Deuterium Isotope Effects of Dimethyl labeled proteomes in Capillary Zone electrophoresis and UHPLC analyzed proteomes
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ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Escherichia Coli (ncbitaxon:562)
SUBMITTER: 
Matthew Champion
PROVIDER: MSV000084889 | MassIVE | Mon Feb 03 10:57:00 GMT 2020
REPOSITORIES: MassIVE
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Project description: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.
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Project description:Capillary zone electrophoresis (CZE) is a relatively new serum protein electrophoresis method with higher resolution than other electrophoretic techniques. Hypercholesterolemic dogs exhibit a peculiar CZE pattern. Specifically, they have a shoulder or peak immediately next to the albumin peak. We investigated the prevalence of this spurious peak in hypercholesterolemic dogs and its correlation with the serum cholesterol concentration. Moreover, possible discrepancies between the CZE and spectrophotometric (bromocresol green [BCG] method) albumin concentrations in those animals were evaluated, as well as the accuracy in measuring albumin by a different CZE fractionation system. We retrospectively enrolled 500 hypercholesterolemic and normotriglyceridemic dogs. Each electrophoretic curve was inspected visually to identify a spurious peak (prevalence of 68.8%). We chose 120 dogs to further investigate the albumin concentration; CZE albumin was significantly higher than measured using the BCG method. A weak but significant correlation (r = 0.412; p <0.0001) was observed between the magnitude of the spurious peak and the serum cholesterol concentration. Finally, the significant difference between CZE and BCG albumin measurement disappeared (p = 0.92) when the spurious peak was considered as α1-globulins instead of albumin.
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