Using more than one calibration curve in a single skyline document

Using more than one calibration curve in a single skyline document hogan  2019-10-30


I have been using skyline smoothly to run a quantitative small molecule assay with a calibration curve and check standards and the whole nine yards for a few months now and it is great!!! Thank you all so much for your hard work in bringing small molecule functionality into this great platform!

Now the issue.
Skyline works fine for 1 analyte with 1 standard curve. I have recently tried to add a second analyte with a separate calibration curve to the same skyline document and I am only able to input one value in the "Analyte Concentration" column of the document grid for each replicate that is set as a "Standard". (I have multiple analytes in my calibration curve and their concentrations are different in the CAL levels). I can only load up one analyte curve worth of values and then they repeat over and over again in the grid even if the "Molecule Name" or "Precursor" are set to different values.

Is it possible for skyline to have multiple calibration curves set in the same document? Can you add a custom sample type like "Standard2", "Standard3", etc. and assign separate calibration values there?

My current workaround is to pull the data files into two separate skyline documents, with different analytes. I then import the annotations separately in each one and combine the calculated concentration data afterward using the replicate name as the index to join them on. Please let me know if there is another way to do this or if there is something in the works!

Again. Thanks so much for your work!


Nick Shulman responded:  2019-10-30
There is a "Concentration Multiplier" that you can set on each peptide or molecule in your document.
The Concentration Multiplier gets multiplied by the analyte concentration.
This works in the case that your peptides were all spiked in at different starting concentrations, but the dilution series for each peptide was the same.

You can find more information about Concentration Multiplier in this recent support request:

-- Nick
hogan responded:  2019-11-05
Thank you for your Response Nick! I got what I needed from that recent support request. It appears that I do not currently have my calibrator standards set up to operate using the Concentration Multiplier as they are not generated as a dilution series from a single stock concentration.
Brendan MacLean responded:  2019-11-06
Can you give us more detail on how you achieved your sample concentrations? How many analytes? How many different dilution series, i.e. how many different slopes are expected among your analytes? A mixture with a single dilution series would have 1 slope, while I have seen at least one paper where a controlled mixture was created out of multiple dilution series (2 or 3) spiked into a background.

It would be useful to us to understand your situation and whether it is similar with just a handfull of dilution series or whether you truly have a different expected dilution slope for 10s or more analytes.

Thanks for posting your feedback on the Skyline support board.

nfoulon responded:  2020-02-20

The Hoofnagle Laboratory is trying to use Skyline to quantify small molecules, and we are running into the same problem as Kyle is above. We are adapting a clinical assay (Vitamin D and metabolites) for research testing and have five different calibrators. Our calibration curves run at different slopes for each of five analytes, and the actual concentration of these standards is quantified spectrophotometrically, so the concentrations are not "clean" dilutions of one another but rather experimental values (see attachment). Therefore, it seems as though the concentration multiplier fix suggested above will not work for this application. The question ultimately boils down to whether or not Skyline can create calibration curves using unique values for each standard across multiple analytes.

Thank you.