There is a column that you can edit on the Document Grid called "Concentration Multiplier" in order to specify, for each Peptide, what the
"Analyte Concentration" of the replicate should be multiplied by so that it accurately represents the concentration of that Peptide in that standard.
Typically, you would set the "Concentration Multiplier" for each peptide to the concentration in the highest concentration external standard. Then, you would set the "Analyte Concentration" of that external standard Replicate to "1" and the other external standards would have lower Analyte Concentrations.

The Units that you specify on the Quantification tab at "Settings > Peptide Settings" can be any text that you want. Skyline does not interpret that text, but displays it next to certain calculated values.
-- Nick

Thank you!

I have attached few screenshots, could you please have a look!
Just want to make sure.
I am using the multiplier, so do i still have to use the molar mass of the peptide to express the results in molar terms (i.e., nM or pmol/mL)?

Thank you.

It might be helpful if you could 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 see that you have set "Standard Type" to "Surrogate Standard" in that first screenshot.
You would typically only do that if there is another molecule in the document whose Normalization Method is "Ratio to <that surrogate standard>".
You can read about Surrogate Standards here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=Surrogate%20Standards

There are two different ways of doing absolute quantification in Skyline.
One way is to have external standards of known concentration. You use the "Analyte Concentration" and "Concentration Multiplier" fields to tell Skyline what the concentration of your analyte is in the external standards (i.e. calibration samples). Skyline tells you what the concentration is in your unknown samples by comparing the signal in the external standards with the signal in the unknown samples. If you are using an internal standard for normalization, the internal standard needs to be spiked in at the same concentration in both the external standards and the unknown samples.
In this case, the actual concentration of the internal standard is irrelevant because it appears in the denominator of the normalized area in both the external standard and the unknown samples.

The other way to do absolute quantification is without any external standards and just an internal standard. In this case, you would tell Skyline the concentration of the internal standard by specifying the "Internal Standard Concentration" value. In this case, the Calculated Concentration ends up being the Normalized Area times the Internal Standard Concentration value (where the "Normalized Area" is the ratio of the Light area to the Heavy area because of the "Normalization Method" chosen on the Quantification tab at "Settings > Peptide Settings").

If there are any external standards in your document then Skyline assumes you want to calculate concentrations using the calibration curve so the "Internal Standard Concentration" value ends up being ignored.

I am not sure I understand your original question. It might make more sense after I see your Skyline document.
-- Nick

Hi Nick,

So based on the calibration curve, I have spike fixed amount of heavy labelled peptides to my CSF samples. So my Control and disease CSF is constant and I have spiked 128fmol/ul of heavy labelled peptide of my select protein to all the samples.
I have attached my file. Please have a look.

Thank you.

As my file size was big, I have uploaded inthe larger file section. Please have a look.

Thank you for sending that Skyline document.
I see that you do not have any external standards so Skyline is calculating the concentration by multiplying the light:heavy peak area ratio by the Internal Standard Concentration that you have specified (i.e. 128).
Skyline tells you that the slope of the calibration curve is 7.8125E-E which is 1 divided by 128, which makes sense.

Yes, I would expect that the numbers that you see for the calculated concentration for your peptide will be the right numbers in fmol/ul.
The reason for this is that you spiked your internal standard in at 128fmol/ul, and you would expect that the light and heavy peptides would produce the same amount of signal. Therefore, multiplying the light:heavy peak ratio area ratio by 128 should give you the light concentration in fmol/ul.
-- Nick

Thank you Nick!

As I have 3 peptides corresponding to 1 protein, do you think for protein level quant, I can simply average the values of 3 peptides from the quantification data. I know Skyline does that too. But if I want to calculate the amount of protein per patient wise; can I simply average the 3 peptide quant or shall Add the values in the excel after exporting all peptide level quant from Skyline?
Also shall i consider the Molar mass of peptides (g/mol) for further calculation as I believe, I can directly pick the results provided by Skyline in the quant tabel.

Thank you.