Need to get normalized area for SRM data

support
Need to get normalized area for SRM data kerry hassell  2018-05-09 12:37
 

The software is calculating the peak area in replicate comparison but not in the results grid (showing #N/A). It is SRM data from the triple.

 
 
Nick Shulman responded:  2018-05-09 13:07
What normalization method are you trying to use?
The Normalized Area column uses the normalization method that you have specified in:
Settings > Peptide Settings > Quantification

You can also specify different normalization methods for different peptides in the Document Grid using the "Peptide Quantification" view.

The normalized area will be "#N/A" if that replicate is missing values for some of the transitions. The idea is that it does not make sense to compare areas across replicates if some of those replicates have transitions that the other replicates do not.

I do not see any red dots next to the transitions in the Targets tree, so it must be something to do with the normalization method that causes those values to be "#N/A".

One thing that you should make sure of is that you have a checkmark next to the menu item "Settings > Integrate All". When Integrate All is turned off, Skyline will aggressively give you #N/A when peaks are slightly off-center. However, since you do not have the red dots in your Targets tree, it is unlikely that Integrate All is the cause of your problem.
 
kerry hassell responded:  2018-05-09 13:14
Weird think is when you click the precursor a Total area will populate?

I do have the Integrate All clicked
 
Nick Shulman responded:  2018-05-09 13:32
The reason that you are seeing #N/A probably has something to do with the Normalization Method that you have selected in:
Settings > Peptide Settings > Quantification

If you would like, you can send us your Skyline document.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files, including extracted chromatograms.

If that .zip file is less than 50MB then you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url