Using Surrogate internal standard

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Using Surrogate internal standard jihyunk  2024-10-31 14:14
 

Hi,

I'm currently quantifying small molecules using Skyline. For some analytes, I have labeled internal standards, but for others, I don't. So, I'm considering using surrogate internal standards from different molecules. I'd like to assign these individually but haven't found instructions on how to do this.

Could you please guide me on where to find this information?

Thank you.

 
 
Nick Shulman responded:  2024-10-31 21:08
There is some information about how to use surrogate internal standards here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=Surrogate%20Standards

The first section is about surrogate internal standards.
The second section is about surrogate external standards which is not what you want.
-- Nick
 
Harrison Kim responded:  2024-11-19 06:11
I also had a similar query as I'd like to normalize the peak area of a small molecule to a surrogate internal standard that has a different retention time. I tried a workaround of editing my transition list to give the surrogate the same name as my molecule but with a different explicit RT and different precursor/product m/z values but it seems like Skyline does not like integrating two different RT windows for the same compound.

Perhaps I misinterpreted the documentation on surrogate standards but it does not seem like there is the ability to assign surrogate internal standards?

I have also encountered an issue where the retention times for our internal standards is slightly shifted from their respective compounds. Is there any way I can individually adjust the RTs for a product or precursor m/z without adjusting it for the entire molecule?

Thanks!
 
Nick Shulman responded:  2024-11-19 07:45
Harrison Kim,

It sounds like you were not able to get surrogate standards to work the way that they are supposed to.
I have attached a couple of screenshots showing how to do it.
First, you right-click on the surrogate standard in the Targets tree and choose "Set Standard Type > Surrogate Standard".
Then, you view the Document Grid and choose "Molecule Quantification" in the Reports dropdown.
Then, you set the "Normalization Method" of the molecule that you would like to have use the surrogate standard.

Does that help? Let me know if there is some place where the instructions were not clear and I will update the documentation.

I think if you had surrogate standards working correctly then you be able to give your analyte and its standard different molecule names and then their peak boundaries will be independent.

It is possible to adjust the peak boundaries of a single transition to be different from the rest of the molecule, but Skyline intentionally makes it difficult because it is usually not what you want to do. You would need to select the transition in the Targets tree and then choose "View > Transitions > Single" on the chromatogram graph right-click menu. Then, when you are looking at only a single transition's chromatogram if you adjust the peak boundaries it will affect only that one transition and not the rest of the molecule.
-- Nick
 
Harrison Kim responded:  2024-11-19 09:56
Thanks Nick, this is incredibly helpful. I was able to set the surrogate samples through the normalization method. I think that in hindsight, the documentation is clear however it did help to know that I can set surrogate standard in the "Molecule Quantification" report.

With regards to adjusting the peak boundaries of a single transition, I have two precursor ions for each molecule; a heavy and light isotope. I am able to change the peak boundaries of the chromatograph for an individual transition without affecting the boundaries for the other transition under the same precursor ion. However, while the change in the peak boundary does not affect the transition under the same precursor, it does affect the boundaries of the transitions under the second precursor.

Thanks again for all your help.