Study of disulfide reduction and alkyl chloroformate derivatization of plasma sulfur amino acids using gas chromatography–mass spectrometry

Abstract

Four disulfide-reducing agents, dithiothreitol (DTT), 2,3-dimercaptopropanesulfonate (DMPS), and the newly tested 2-mercaptoethanesulfonate (MESNA) and Tris(hydroxypropyl)phosphine (THP), were investigated in detail for release of sulfur amino acids in human plasma. After protein precipitation with trichloroacetic acid (TCA), the plasma supernatant was treated with methyl, ethyl, or propyl chloroformate via the well-proven derivatization-extraction technique and the products were subjected to gas chromatographic-mass spectrometric (GC-MS) analysis. All the tested agents proved to be rapid and effective reducing agents for the assay of plasma thiols. When compared with DTT, the novel reducing agents DMPS, MESNA, and THP provided much cleaner extracts and improved analytical performance. Quantification of homocysteine, cysteine, and methionine was performed using their deuterated analogues, whereas other analytes were quantified by means of 4-chlorophenylalanine. Precise and reliable assay of all examined analytes was achieved, irrespective of the chloroformate reagent used. Average relative standard deviations at each analyte level were ≤6%, quantification limits were 0.1-0.2 μmol L(-1), recoveries were 94-121%, and linearity was over three orders of magnitude (r(2) equal to 0.997-0.998). Validation performed with the THP agent and propyl chloroformate derivatization demonstrated the robustness and reliability of this simple sample-preparation methodology.

Redox‐dependent lipoylation of mitochondrial proteins in Plasmodium falciparum

“…The lower activity with TCEP (also observed in Fig. A) probably results from the instability of TCEP in phosphate buffer rather than a redox effect (Hansen and Winther, ; Svagera et al ., ). The other strong reducing agent THP only decreased LipL1 activity relative to DTT when lipoate, but not 6,8‐diClO was used as a substrate, indicating a redox‐specific effect on lipoate itself (compare lanes 3 and 7 from Figs A and C).…”

“…A). The decreased lipoylation of H‐protein in the presence of TCEP as compared to THP is likely due to the reactivity of TCEP in phosphate buffer (Hansen and Winther, ; Svagera et al ., ). In contrast to the first pathway, the lipoylation of BCDH and KDH by the second pathway only occurs in the presence of TCEP or THP (Figs B and http://onlinelibrary.wiley.com/10.1111/mmi.12753/suppinfo).…”

Changes in melanocyte RNA and DNA methylation favour pheomelanin synthesis and may avoid systemic oxidative stress after dietary cysteine supplementation in birds

“…To investigate the effect of experimental treatment on systemic cysteine levels, we measured the levels of cysteine in erythrocytes following the method developed by Švagera, Hanzlíková, Šimek, and Hušek () for plasma. To induce cell lysis and thus facilitate the extraction of intracellular cysteine, erythrocytes were first diluted to 1:10 with a carbonate‐buffered saline (5 m m Na 2 CO 3 in saline).…”

“One-pot” ethyl chloroformate derivatization and liquid-liquid extraction of reduced glutathione in erythrocyte and its quantitative GC–MS analysis

“…In the present study, the instrumental conditions to detect and quantify GSH ethyl chloroformate derivative in erythrocytes with the highly-specific mass detector coupled with gas chromatography (GC-MS) were investigated. Furthermore a novel "one-pot" derivatization and LLE procedure was developed for GSH in erythrocytes, in accordance to P. Šimek and his coworkers' studies on chloroformates [28][29][30][31].…”