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Response of CE optimization (attached files)

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Response of CE optimization (attached files) hjl  2021-01-27 20:42
 

Dear Skyline team,

As you were asking a sample files of Skyline on my question of CE optimization, I prepared the files.

My workflow of CE optimization is described as below.

1.Specified 1 transition per compound

2.Based on specified transition, set methods for 1st CE optimization( step size 6, step count 3) = 1st analysis
Before) I used 4 step size and 6 count, I thought many step counts may draw some confusion to measure the best transition, I shorted it as 3 instead increasing step size( 4 to 6)

3.Analyzed and clarified the best CE for each transition

4.On the basis of 1st result, set methods for 2nd CE optimization( step size 2, step count 3) = 2nd analysis

5.Analyzed and acquired the final optimized CE for every transition each

6.Compared two methods( one = original method ; the other = obtained by CE optimization)

  • in the process of optimization, I specified each compound's RT as dragging the peak on the chromatogram, but in the sample file I attached, some of compounds might not be assigned for its peak. Please consider that flaws of those files.

My question is on the 1st optimization, I could clearly see that the CE optimization is needed for every compounds
then, after setting the optimized method( on the comparison step) Original data showed far better results than the other.
If the original data is better than modified one, what made the optimization result (especially that of 1st optimization) different ?

Thank you for your help as always.
 
 
Nick Shulman responded:  2021-01-27 20:52
Can you send us .sky.zip files instead of these .sky files?
The .sky file contains everything that you see in the Targets tree, but a sky.zip file will contain supporting files including extracted chromatograms.

In Skyline you can use the menu item:
File > Share
to create a .sky.zip file containing your Skyline document and supporting files including extracted chromatograms.

If those .zip files are less than 50MB you can attach them to this support request.

Otherwise, you can upload them here:
https://skyline.ms/files.url

-- Nick
 
hjl responded:  2021-01-27 21:04
I attached the zip file you requested :)

please chech the upper writing.

Thank you
 
Nick Shulman responded:  2021-01-27 21:48
Thank you for attaching those .sky.zip files.

I am not sure what we are supposed to look at. Can you attach a screenshot of what you are seeing that looks suspicious?
-- Nick
 
hjl responded:  2021-01-28 00:42
Major question is there is mismatches of results between the skyline files.

For example, In the routine_1transition_1st file, Meldonium, S6b_T.Cathine/[Mo_S6]Phenylpropanolamine, oxymorphone( etc...) showed best results on the original CE.

In the second optimization, I just exported the transition from the first file and applyed the second CE optimization setting( step size 2, count 3)
then, the 2nd result showed all the compounds I suggested as an example, are turned out to be needed minor modification( actual graph showed the best results on the higer CE)
The another example was shown in my question before also( posted on 22nd Jan)

I carefully thought that those mismatches caused the results that showed better sensitivity on the original compared to the CE optimized.

I considered if the original CE was the best one, the 2nd(minor optimization) has to be suggested the same result..

Thank you for your active response.
 
Nick Shulman responded:  2021-01-28 09:44
Here is what I see when I look at S6b_T.Cathine/[Mo_S6]Phenylpropanolamine in your files.

In the image CE_Optimization_Run.png shows a CE Optimization run where several different collision energies were used in a file called "Z:\G16\Skyline\DATA\기존물질\NewMethod\1st\PCU3.raw". It shows that "Step -1" had the best response with an intensity of 5x10^6, compared to step 0 which was 2x10^6.

In the next file, Comparison.png shows some other runs which were normal (i.e. not optimization) runs, where each molecule gets only one chromatogam.
The two replicates "optimized1" and "optimized2" each have peak intensities around 1.5x10^6, and "original1" and "original2" are both around 4x10^6.

The names of the result files are:
Z:\G16\Skyline\DATA\기존물질\NewMethod\3rd\PCU_optimized1.raw
Z:\G16\Skyline\DATA\기존물질\NewMethod\3rd\PCU_optimized2.raw
Z:\G16\Skyline\DATA\기존물질\NewMethod\3rd\PCU_original1.raw
Z:\G16\Skyline\DATA\기존물질\NewMethod\3rd\PCU_original2.raw

I imagine the next step is to look at these .raw files and see whether they are actually using the collision energies that you think they are.
I am not sure whether we on the Skyline team have programs installed on our computers that can figure out what the collision energy in a raw file is. We might have the right software, or we might have to ask you to send us screenshots of what these files look like in your software.

Can you send us all of these raw files?
PCU3.raw
PCU_optimized1.raw
PCU_optimized2.raw
PCU_original1.raw
PCU_original2.raw
-- Nick
 
hjl responded:  2021-01-28 17:12
Sure, but One thing I want to clear is I did CE optimization process two times.

There are two files named PCU3( 1st optimization step size 6, step count 3, 2nd optimization step size 3, step count 2)

I will attatch 2 transtion list that I exported from the skyline
( 1st step was extracted 1st optimization setting based on the original transition that we have used for a while(file name: 1st_1tran_003 as you mentioned S6b_T.Cathine/[Mo_S6]Phenylpropanolamine, I picked the one that contain that compound;
2nd step was based on the transition from 1st skyline file (just applyed Use optimization values when present) and imported to 2nd skyline then, exported after adapting the 2nd optimization setting, file name: 2nd_1tran_003)

And other file you requested are attached below.

Thank you so much to give me these active response.
Hope you have a great day.
 
Nick Shulman responded:  2021-01-28 19:20

I think Skyline might have been confused by the fact that your 1st_1tran__0003.csv only has 6 steps for each compound (-2 to +3), instead of the usual 7.

So your transition list looks like this:
|Compound|Retention Time (min)|RT Window (min)|Polarity|Precursor (m/z)|Product (m/z)|Collision Energy (V)|
|-------|--------|----------|------------|-----------|-----------|------------|----------|
|S6b_T.Cathine/[Mo_S6]Phenylpropanolamine(+1).-2|1.95|1.2|Positive|152.1|117.08|6|
|S6b_T.Cathine/[Mo_S6]Phenylpropanolamine(+1).-1|1.95|1.2|Positive|152.1|117.09|12|
|S6b_T.Cathine/[Mo_S6]Phenylpropanolamine(+1)|1.95|1.2|Positive|152.1|117.1|18|
|S6b_T.Cathine/[Mo_S6]Phenylpropanolamine(+1).1|1.95|1.2|Positive|152.1|117.11|24|
|S6b_T.Cathine/[Mo_S6]Phenylpropanolamine(+1).2|1.95|1.2|Positive|152.1|117.12|30|
|S6b_T.Cathine/[Mo_S6]Phenylpropanolamine(+1).3|1.95|1.2|Positive|152.1|117.13|36|

However, when Skyline extracts chromatograms from that, Skyline does not know that it was supposed to start a -2 and go to +3. Instead, Skyline thinks it went from -3 to +2, and the +3 chromatogram is missing.

So, the chromatograms in Skyline look like the attached screenshot.

It looks like Skyline is showing you all the chromatograms from step -3 to +3, but the yellow +3 chromatogram is actually missing.

Skyline ends up thinking that Step -1 is the best chromatogram, and so, when Skyline exports your transition list for the second round of optimization, the CE values that Skyline exports are all centered around 12, because that's what Skyline though yielded the biggest chromatogram.

However, in reality, since Skyline was confused about which chromatogram had which collision energy, the best chromatogram had a collision energy of 18.

The thing to keep in mind about Skyline and CE optimization is that Skyline has no idea what the collision energies in your raw file are. Skyline notices a set of chromatogams with identical Q1 values and where the Q3 number differs by 0.01, and then Skyline has to make assumptions about what their collision energies were.

-- Nick