different heavy IS concentration

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different heavy IS concentration Wael  2020-07-02
 

Dear Skyline team,
as we do multiplexed analysis we might need to adject the amount of spiked IS standard depending on the endogenous peptide level to be within a defined (SIS/EN) range. Is it possible to feed the results grid with different IS standard concentration for each peptide individually, or we should create a different skyline folder for each analyte to be able include the particular SIS concentration?

The issue also applies when creating a standard curve using range of different EN concentrations since the dilution curve concentration range vary depending in the analyte.

Best regards
Wael

 
 
Nick Shulman responded:  2020-07-02
There is a column in the Document Grid called "Concentration Multiplier" which you can set for each peptide.
You can use that in combination with the "Analyte Concentration" that you set on each replicate so that the two numbers multiplied together equals the actual concentration of that peptide in that replicate.

In addition, there's a column called "Dilution Factor" on the Replicate. You can specify the "Dilution Factor" on your Unknown replicates if you had to dilute that particular replicate in order to bring it into the linear range.

Also, if you are using Skyline-Daily, there is a new column called "Explicit Analyte Concentration" on the Peptide Result which you can use to have complete control over peptides, replicates and concentrations, in case your dilution series for each peptide is completely different, and not a simple multiple.
-- Nick
 
Brendan MacLean responded:  2020-07-02
All of what Nick says is true, but there is also a "Peptides > Internal Standard Concentration" value which can be set in the "Peptide Quantification" default report. So, yes, it is peptide-specific. We generally do not recommend using this value for single-point internal standard calibration (analyte concentration = standard concentration * analyte area / standard area). Though, we recognize that many people still do this. So, we made it possible in Skyline.

Instead, we would recommend that you use external calibration with either a multi- (calibration curve) or single-point external calibrator. Look for videos of lectures by Andy Hoofnagle on this, like this one:

https://youtu.be/ZDKon8v9P4Q

With external calibration the internal standard is use for normalization only, and Skyline does not require any knowledge of its concentration. The (analyte area / standard area) ratio is used to calibrate against the external curve or single-point.

So, while I think what you are asking for is possible in Skyline, we would recommend you consider external calibration instead, which is also very flexible, as Nick described.

Thanks for posting to the Skyline support board.

--Brendan
 
Wael responded:  2020-07-02
thanks for the answer and for the suggestions.
Best,
Wael