Small molecule (non-global) standard concentrations

Small molecule (non-global) standard concentrations jrenders  2020-09-01

Hi there,
Thanks so much for adding the ion ratio features to skyline - we are currently rebuilding all of our small molecule methods in skyline and have unfortunately hit a snag. I think you previously made mention of (in the future) adding the ability to assign calibration curve regression and weighting on a per-analyte basis. Right now, via the "Document grid --> Reports --> Replicates" report we can assign analyte concentrations, but only globally. However, we have methods where our calibrators do not have all analytes at the same concentration. Is there plans to allow for a per-analyte concentration assignment as well in the future? Or perhaps there is already a way to handle this via the document grid or pivot table? Thanks for any guidance you can suggest.

Nick Shulman responded:  2020-09-01
In Skyline-Daily, there is a new feature where you can set the concentration of each molecule in each replicate independently.

The column in the Document Grid is called "Explicit Analyte Concentration" and it can be found on the "Molecule Result":
Molecule Lists > Molecules > Molecule Results
Proteins > Peptides > Peptide Results

-- Nick
jrenders responded:  2020-09-01
Thanks Nick! I probably should have been able to figure that one out on my own, but I appreciate the help!
abrell responded:  2021-01-15
Hi, is there a way to extend the concept of explicit analyte concentration to heavy standards?

Not all of our calibrators have the same amount of IS. I've tried using "Concentration Multiplier", as well as treating the labeled- and un-labeled calibrants as separate spp (i.e. not heavy and light) and thus far I've been unable to handle this situation. Thanks.
Nick Shulman responded:  2021-01-15

Do you have a calibration curve (even if it's a single point calibration curve with one external standard)?
If your different calibrators have external standards in addition to the samples, and you would like certain samples to use certain external standards, you can set the "Batch Name" column on the Replicates in the Document Grid. A particular replicate will only use the external standards with the same Batch Name.

Otherwise, it might be that you need to have your different replicates be in separate Skyline documents.
-- Nick
abrell responded:  2022-03-09
Hello, picking up this question again for small molecules work.
I may not have explained clearly what I'm looking for in Skyline, so I'll try a little bit more.

In our workflow we measure (using mass and density) the individual amount of labeled standard that goes in to every (i) calibrator, and (ii) replicate unknown sample; aka isotope dilution method. I have not found a way for Skyline to consider these internal standard values. Adding them in EXPLCIT ANALYTE CONCENTRATION column seems like where they should go, but I see their value there has no effect.

In a separate thread titled "different heavy IS concentration" I read from Brendon: "With external calibration the internal standard is use for normalization only, and Skyline does not require any knowledge of its concentration. The (analyte area / standard area) ratio is used to calibrate against the external curve or single-point."

In newest Skyline, is there a way to consider the internal standard concentration?
jrenders responded:  2022-03-09
Hi Abrell,
I can appreciate you are probably hoping for a Skyline response, but I wanted to chime in. I do a lot of external calibration with internal standardization small molecule quantitation (i.e., isotope dilution). I know via using many vendor platforms (e.g., Agilent, Waters, SCIEX, Thermo) for this type of analysis that there is often a placeholder where you can input the concentration of the IS, however, I'm not aware that this placeholder ever has any mathematical use (as Brendan has mentioned in your quote). It's not clear to me what you hope that Skyline will do with this IS concentration? If you are trying to use this IS addition to your samples to perform calibration of some sort (e.g., internal calibration) this can be handled a separate way in Skyline, I believe.
Nick Shulman responded:  2022-03-10

I do not think I understand your question.
Can you post a picture or a spreadsheet illustrating what you are trying to calculate?
It might also be helpful if you could send us your Skyline document.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
Files which are less than 50MB can be attached to this support request.
You can upload larger files here:

-- Nick