Instructions for normalizing with TIC dave lee-2  2023-02-02 04:29
 

Hi all,

I have a PRM experiment where I am trying to normalize each sample according to their respective TIC.
I have looked in the Skyline forum where the suggestion is to take modify the document settings and enable calculation of the Total Ion Current Area.
Subsequently, setting the "Explicit Global Standard Area" column to the TIC area

However, I have come across a couple of issues -> The "Explicit Global Standard Area" is under the "calculated" tab rather than "editable" in the document settings so I can't seem to copy and paste.

Ultimately, what I would like is when I view the peak areas of my peptides (with respect to the other replicates); I would like to normalize against the TIC -> Currently, the only options available are "Total", "Maximum" and "None"
And then to take these values through to MSstats

Is there any resource that provides step-by-step instructions in what I am attempting to do?

The version of Skyline I'm using is:
20.2.0.343
 
 
Nick Shulman responded:  2023-02-02 07:54
Your screenshots show that you have been trying to "Define an annotation". That is very different from "add a column to a report".

If you want to learn about adding columns to reports you should look at the Custom Reports tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

Is there a reason that you are using such an old version of Skyline? The current version of Skyline is 22.2. The feature of being able to normalize to TIC without having paste numbers into the "Explicit Global Standard Area" was added in version 21.1. We usually recommend that you use the most recent version of Skyline whenever possible unless you are trying to reproduce numbers from an older experiment that used a specific version of Skyline.

There is no straightforward way to use either the "Explicit Global Standard Area" or the total ion current in MSstats. Skyline has its own Group Comparison feature, and you can use TIC normalization there, but if you wanted to do something like that in MSstats you would probably need to write your own R code to get the numbers to be what you want them to be before you pass them to MSstats.
-- Nick