Normalizing Unknowns?

Normalizing Unknowns? whale  2022-04-29 12:30


I am running a peptide quant experiment and spiked the same amount of ISTD into each unknown. . However, according to the BCA assay, a few of the unknowns had a very small amount of protein (likely due to treatment) so I was unable to inject the same amount of total protein for each sample. Is there a way to normalize these small amounts (i.e. I injected 10x less material for unknown 1 vs. unknown 2)?

Many thanks!

Nick Shulman responded:  2022-04-29 14:06
In the Document Grid there is a column on the Replicates called "Sample Dilution Factor".
You can use this column to tell Skyline that you have changed the concentration of your unknown samples in order to bring them into the linear range of the mass spectrometer.
-- Nick
whale responded:  2022-04-29 14:12
Thank you so much, Nick!
whale responded:  2022-04-29 14:42
So sorry, hopefully just one more question. I didn't see it in the document grid's Replicates. So I went to the doc grid settings and tried to add sample dilution factor (try1 photo). When I did this, it added a column of 1s, but I am unable to edit them (try2). I am sure I am doing something wrong and missing something very obvious. How can I get the sample dilution factor column to add to the replicates part of the grid and/or how to make the values editable?

Many thanks again!
Nick Shulman responded:  2022-04-29 15:32
If you want to make a column visible in the Document Grid, you should use the "Reports" dropdown at the top of the Document Grid. Towards the bottom of that dropdown menu is an item that says either "Customize Report" or "Edit Report" which will bring up the Report Editor which will let you choose any available column.

If you would like to learn more about the Document Grid you should look at the Custom Reports tutorial:

By the way, the "Calculated Annotation" dialog that you have in your second screenshot is an obscure feature you might use if there was some data that Skyline knows how to calculate but which you needed to make available after you have uploaded your Skyline document to the Panorama website.
-- Nick
whale responded:  2022-05-02 05:58
Thank you again!!