Quantification naimamuntu  2024-04-10 23:44


I am looking at the quantification of the two peptides R.IEDEQALGSQLQK.K [1093, 1105] and R.ILNPVAIPEGQFIDSR.K [725, 740] in disease (GoH) and healthy hearts (NF). I have n=3 for GoH disease (2LV, GoH 5 LV, GoH 9 LV ), and I ran each sample in triplicate. The healthy group, n=5, NF_4LV, NF_13LV, NF_14LV, NF_18LV, NF_20LV and ran in triplicate.
I ran a multi-point calibration point for IEDEQALGSQLQK labeled 1 through 8 and ILNPVAIPEGQFIDSR labeled M6 through 8
Which transition to choose? How do we proceed to quantify these two peptides across disease and health?
I've attached my document as Skyline_Nitha_41024_

Thank you so much.

Nick Shulman responded:  2024-04-11 00:00
I see that you uploaded a file called "Skyline_Nitha_41024_.skyd" but what I would really need to see would be a .sky.zip.
The .skyd file contains extracted chromatograms but it is very difficult to interpret without the .sky file that goes with it.

You can create a .sky.zip file using the Skyline menu item:
File > Share
This .sky.zip will contain all of the necessary files for someone else to be able to open your Skyline document in Skyline.

If that .sky.zip file is less than 50MB you can attach it to this support request.

You can always upload larger files here:
-- Nick
naimamuntu responded:  2024-04-11 05:25
Hi Nick,

Thanks. I've uploaded the correct format.

Nick Shulman responded:  2024-04-11 07:23
Thank you for uploading that file.
I see that you are using Skyline version 21.1.
The latest released version of Skyline is 23.1. We generally recommend that you always use the latest version of Skyline unless you are trying to reproduce results from old experiments.

The Group Comparison tutorial will show you how to compare quantities between your groups of replicates:

The steps you will need to do are:
1. Go to "Settings > Document Settings > Annotations" and define a replicate annotation (this annotation is usually named "Condition" but you can name it whatever you want)
2. Use the "Replicates" report in the Document Grid to set the value of that replicate annotation on your replicates to tell Skyline which group they belong to.
3. Go to:
View > Other Grids > Group Comparisons > Add...
and tell Skyline how to perform the group comparison.

Your calibration curve will not be used in this group comparison. The reason is that the Group Comparison feature only cares about the relative abundance between replicates. Even if the observed values were corrected by using the calibration curve, the ratios of these corrected values between the replicates would still be the exact same number.
-- Nick