Premium
Quantitative analysis of γ‐glutamylisoleucine, γ‐glutamylthreonine, and γ‐glutamylvaline in HeLa cells using UHPLC‐MS/MS
Author(s) -
Thacker Jonathan B.,
He Chenchen,
Pennathur Subramaniam
Publication year - 2021
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.202001266
Subject(s) - chromatography , chemistry , formic acid , benzoyl chloride , sample preparation , hela , derivatization , analyte , quantitative analysis (chemistry) , mass spectrometry , tandem mass spectrometry , high performance liquid chromatography , biochemistry , cell , medicinal chemistry
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 ultra‐high‐performance liquid chromatography‐tandem mass spectrometry. 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.