Hi, thanks for your question! Without knowing anything about the accuracy or precision of your measurements, or if your standards are well calibrated it is hard to say what the cause is. How well do you know the peptide standard concentrations?

That said, even if they were perfect measurements, you shouldn't expect two peptides for the same protein to necessarily give the same quantities. This paper provides a little context on our perspective. https://pmc.ncbi.nlm.nih.gov/articles/PMC8976764/

Any two peptides could be spanning different combinations of proteoforms. We definitely don't expect different peptides from the same protein coding gene to give the same results. I always like this figure of different peptides from Tau (https://pmc.ncbi.nlm.nih.gov/articles/PMC10104800/)

Cheers, Mike