Stable heavy-isotope labeling is commonly used in quantitative proteomics. Several common techniques incorporate deuterium (2 H) as the heavy isotopic label using reductive amination with formaldehyde. Compared with alternatives, dimethyl labeling reagents are inexpensive and the labeling chemistry is simple and rapid. However, the substitution of hydrogen by deuterium can introduce subtle changes in peptides' polarities, leading to a shift in chromatographic retention times between deuterated and nondeuterated peptides that can lead to quantification deviations. Capillary zone electrophoresis has emerged as a complementary separation for ESI-MS-based proteomics, including targeted and quantitative approaches. The extent to which the deuterium isotope effect impacts CZE-based proteomics, which separates peptides based on their S/N ratios, has not been investigated. To address this issue, CZE was used to analyze dimethyl labeled E. coli tryptic digests in 100 min single-shot analyses. The median migration time shift was 0.1 s for light versus heavy labeled peptides, which is 2.5% of the peak width. For comparison, nUHPLC-ESI-MS/MS was used to analyze the same sample. In UPLC, deuterated peptides tended to elute earlier than nondeuterated peptides, with a retention shift of 3 s for light versus heavy labeled peptides, which is roughly half the peak width. This shift in separation time did not have a significant effect on quantitation for either method for equal mixing ratios of the light-intermediate-heavy isotope labeled samples.
Keywords: Capillary zone electrophoresis; Dimethyl labeling; Isotope effect; Nanospray interface for capillary electrospray; Protein quantification.
© 2020 The Authors. Electrophoresis published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.