Small molecule: calibration curves for individual targets

Small molecule: calibration curves for individual targets alejandro.cohen  2020-11-18

Hi !

I'm developing a targeted SIM method for 18 steroid hormones using Skylines Small Molecule interface. So far so good.
I have prepared calibration curves for each standard (3 concentration levels) separately and run them on a LC-MS (QExactive). What I mean is that I did NOT pool all standards together in the same tubes, rather, have a separate calibration curve for each standard.

Replicate 1: Std X at 0.1ng
Replicate 2: Std X at 1ng
Replicate 3: Std X at 10 ng
Replicate 4: Std Y at 0.1ng
Replicate 5: Std Y at 1ng
Replicate 6: Std Y at 10 ng
Replicate 7 :Std Z at 0.1 ng, etc etc

In the Replicate Tables, I have already classified them as Standards and their concentrations as in tutorial.

Is there any way to inform Skyline which Molecule each Standard corresponds to? By default, Syline tries to find ALL Molecules in each each Replicate. Manually removing peaks from each Replicate is very time consuming.

All tutorials I have read so far combine all standards into single replicate injections.



Nick Shulman responded:  2020-11-18
I cannot think of a good way to do what you are asking. I think that you should have a separate Skyline document for each of your set of standards.

You could try using the "File > Import > Document" feature in order to combine your separate Skyline documents together after you have made the separate documents. Importing another document brings up a dialog which gives you options about how to combine replicates together.

Skyline has the ability to combine multiple result files into a single replicate. The most common reason that people do this is that the same sample was injected into the mass spectrometer multiple times because the mass spectrometer did not have enough bandwidth to collect data for all of your targets at the same time. However, the only way that Skyline would know not to look for a particular molecule in a particular result file would be if the result files had different SRM chromatograms or different MS2 isolation windows in them. If all of your result files have the same set of isolation windows in them, then Skyline is not going to know which result file to look in for which molecule.

-- Nick
alejandro.cohen responded:  2020-11-18
Thanks Nick for your quick response!

Your suggestion would work, but would add considerable postacquisition editing and stitching! (18 Skyline docs!).

I think I will just pool the standards together into three replicates (Replicate X to Z, conc 0.1ng; Rep X to Z, conc 1ng; Rep Z to Z, conc 10ng). A little more work for me know upfront, but less data stitching at the end.

However, this could be an idea for an Feature Request. Maybe have the Replicate table to have an additional column to set which Molecules are present in each replicate. That would allow more flexibility for users to decide how to set up their calibration curves.