This is what I did:
I serially diluted my protein of interest in human serum. Concentrations were measured with an immunoassay. The protein was then captured using immunoprecipitation. Eluates were separated by SDS-PAGE. In-gel digestion was performed and several peptides of the protein were relatively quantified using a targeted-SIM method.
I expected a linear relation between the protein concentration and the AUC of my precursor chromatograms. Instead, I got a quadratic relationship (see attachment, note that both axes are log-transformed). Does anyone have an explanation for this or recognise this? Was I wrong in expecting a linear relation or do you believe something might have gone wrong experimentally?
Hi Alexander,
I agree with Tjorben. Which instrument are you using? It seems to me that 1000000 is too much concentration for target SIM...
Non-linearity may arise in the immunoprecipitation and/or in-gel digestion steps, too.
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