Bias due to isotopic incorporation in both relative and absolute protein quantitation with carbon-13 and nitrogen-15 labeled peptides

Mass spectrometry provides considerable benefits over other analytical techniques due to advantages imparted by the use of isotopically labeled internal standards. In some analytical approaches, the internal standard provides reference points for quantitative data reduction. However, there is an important phenomenon that occurs in the development of an internal standard, namely the distribution of naturally occurring isotopes is altered by artificial labeling. As a result, the number of molecules measured in each isotopic state (e.g., +0, +1, +2) varies between the manufactured internal standard and the naturally occurring unmodified analyte. This can create inaccuracies that are a function of the abundance, type and location of the isotopic labeling when internal standards act as the calibration material. In this study, theoretical examples calculated with naturally occurring isotopic incorporation, manufactured isotopic enrichment, and experimental data from comparative analysis of naturally and artificially labeled peptides were explored to demonstrate the variation in measurement between analytes and their stable, isotopically labeled internal standards.