we fool it into thinking they're three different transitions. The assumption is the difference from the actual fragment m/z is so small as to make no actual difference to the analysis. I hope that makes sense?

Best regards,

Brian
 
Peter Jackson responded:  2022-01-21 12:17

Thanks for the explanation, Brian! Yes that makes sense to me, but perhaps it's a vendor-specific issue? I edited the transition list so that all the product ions were the same and I am able to run them all as separate transitions on my Agilent 6470. The data processing side of things works fine as well, even with the product ions being identical (at different collision energies).

Appreciate it,

Peter
 
Nick Shulman responded:  2022-01-21 12:30
Peter,

The other part of the reason that Skyline changes the Q3 values slightly when exporting a transition list with CE optimization is so that Skyline can tell them apart when you later do "File > Import > Results".

Skyline does not know how to read CE values from a .raw file.

Instead, when you do "File > Import > Results", Skyline asks you whether you were doing CE optimization. If you were, then Skyline looks for groups of chromatograms with the same Q1 value and where the Q3 value differs by 0.01 units. When Skyline finds a group of chromatograms like that, Skyline assumes that the chromatogram with the middle Q3 value used the predicted CE value, and the other chromatograms used CE values that were increments of the Step Size that you have specified at "Settings > Transition Settings > Prediction > Collision Energy > Edit Current".

If you change the Q3 values in your method, Skyline will not be able to figure out what CE values were used.
-- Nick
 
Brian Pratt responded:  2022-01-21 12:35

Well, the other half of that slightly-different-m/z scheme is about Skyline reading the results back in as part of the optimization process. If you edit them to be all the same Skyline won't be able to tell them apart when it reads the mass spec result, so it won't be able to decide which CE is optimal for the actual transition. (And you are correct, we do it this way so as to be independent of instrument vendors since not all formats report CE in the results.)

Is that tiny difference in fragment m/z actually making any difference to your results?
 
Peter Jackson responded:  2022-01-21 12:35

Thanks to you both!