Fractionation of Isotopically Labeled Peptides in Quantitative Proteomics

The goal of quantitative proteomics is to examine the expression levels of all of the proteins in a biological system and recognize those that change as a function of some stimulus. Quantification is now frequently based on derivatization of peptides with isotopically distinguishable labeling agents. This study examines the extent to which isotopic forms of peptides having the same amino acid sequence are resolved by reversed-phase chromatography and assesses the degree to which resolution of these isotopically different forms of a peptide impact quantification. Three derivatizing agents were examined, the d 0 and d 3 forms of N-acetoxysuccinimide, the d 0 and d 4 forms of succinic anhydride, and the d 0 and d 8 forms of the commercial ICAT reagent. Peptide mixtures from control and experimental samples were derivatized individually, mixed, subjected to reversed-phase chromatography, and analyzed by ESI-MS. When partial resolution of the isotopic forms of a peptide occurs, the largest error in assessing the true isotope ratio in a sample occurs when sampling at the extremes of a peak. Early in the elution of a peak, the sample will be enriched in the deuterated species, whereas the opposite is true at the tailing edge of a peak. Acetylated peptides showed the lowest degree of separation. Resolution of the deuterated and nondeuterated forms in this case was 0.023. This amounts to slightly over a 1-s difference in their peak maxima and can cause a typical error of ±6% at the leading and tailing edges of a peak. In contrast, resolution of the deuterated and nondeuterated forms of the ICAT reagent were calculated to be 0.45. This means that in a peak of 1-min width (W1/2), the peak maxima will vary by ∼ 30 s, and measurement errors of −83 and +500% can occur at the leading and tailing edges of a peak. It is concluded that resolution of isotopic forms of a peptide can cause substantial quantification errors in quantitative proteomics.