When trying to quantify proteins using IDMS, the recommended method is to use an amino acid containing an isotope in your reference peptide. Can somebody please explain why this step is necessary?
Because you create a chemically identical internal standard in you sample that behaves exactly the same as the endogenous light peptide, but ideally has no or little background and thus allows you to use the linear dynamic range as much as possible. If you only have the regular unlabeled peptide in hand, you can still quantitate using either an external calibration curve or a technique called standard addition, however these moethods will not be as accurate as using an internal heavy standard.
The advantages of using isotopically labeled peptides during isotope dilution mass spectrometry is the fact that these isotopically labeled compounds will have simialr physicochemical properties as the non-labeled counterparts. and that will permit you to quantitate at the same linear range. Also the analysis will be more accurate and precise.
I agree with the comments here. For me the main aspects are:
1. Identification, it is very straightforward to confirm that the signal you are looking at comes really from the peptide you think it is
2. Normalization: You cancel all the variance that is connected with run-by-run differences or even in-run fluctuation. It also makes it easier to compare between experiments performed at different times
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