The Blank replicates are supposed to have the usual amount of heavy stuff spiked in, and have none of the light analyte.

The data you are showing looks more like what we would call a "Double Blank", which contains neither heavy nor light stuff.
Only the Blank is useful for calculating limits of detection in Skyline. Double Blanks do not provide useful information in Skyline, since you end up dividing by a number which is close to zero, and which also is not a meaningful quantity.

Skyline does require that your chromatograms have reasonably shaped peaks. Another scenario where Skyline does a bad job is "direct infusion", where, instead of having a chromatographic column where your analyte elutes at a particular retention time, your analyte is injected into the mass spectrometer at a constant rate. Skyline does a bad job calculating peak areas in direct infusion experiments, for the same reasons that you are seeing here.

By the way, if you would like to learn more about how Skyline calculates background, there are some diagrams here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tip_peak_calc

-- Nick

Hi Nick,

Thank you for the information that is very helpful. Regarding the 'blank' versus 'double blank'. Does Skyline use the 'blank' and/or the 'double blank' for the 'background' peak area/height calculation that is then substracted from the peak area measurement in our analytes of interest?

-Brianna

No, the blank replicates do not get used for calculating background levels.

When Skyline is calculating the background level for a particular chromatogram peak, the only thing that Skyline is looking at is that particular transition's chromatogram in that particular replicate.
If you select a Transition in the Targets tree, then Skyline will shade a rectangle on the chromatogram graph indicating the background level for that particular transition peak. The background rectangle will be gray, and will have a height which is the lower of the chromatogram intensity at the start and end of the peak integration boundaries.

The thing that Blank replicates can be used for is the "Calculate LOD by" setting at "Settings > Peptide Settings > Quantification". You can tell Skyline that the reported LOD should be "Blank plus (two or three) times the standard deviation".

-- Nick

Hi Nick,

Thank you for the clarification. The reason I was concerned about the background is that based on the provided documentation my peak ratios do not appear consistent with what I would expect them to be visually, so I was curious if the 'blanks' or 'double blanks' were being considered for the 'background'.

I've attached some screenshots here as an example where (1) the L/H ratio seems larger than expected and (2) the background calculated is much larger than I would anticipate.

Whether I use the values provided in my PowerPoint, the 'area' values provided by Skyline, or the 'area' - 'background' I can't reproduce the value Skyline is giving me for L/H ratio.

Thanks for all the help and quick responses!

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 including extracted chromatograms.

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

Hi Nick,

I have gone ahead and attached that here.

When Skyline is calculating peak areas, Skyline slices the chromatogram into a bunch of narrow rectangles, and then adds up all of the chromatogram heights times the width of the rectangle in seconds.

In your screenshot, the background level of the peak that you are integrating for the precursor is 60,000 units. The peak boundaries go from approximately 11.31 minutes to 11.415 minutes. That means the peak width is 6.3 seconds wide.
6.3 times 60,000 is 378000 which is pretty close to the background area in your screenshot which is 372245.

Figuring out the area of the (non-background) peak is a little trickier because that peak has a complicated shape. If that peak were a triangle with a height of 8.8 million and a width of 6.3 seconds, then it would have an area of 27 million. Skyline is saying that the area is 11.6 million, which I guess makes sense since the peak is shaped like a triangle which has been pinched on the sides.

-- Nick

Hi Nick,

Ok that makes sense in terms of the calculations. Thanks for taking a look for me.

Do you have any input on the L/H ratio that it is reporting?

Whether I divide the areas with background subtraction (1) or two reported areas as reported by Skyline (2) I do not get the 6.067 it is reporting. Is there more that goes into that calculation than the area and background area?

(1) (11,587,067 - 372,245) / (2,721,393 - 0) = 4.12
(2) 11,587,067 / 2,721,393 = 4.25

-Brianna

I see that you have "Simple precursor ratios" checked at "Settings > Molecule Settings > Quantification"

When you have "Simple precursor ratios" checked, when Skyline wants to calculate the light to heavy ratio, Skyline adds up all of the light transition peak areas and divides that by the sum of the heavy transition peak areas.
Your light Isoleucine precursor has three transitions: the precursor transition and two fragment transitions. All three of those transitions contribute to the numerator of the light to heavy ratio. The sum of those three transition peak areas is 16.5 million. When you divide that by the heavy total peak area (2.7 million) you get 6.067.

If you were to go to "Settings > Molecule Settings > Quantification" and uncheck "Simple precursor ratios", then Skyline would notice that the two light fragment transitions do not have corresponding heavy transitions, so their peak areas would not be part of the numerator in the ratio calculation. Then you would see that the ratio is 4.25 as you are expecting.
-- Nick