Accurate Values for peak intensity

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Accurate Values for peak intensity sa825  2019-07-23
 

Good morning,

I am using Skyline 19.1.0.193 on Windows 10
I wanted to ask how you would accurately read off the intensity of a peak/peptide in skyline?
I can read it off by eye but II feel as though there is probably a more accurate method?
I have attached an image so you can see what I mean.

Also, how do I view values for the area of the peak?

Thanks,

Shimon

 
 
Brian Pratt responded:  2019-07-23

Hi Shimon,

You can get those and many other values from the Document Grid.

Best,

Brian Pratt

 
Brendan MacLean responded:  2019-07-23

Or the Results Grid. You may want to refer to the Custom & Live Reports tutorial:

https://skyline.ms/tutorial_custom_reports.url

 
sa825 responded:  2019-08-02

Thank you for your response. The document and results grid was unable to give me the data I wanted.
But the Custom & Live Reports tutorial was extremely helpful.

Thanks,

Shimon

 
Brendan MacLean responded:  2019-08-02

True enough. In your screenshot, you are looking at the summed chromatograms for your light and heavy precursors, and requesting the maximum height between the integration boundaries of the heavy precursor. This is not a metric that Skyline stores or gives access to in its reports. The closest proxy would be Precursor Results.Max Height, which I should note is a background subtracted height for the most intense transition in the precursor (light or heavy). So, it will not correspond exactly with the height you see in the summed total chromatogram. It should correspond more closely with the height you see when you are showing all transitions of the precursor (though, it will have background subtracted).

In general, we don't make a lot of use of peak heights, but would generally direct you toward using peak area, and striving for more points across your peaks than one can see in your screenshot. Even peak heights will be negatively impacted in their accurate representation of the underlying peak when the number of points across a peak are insufficient. (See figure 5 in Lange, Mol. Sys. Biol. 2008 https://www.embopress.org/cgi/doi/10.1038/msb.2008.61)

Glad the tutorial was useful. Thanks for using Skyline in your research.

--Brendan

 
sa825 responded:  2019-08-02

However I do have another issue.

I have been able to view the height of my peaks using a "Custom Report" I created.
However, the Report is showing three different readings for the height and area of the light and heavy versions of the same peptide, how do I know which of the three is the correct one?

My column settings for the Custom Report are attached as a screenshot.

Thanks,

Shimon

 
sa825 responded:  2019-08-12

Hi,

Just sending this as a reminder incase you missed my question.

Thanks,

Shimon

 
Brendan MacLean responded:  2019-08-12

Hi Shimon,
First, you should definitely consult the tip that explains how all the peak statistics are calculated:

https://skyline.ms/wiki/home/software/Skyline/page.view?name=tip_peak_calc

Then, you can find your columns in the tree on the left by double-clicking on them, and you will find that "Area" and "Height" are TransitionResult values, which means you will get a row per transition, and you would likely want to include at least the "Transition" column to help understand which transition each "Area" and "Height" value a row is reporting.

If instead you want something that is a "PrecursorResult" value, then you would want something like "Total Area" (or "Total Area MS1") and "Max Height", but you should also note that "Max Height" will not correspond perfectly to what you are seeing in the chromatogram graph you have pictured, which would have to be some new kind of value like "Total Height" (since the chromatogram you see is summed from all the underlying transition chromatograms) and even then our "Height" values are background subtracted, and unfortunately, we don't supply a "Background Height" value. So, it is not actually possible to get at the un-background-subtracted height.

So, it is not at all easy to get something that looks just like the height you see in the total chromatogram graph, which you are showing. But, Precursor Result - Max Height is probably a resonable proxy, which should look like the background subtracted height you see for the most intense transition chromatogram, when you are looking at all the transitions (selecting a single precursor or using Split Graph).

Hope this helps. Sorry for not answering more quickly.

--Brendan

 
sa825 responded:  2019-08-20

Thanks you so much for your response. I have successfully been able to use the Total Area value and Total Area Ratio (Light Total Area/Heavy Total Area) value.

I was wondering if there was a way I could automatically get Skyline to select peptides above or below a particular Total Area Ratio.
For example, could I ask Skyline to only keep peptides that had a Total Area ratio of less than or equal to 0.02? And remove any peptides without this ratio from my protein list?

Thanks,

Shimon

 
sa660 responded:  2019-08-26

Sorry,

Just checking if you saw my last response/question?

Thanks,

Shimon