Ratio and area calculations in skyline

support
Ratio and area calculations in skyline vabitsika  2017-02-13
 
I am using Skyline (64 bit) 3.6.0 10162.
I am having some trouble to understand the calculations performed by Skyline, if I am using the correct exported light to heavy ratio and how can I export the raw areas. In the “export report” window I export the “Peptide Ratio results” and use the “ratio to standard” as the main quantification value. But how are the “ratio to standard” and the “quantification” values calculated?
I read your comment on the total area: that it is calculated as follows:
TotalRatio = LightArea1 + LightArea2 + LightArea3
             ------------------------------------
             HeavyArea1 + HeavyArea2 + HeavyArea3
How can I export this total ratio? In the precursor results summary: I only find the total area and the total area ratio. How are these calculated? FYI I checked this glossary (https://skyline.ms/_webdav/home/software/Skyline/%40files/tutorials/CustomReports-2_5.pdf#page=32) and not enough information on the exact calculations is included.
Also how can I export the raw Light area 1, Light area2 …heavy area1 etc ( in the quantification setting, I have selected in regression fit: none and in normalization: none).
- One more point I would like to make is that the values in the “quantification” column do not seem to match with the observed areas of the transitions peaks: eg the highest transition is around 3.5 * 106 and in the respective exported file the quantification value is 1.16 * 107
- The final point I would like to make is that I get some NA in the quantification column of the heavy peptide when the spectrum of the heavy peptide looks good and ratio to standard is calculated fine (no NA). I can send you a report to see for yourself if that would be more helpful. How can this be solved?
Thanks a lot,
Vasiliki Bitsika
email: vabitsika@bioacademy.gr
 
 
Nick Shulman responded:  2017-02-13
You probably do not want to be looking at the things on the "precursor results summary". The columns in there are the average across all of your replicates.

The "Ratio to Standard" and the "Total Area Ratio" are often the same value.
The "Ratio to Standard" lives on the "Peptide Result", and is the ratio of the light precursor areas to the heavy precursor areas.

The "Total Area Ratio" lives on the "Precursor Result", and is the ratio of that precursor's area to the heavy form of that precursor.

If your peptide contains only one light and one heavy precursor, then the "Total Area Ratio" will be the same as the "Ratio to Standard".
If your peptide contains precursors with different charge states, then "Total Area Ratio" will be slightly different, since it will only use the one charge state.

Yes, you are correct that the TotalAreaRatio is calculated as the sum of the light areas divided by the sum of the heavy areas.
Those areas are the Transition Result Area values (that is, the "Area" column on the "Transition Results" view). You can find that column in the "Customize View" dialog here:
Proteins > Peptides > Precursors > Transitions > Transition Results > Area

I do not understand your questions about the Quantification column.
Can you post a picture of what you are seeing? Alternatively, you could post 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.
If that file is less than 50MB, you can attach it to this support request.
I will also send you an email with other instructions for how to send us your files.
 
vabitsika responded:  2017-02-14
Dear Nick,
thank you very much for your prompt reply. Your response was very helpful.
1. I would also like to perform quantification using only the area of the highest transition. Is there a setting to select this?
2. Regarding the "Quantification" information (from peptide results), how is it calculated? What does it represent?
3. Also how exactly is the area of each transition peak calculated? The surface of the peak? With which units?
4. I understand that for the calculation of the “Ratio to standard” only transitions that were detected in both light and heavy precursors are used. You told me how the Area of each transition is exported (Transition Result Area). Is there a way to export only the areas the light and heavy precursor used for the ratio calculation?
I am sending you one skyline file so that you can see what kind of analysis we are doing.
thank you very much,
Vasiliki
 
Nick Shulman responded:  2017-02-14
The Quantification column in the Document Grid uses the settings that you might have set in:
Settings > Peptide Settings > Quantification

You would use that if you had external standards that contain a known amount of your analyte and you want Skyline to use a calibration curve to calculate the absolute quantity of your peptide.