Proteomics tools reveal startlingly high amounts of oxytocin in plasma and serum

The neuropeptide oxytocin (OT) is associated with a plethora of social behaviors, and is a key topic at the intersection of psychology and biology. However, tools for measuring OT are still not fully developed. We describe a robust nano liquid chromatography-mass spectrometry (nanoLC-MS) platform for measuring the total amount of OT in human plasma/serum. OT binds strongly to plasma proteins, but a reduction/alkylation (R/A) procedure breaks this bond, enabling ample detection of total OT. The method (R/A + robust nanoLC-MS) was used to determine total OT plasma/serum levels to startlingly high concentrations (high pg/mL-ng/mL). Similar results were obtained when combining R/A and ELISA. Compared to measuring free OT, measuring total OT can have advantages in e.g. biomarker studies

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

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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/169280/1/jssc7308.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/169280/2/jssc7308_am.pd