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.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url

I am not sure why you would be seeing NaN as the slope and intercept for the calibration curve that Skyline has calculated. "NaN" stands for "Not a number" and it is the value that the computer returns when doing things like dividing by zero. Probably one of the standards is causing an infinity to be part of the linear regression, which results in the NaN result. I do not know where that infinity might be coming from. One common source of infinity would be if you specified "0" as the analyte concentration of one of the Standards and had chosen "1/x" weighting. Skyline is supposed to notice that and just exclude that particular standard. Another source of infinity would be if you are doing ratio to heavy normalization and the heavy area is zero. I thought Skyline would also exclude infinities caused by that too.

After I see your Skyline document I will probably be able to tell you what is going wrong.

The most common settings for calibration curves is "Linear" regression fit and "1/(x*x)" weighting like you have.

"Bilinear" is a bit of a novel technique that Skyline does where the lower part of the calibration curve is horizontal in order to better model data below the limit of detection. You can read about that in this paper:
https://pubmed.ncbi.nlm.nih.gov/32037841/

If you want to calculate limit of detection, there are two techniques which Skyline offers.
If you have multiple blank samples, then you can use either the Blank plus two standard deviations or Blank plus three standard deviations options for "Calculate LOD By" on the Quantification tab at "Settings > Peptide Settings".
Note that if you have some standards whose analyte concentration is zero, their sample type should be specified as "Blank" not "Standard".

If you do not have blank samples then you could use "Bilinear" as the regression fit and choose "Bilinear turning point" for the LOD calculation. This will cause the LOD to be reported as the point where the piecewise linear calibration curve switches from being horizontal to having a positive slope.
-- Nick

Thank you Nick for the reply. I have shared the skyline zip file in the folder with the file name "CC-Skyline-PRM_10282024 - Copy.sky.zip". Can you have a look into it and provide any suggestions on how to deal with those peptides?

Hi Nick,

I am not getting LOD value as well. Please help. I have already shared the file.

Thank you.
Akhila

Sorry for the slow response.

The heavy and light precursors for VFSNGADLSGVTEEAPLK have a different set of transitions.
When Skyline calculates the ratio of light to heavy, only those transitions that are in common between the light and heavy precursors get used, so that's y10, y4, y3, y1 and b2.
Also, because the "MS Level" at "Settings > Peptide Settings > Quantification" is set to "2", the precursor peak areas are not part of the denominator.
The following replicates end up having zero heavy MS2 peak area which is what causes the NaN values in the calibration curve:
CC_CRC_Std1_R1
CC_CRC_Std1_R2
Usually you can right-click on a standard point in the Calibration Curve window and choose "Exclude Standard". Unfortunately, because the value for these points is not a number, there is nothing to click on in the Calibration Curve window. One thing that you could do is right-click on the chromatogram graph for these two replicate and choose "Remove Peak".
-- Nick

Thank you, Nick. It worked. Where do we get the MS2 peak area? I need to check a few other peptides that have the same issue.

Still I am not getting LOD values. Is it because I have imported single blank raw file and have selected Blank plus 2 * SD in the quantification settings? What can be done if I have single blank raw file?

A different way to have Skyline calculate limit of detection is to choose "Bilinear" as the "Regression fit" at "Settings > Peptide Settings > Quantification".
When the Regression Fit is "Bilinear" you will be able to choose "Bilinear turning point" for "Calculate LOD by:".

With the Bilinear regression method, Skyline fits a curve to the data which consists of a horizontal portion through the points below the limit of detection and a upward sloping linear region through the rest of the points.
-- Nick

Hi Nick,

Thank you for the reply. Can you also let me know how to get the MS2 peak area?