Endogenous peptide overlaps with minor peak of heavy

Endogenous peptide overlaps with minor peak of heavy jjotto  2019-03-26


I am using a heavy-labeled peptide (13C(6)15N(2) on Lysine). The neat heavy-labeled peptide shows multiple peaks all within a few minutes of each other with one major peak. I believe some of the peaks after the main peak are the result of deamidations. (the peptide is not ideal because of these modifications, but it has been selected for other reasons) There are also a few minor peaks before the major peak. When spiking the heavy-labeled peptide into real samples, the endogenous peptide seems to consistently overlap with a minor peak before the major peak of the heavy-labeled peptide. Other spiked heavy peptides and endogenous peptides within the same samples show very clear and complete overlap. I am trying to compare the endogenous peak area to the known amount of spiked heavy peptide, but this is causing problems. I'm attaching a few images that I think will help illustrate what I'm seeing. Any help would be appreciated.

Thank you