| Skyline to calculate LOD/LOQ from PRM data | floriana | 2025-11-03 03:43 | |||||||||
Hi Skyline team, We are using Skyline to analyze PRM data to build a peptide calibration curve and calculate LOD and LOQ. We spiked a constant amount of heavy into serially diluted light peptide. We also acquired two blanks in which only the heavy peptide is present at the same amount as the serially diluted samples. Our quantification settings are as follows: Regression fit = Linear in log scale, MS2 level, LOD = blank + 2SD, LOQ: Max LOQ bias = 20%, Max LOQ CV =20%. We are using the ratio to heavy from the Peptide Ratio report to build calibration curves in R and calculate LOD and LOQ using blank and regression methods. The LOD/LOQ values that we obtain with our script differ from those calculated by Skyline. Is there a document we can refer to that describes the steps Skyline uses to build peptide calibration curves and assess LOD/LOQ (is skyline using all fragments, are the fragments used always the same in different samples, are ratio L/H ratio calculated first for each transition and then summed, etc)? In case LOD are negative values what is the recommended approach? Many thanks for your support, Floriana |
|||||||||||
| |||||||||||