calibration curve with spiked ISTD for multiple peptides DeWi  2019-03-29

Hey there,

I'm quite new in working with skyline.
I've got an issue or I didn't get how it works with skyline, however:
I tried to create my calibration curves with spiked ISTD (5nM) of 18 pepiteds ( à 3 Transitions) simultaneously.
So, I already found the option to define the calibration points of only one peptide in the menu View-> document grid-> replicates.
And the other option to define the spiked-in concentration of the ISTD for all peptides in the documentation grid -> Peptide Quantification.
But I have not found a solution where I can definde both (and for all peptides instead of just one, because every peptide has a slightly different concentration in each calibration point).

Is this possible to do both with skyline?
Maybe you can help me.

Thank you!


Brendan MacLean responded:  2019-03-29

Hi Denise,
Yes, it is possible. Generally, you would use the Concentration Multiplier property in the Peptide Quantification view in the Document Grid. As long as you created your response curve as a single dilution series from a set of starting concentrations this should work just fine. If you like then, you can just use 1 as the Replicate > Analyte Concentration for your most intense concentration and then use fractions for the dilutions you made, like 0.5, 0.25, 0.125, etc. Then you can just enter the starting concentration for each peptide as its Concentration Multiplier.

I think we also made it possible to achieve the case where you had multiple dilution series that you mixed together but I can't remember the details of that, and it is much less common. Please let me know if this is what you have done or whether the single dilution of a mix of different concentrations solves your case as it has for most people.

Thanks for posting to the Skyline support board. We hope you will enjoy using Skyline as you learn more.


DeWi responded:  2019-04-01

Hey Brendan,
thanks for your response.

Unfortunately I'm really a beginner and have 2 more questions.

1.)Is it possible to combine 2 calibration curves? I have one stock solution for the lowest concentrations and one for the highest. Now I understand how to generate the curves separately, but it would be more practically if there is an option to combine them, although they have 2 different stock solutions.

Further, I just want to be sure how skyline is programmed for the dilution series.
Is the dilution factor always be based on the stock solution (so all 10 concentrations are diluted directly from the stock) or recognized skyline the order of the concentrations and can calculate the factor between dilution3 and dilution4, like the series is generally diluted?

For Example: 1mg/mL stock solution and solutions of 0.5mg/mL and 0.25 mg/mL should be made with a Volume of 200µL
First: 100µL of the stock solution + 100µL water (solution1). Factor = 0.5 this is clear.
Now, what factor does skyline need: 100µL of the solution1 +100µL water (solution 2). factor= 0.5 (then you have for the concentration Multiplier column 0.5, 0.5)
50µL from the stock solution + 150µL water. factor= 0.25 then you have for the concentration Multiplier 0.5, 0.25 ?

Hope you can understand what I mean.

Thank you & best regards from Germany

DeWi responded:  2019-04-08

Hey Brendan,

I'm really interested in the answers, especially for the first one but also for the 2nd.

Thank you & best regards


Nick Shulman responded:  2019-04-09
Hi, Denise,

I am not sure that I understand your question, but I will try to answer it.

For your external standards, where you are spiking in a known amount of the analyte, you should not use the Dilution Factor. The Dilution Factor is only useful on unknown samples, where you had to dilute the sample a certain amount in order to bring the measurement into the linear range of your mass spectrometer.
For external standards, you do not need to specify a Dilution Factor, since you are already specifying the Analyte Concentration.

I might be able to answer your questions better if you send us 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 including extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request.
Otherwise, you can upload it here:
-- Nick
DeWi responded:  2019-04-09
Hey Nick,

I'm sorry, but in the next days I'm not able to send you the data.

Let me try to explain it another way.

I have two separate stock solutions for the same substances for the calibration curve (each point have 18 peptides + 18 ISTDs รก 3 Transitions). One for the very lowest concentrations and the other for very high concentrations. The first question is, if it's possible to combine these 'two calibration curves' in skyline.
Each calibration point have a spiked-in ISTD of 5nM, as well as the unknown samples have.

In the software I used so far it was possible to define the start concentration for each substance in the highest calibration point, as well as the concentration of the ISTDs.
How Brendan explained, it is possible to define both in skyline - see above.
But I want to know if skyline is able to recognize the dilution factor like a normal dilution series is made or if you have to calculate the factor from the stock solution. See picture.

Best regards
Nick Shulman responded:  2019-04-10
The dilution factor would be the factor from your starting stock solution.

For your other question, it sounds like you have two different stock solutions, and in each of those stock solutions the ratio of the peptides to each other is not the same. For this reason, you are unable to tell Skyline what amount of each peptide is in each external standard, since all that Skyline allows you to specify is the Analyte Concentration on the replicate, and the Concentration Multiplier on the peptide.

Several people have asked for the ability to specify this "full matrix" of peptide concentrations across several peptides and several replicates. So far, we have not implemented this, because no one has sent us a dataset that required this. If this is what you need and you can send me your dataset I can try to implement this in a future version of Skyline.
-- Nick
DeWi responded:  2019-04-14
Hey Nick & Brendan,

"it sounds like you have two different stock solutions, and in each of those stock solutions the ratio of the peptides to each other is not the same"

Give me some days and I will check if its possible to make some special dataset just for you. Maybe it will be a bit smaller, actual there are 18 Peptides + their ISTD in one calibration point.

To whom should I send the dataset?

Maybe you can also check if it's not possible to add the function to combine two calibration curves for the same substances in two different ranges.

Best regards

Nick Shulman responded:  2019-04-15
You can upload files here:

Are you saying that you are doing a single point calibrator? If you have a single point calibrator, then you do not need any extra features in Skyline. You would set the analyte concentration of your external standard replicate to 1, and you would set the Concentration Multiplier of each of the peptides to whatever number that it needs to be.

It's only if you have multiple calibration points where each calibration point is a different ratio of your two different stock solutions where you would need to be able to specify the "full matrix" of peptide concentrations versus replicate.
-- Nick