Yes, those settings look good.

There is a column in the Document Grid called "Normalized Area" which is the area normalized according to the normalization method specified on the Quantification tab of the Molecule Settings dialog.
You can use the binoculars button at the top of the Report Editor to search for the column named "Normalized Area".

The non-normalized values are available in different columns in the Document Grid. There is the "Total Area" column. which is the sum of the transition areas under the each precursor. The light and the heavy molecules both have their own total area, so if you include the Total Area column in your report the number of rows will double unless you check the "Pivot label type" checkbox in the Report Editor.

If you do not want to use all of the transitions for quantification, you can right-click on some Transitions in the Targets tree and uncheck the "Quantitative" menu item.

If you have more questions about quantification, we might be able to give you better answers if you send us your Skyline document.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
Files which are less than 50MB can be attached to these support requests.
You can always upload larger files here:
https://skyline.ms/files.url
-- Nick

Hi Nick,

I am doing a similar peptide analysis and wish to clarify a few doubts. Do we have to mention the heavy peptide concentration before normalizing with the heavy standards? What are the columns that are essential to export to find the sensitivity and specificity of the proteins?

Thank you.
Akhila

Akhila,

You do not need to tell Skyline the concentration of the heavy standard.
Skyline assumes that you have used the same concentration to spike the heavy standard into each sample. Therefore, the "Normalized Area" value is a number which can be used to compare the amount of light peptide in each replicate.
There is a column "Internal Standard Concentration" which you can provide a value for in the Document Grid.
If the Internal Standard Concentration has been specified then the column "Calculated Concentration" will contain the Internal Standard Concentration multiplied by the Normalized Area.

I am not sure what you are asking about sensitivity and specificity of proteins. You might find the answer to that in the Group Comparison tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_grouped

When you do a Group Comparison in Skyline, it performs T-tests to figure out which proteins or peptides have larger between-group variation compared to within-group variation.
-- Nick

Thank you for clarifying.

Hi Nick,

Thank you so much for the explanation!

I opened the Document Grid, but I wasn’t able to find a column called “Normalized Area.”
The only thing I did find was the “Ratio Results” column, which shows the ratio to the standard.

I’d really appreciate your help in understanding where I can find the normalized area values—maybe I’m missing a step?

Also, just to confirm: I only defined one fragment in the method.
That means the normalization is based on the ratio between the light and heavy versions of that same fragment, right?
This is important to me since that specific fragment has a very clean and strong signal.

Thanks again—I really appreciate your help!

Best,
Michal

In the Report Editor, there is a binoculars button at the top which you can use to search for a column by name.

Can you send us your Skyline document?
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files.
Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url

The rules for how Skyline calculates the light:heavy ratio are a little complicated and it would be easiest to explain them while looking at your Skyline document.
-- Nick

Hi Nick,

Thanks so much for offering to help — I really appreciate it!

I'm actually having trouble finding the Report Editor… maybe I'm looking in the wrong place or just missing something obvious.

In the meantime, I’ve attached my Skyline document, which includes some pooled sample examples with 3-fold and 10-fold dilutions. I’d love your advice on how best to include and interpret that information in the analysis.

I know the data isn’t super stable yet — which is exactly why I’m hoping the internal standard will help normalize things and make the results more consistent.

Thanks again for all your support!

Best,
Michal

The Report Editor is the thing that comes up when you choose "Customize Report" (or "Edit Report") from the Reports dropdown at the top of the Document Grid.
In Skyline 24.1 you can get to the Document Grid with the "View > Document Grid" menu item.
In Skyline-daily that menu item has moved to:
View > Live Reports > Document Grid.

You can learn more about the Document Grid and the Report Editor here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

Thank you for attaching that Skyline document.
I see that you have given different names to your fragment transitions.
The light one is named "Frg_255.211" and the heavy one is named "Frr_260.2432".
Unfortunately, because those names are different, Skyline thinks you would not want to calculate the ratio between those two transition peak areas because they have different names.
Fortunately, it is easy to change the name of those transitions. You can right-click on each of those transitions in the Targets tree and choose "Modify" and then change the names to either blank or the same value as each other.

Assigning names to transitions is a great way to tell Skyline exactly which light transition corresponds to which heavy transition.
If names have not been assigned to transitions, then Skyline matches them up by their position in the list that you see in the Targets tree.
The "Simple precursor ratios" checkbox on the "Quantification" tab of Molecule Settings tells Skyline to not try to match up transitions at all, and just calculate the ratio of the sum of light areas to the sum of heavy areas. However, when "Simple precursor ratios" is checked, Skyline will no longer be able to tell you the ratio between individual transitions' peak areas. For this reason, it is better to make it so the light and heavy transition Names match.
-- Nick

Hi Nick,

Thank you so much for your suggestion to share my document – I really appreciate it! I would never have realized that the fragment names were mismatched, and that’s why Skyline couldn’t identify my internal standard for normalization. That was super helpful.

And yes – I do work with Skyline daily, thanks again!

In the meantime, I’m attaching a screenshot because I still can’t figure out how to add the 'Normalized Area' column. I’m also attaching the updated document with the corrected fragment names, as you showed me.

If there’s any chance you could explain how to include the dilution pools information, that would be amazing. I usually clean this part up in R afterwards, but I’d love to know if there's a way to handle it in Skyline directly.
Also – will that information appear in the regular “SampleInColumn - Daily” report?

Thanks again for all your help!
Michal

If you want to learn how to do calibration curves in Skyline you should look at the Absolute Quantification tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_absolute_quant

I'm not sure what you're asking about in terms of adding the "Normalized Area" column to a report.
You will probably find the answer in the Custom Reports tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports
-- Nick

Hi Nick,

Thank you so much for the detailed explanation — I really appreciate it.

I was able to find where to add the normalized value.

Is there a way to view a bar chart comparison of the newly normalized values, similar to how we use the replicate comparison for the peak areas?

I’m also attaching the report I managed to export. Is the normalized value shown in column D?

I can see the normalization factor, but is there a way to also view the original (pre-normalization) peak area value?

Also, how can I be sure that the value I'm seeing corresponds to the fragment peak area?
In the standard report I usually get both the precursor and the fragment peak areas for each compound, and of course, when applying normalization, I need to know which one it refers to.
I'm only doing relative quantification at this point, but I'd prefer to base it on the fragment.

Thanks again!
Michal