Hi Skyline team,

I am struggling to perform absolute quant using Linear fit and 1/x*x weighing using Skyline. And the reason for this is that I am using 1 amino acid (1AA) K/R and 2 amino acids (2AA) (K/R + amino acid after K/R) heavy peptides to establish calibration curve. so I have three peptides os same sequence but three different masses (Endo light, 1AA heavy and 2AA heavy).

I am using 1AA heavy peptide with varying conc. and 2AA heavy peptide as internal standard (fixed conc.) to establish the calibration curve. This is due lack of surrogate matrix for our sample types. I am analysing the sample (unknown) by spiking same conc of 2AA IS and taking the ratio of Light/2AA to calculate the peptide conc. in unknown. All these works I am doing is in excel by exporting the sum transition peak area from Skyline separately and plotting the calibration curve.

The problem on Skyline is:
Skyline is not giving option to select 2AA heavy as IS and taking ratio of 1AA/2AA heavy to plot the curve. How can I modify the setting to achieve 1AA/2AA heavy ratio for calibration curve and Light/2AA ratio for Unknown analysis in Skyline?

Kind regards,