LC–MS/MS Analysis of Thiol-Containing Amino Acids in Exosomal Fraction of Serum

Abstract It has been suggested that thiol-containing amino acids could be used as biomarkers for diseases associated with oxidative stress. We investigated the thiol-containing amino acids, homocysteine (Hcy), cysteine (Cys), glutathione (GSH) and γ-glutamylcysteine (γ-GluCys), in commercial human serum by using liquid chromatography–tandem mass spectrometry (LC–MS/MS) after precolumn derivatization with 4-fluoro-7-sulfobenzofurazan. This method was applied to determine the composition of thiol-containing amino acids in exosomes prepared from the serum. Hcy, Cys, GSH and γ-GluCys could be detected in the exosomal fraction, and the ratio of each thiol-containing amino acid was similar to those in the corresponding native serum. Cys (94.76%) was most enriched in the exosomal fraction, followed by GSH (2.97%), γ-GluCys (1.59%) and Hcy (0.68%). These findings suggest that thiol-containing amino acids, Hcy, Cys, GSH and γ-GluCys, are included in exosomes in human serum.

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Validation of a rapid, comprehensive and clinically relevant amino acid profile by underivatised liquid chromatography tandem mass spectrometry

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2019-0604 ◽

2020 ◽

Vol 58 (5) ◽

pp. 758-768 ◽

Cited By ~ 1

Author(s):

Rachel S. Carling ◽

Kate John ◽

Richard Churchus ◽

Charles Turner ◽

R. Neil Dalton

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Liquid Chromatography ◽

Amino Acid ◽

Amino Acid Profile ◽

Ion Pair ◽

Analysis Time ◽

Tandem Mass ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

AbstractBackgroundQuantification of plasma amino acids is key to the diagnosis of inherited defects of amino acid synthesis, catabolism and transport, many of which present as clinical emergencies. The utility of this test is limited by the long analysis time and subsequent inability of laboratories to provide results in real-time. Traditionally, analysis has been performed by ion exchange chromatography (IEC) but recently there has been a move towards liquid chromatography tandem mass spectrometry (LC-MS/MS) which provides the potential for faster analysis. However, the necessity to derivatise the sample and/or utilise an ion-pair reagent, combined with lack of commercially available stable isotope internal standards (IS) has prevented laboratories fully exploiting the benefits of this methodology. We describe an underivatised LC-MS/MS method enabling patient results to be reported with an improved turnaround time (<1 h).MethodsMethanolic IS was added to plasma (10 μL) to precipitate protein. Following centrifugation amino acids were analysed by LC-MS/MS using selected reaction monitoring (SRM) for each analyte and corresponding IS.ResultsPatient samples (n = 57) and external quality assessment (EQA) material (n = 11) were analysed and results compared with IEC. Comparable accuracy and precision were obtained with 15-min analysis time.ConclusionsThis method enables the analysis of a clinically comprehensive amino acid profile without the need for derivatisation/ion-pair reagents and benefitting from improved analytical quantitation through multipoint calibration and use of stable isotope IS. The analysis time is fast in comparison to IEC, improves efficiency of laboratory workflow and enables stat analysis of clinically urgent samples.

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