Hello all. I have a question which is not limited to Skyline software. However I would appreciate it if you could have a look. I have developed PRM assays targeting of some proteins of interest. The biological sample into which I apply the PRM assays is bronchoalveolar lavage. I have constructed REVERSE calibration curves to determine LOD and LOQ because the majority of the endogenous peptides I look at are present in most of the samples. Basically the heavy peptides are spiked in, in different dilution in the same BAL sample (or in a pool of BAL samples). In that way our calibrator sample have a constant concentration of the light or endogenous peptide while the heavy counterparts are in different concentrations. What we plot is the H/L ratio over the theoretical concentration of heavy peptide spiked in the sample. The paradox is this: While in this way I manage to "see" the heavy up to a concentration of for example 0.05 fmol/uL (or up to an amount of for example 22 fmol) when I apply my PRM to monitor the light peptides in other BAL samples, following the calculations, it seems that the light peptides exist in lower concentrations than this 0.05 fmol/uL (or in a lower amount than this 22 fmol).

This has confused me very much and I don't even know how to look for this in the literature.