The difference between CV peak area and CV total area

The difference between CV peak area and CV total area Liangshuang  2019-07-24

Hi Skyline team,
When I look at the CV value of my data, why is the CV value of the peak area map in skyline inconsistent with the total area CV value I exported? I have tried non-normalization and normalization methods and none of them is useful. Is there any difference between “peak area” and “total area”? Or is there some problem with the settings of my skyline? Please see the attachment,thanks!


Nick Shulman responded:  2019-07-24
The "area" is a number that you find on the Transition Result in the Document Grid.
The "total area" is on the Precursor Result. It is equal to the sum of the all of the peak areas for the transitions under that precursor.

We do not recommend using the "Total Area Normalized" for anything. It is equal to the Total Area divided by the sum of all of the areas of all of the other things in the replicate. This column existed in Skyline long before Skyline had any features which allowed you to specify different normalization methods.

Skyline does have a useful normalized area column. It's called "Normalized Area" and it is on the Precursor Result
Proteins > Peptides > Peptide Results > Quantification > Normalized Area
That column has the area for the peptide normalized based on your settings at:
Settings > Peptide Settings > Quantification

The CV values that you have in your screenshots are coming from the "Precursor Result Summary" group of columns in the Document Grid.
If you want more control over how the CVs are calculated, for instance, if you want your replicates to be grouped by cohort first, you can take a look at "PivotEditorCalculateCVAcrossCohorts.pdf" on the Pivot Editor tip page:

-- Nick
Liangshuang responded:  2019-07-25
Hi Nick,
     Many thanks for your answer.It is very helpful to me.

Matthias responded:  2019-07-26
Hi all,

I was wondering if the "Area" column which is part of the default MSStats Input format represent the non normalized intensities?
In the help it is written AUC, minus background, for transition peak
Similar to so raw intensities?

When setting MS2 only and None (normalization method) in the Peptide Settings -> Quantification tab does one get the raw intensities including the background for each transition or are they here already calculated for the peptides (summing up all the transitions)?

Best regards
Brendan MacLean responded:  2019-07-26
All areas in Skyline have been background subtracted. To get raw areas you need to add Background. Careful never to subtract Background from Area or you will get:

Raw Area - 2 * Background

Here is our best attempt to explain the Skyline peak statistics: