Quantitative analysis of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells using UHPLC-MS/MS

Published on May 27, 2021in Journal of Separation Science2.878
· DOI :10.1002/JSSC.202001266
Jonathan B. Thacker (UM: University of Michigan), Jonathan B. Thacker5
Estimated H-index: 5
(UM: University of Michigan)
+ 0 AuthorsSubramaniam Pennathur45
Estimated H-index: 45
(UM: University of Michigan)
γ-Glutamylpeptides have been identified as potential biomarkers for a number of diseases including cancer, diabetes, and liver disease. In this study, we developed and validated a novel quantitative analytical strategy for measuring γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline, all of which have been previously reported as potential biomarkers for prostate cancer, in HeLa cells using UHPLC-MS/MS. A BEH C18 column was used as the stationary phase. Mobile phase A was 99:1 water:formic acid, and mobile phase B was acetonitrile. Chemical isotope labeling using benzoyl chloride was used as the internal standardization strategy. Sample preparation consisted of the addition of water to a frozen cell pellet, sonication, derivatization, centrifugation, and subsequent addition of an internal standard solution. The method was validated for selectivity, accuracy, precision, linearity, and stability. The determined concentrations of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells were 1.92 ± 0.06, 10.8 ± 0.4, and 1.96 ± 0.04 pmol/ mg protein, respectively. In addition, the qualitative analysis of these analytes in human serum was achieved using a modified sample preparation strategy. To the best of our knowledge, this is the first report of the use of benzoyl chloride for chemical isotope labeling for metabolite quantitation in cells. This article is protected by copyright. All rights reserved.
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