High‐throughput screening for glutathione conjugates using stable‐isotope labeling and negative electrospray ionization precursor‐ion mass spectrometry

Liao, Simeng; Ewing, Nigel P.; Boucher, Brian; Materne, Olivier L.; Brummel, Christopher L.

doi:10.1002/rcm.6135

Cited by 12 publications

(13 citation statements)

References 23 publications

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“…The methanolic extraction procedure used in our study favors the recovery of polar sulfur metabolites, allowing detection and analysis of GSH and related compounds (Liao et al ., ; Krajewski et al ., ). There are approximately 300 sulfur metabolites assigned to the Arabidopsis metabolome in the KNApSAcK, PlantCyc and KEGG databases.…”

Section: Discussionmentioning

confidence: 99%

Exploring the Arabidopsis sulfur metabolome

et al. 2013

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SUMMARYSulfur plays a crucial role in protein structure and function, redox status and plant biotic stress responses. However, our understanding of sulfur metabolism is limited to identified pathways. In this study, we used a high-resolution Fourier transform mass spectrometric approach in combination with stable isotope labeling to describe the sulfur metabolome of Arabidopsis thaliana. Databases contain roughly 300 sulfur compounds assigned to Arabidopsis. In comparative analyses, we showed that the overlap of the expected sulfur metabolome and the mass spectrometric data was surprisingly low, and we were able to assign only 37 of the 300 predicted compounds. By contrast, we identified approximately 140 sulfur metabolites that have not been assigned to the databases to date. We used our method to characterize the c-glutamyl transferase mutant ggt4-1, which is involved in the vacuolar breakdown of glutathione conjugates in detoxification reactions. Although xenobiotic substrates are well known, only a few endogenous substrates have been described. Among the specifically altered sulfur-containing masses in the ggt4-1 mutant, we characterized one endogenous glutathione conjugate and a number of further candidates for endogenous substrates. The small percentage of predicted compounds and the high proportion of unassigned sulfur compounds identified in this study emphasize the need to re-evaluate our understanding of the sulfur metabolome.

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Section: Discussionmentioning

confidence: 99%

Exploring the Arabidopsis sulfur metabolome

et al. 2013

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“…The total ion chromatograms (TIC), obtained from the screening scans mentioned above, were filtered for the isotopic pattern of 1:1 abundance of ion pairs with a mass difference of 3 Da, using the elemental targeting feature in Analyst software (AB Sciex, Framingham, MA) (Liao et al, 2012; Mutlib et al, 2005). …”

Section: Methodsmentioning

confidence: 99%

Irreversible binding of an anticancer compound (BI-94) to plasma proteins

et al. 2015

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1. We investigated the mechanisms responsible for the in vivo instability of a benzofurazan compound BI-94 (NSC228148) with potent anti-cancer activity. 2. BI-94 was stable in MeOH, water, and in various buffers at pHs 2.5–5, regardless of the buffer composition. In contrast, BI-94 was unstable in NaOH and at pHs 7–9, regardless of the buffer composition. BI-94 disappeared immediately after spiking into mice, rat, monkey, and human plasma. BI-94 stability in plasma can be only partially restored by acidifying it, which indicated other mechanisms in addition to pH for BI-94 instability in plasma. 3. BI-94 formed adducts with the trapping agents, glutathione (GSH) and N-acetylcysteine (NAC), in vivo and in vitro via nucleophilic aromatic substitution reaction. The kinetics of adduct formation showed that neutral or physiological pHs enhanced and accelerated GSH and NAC adduct formation with BI-94, whereas acidic pHs prevented it. Therefore, physiological pHs not only altered BI-94 chemical stability but also enhanced adduct formation with endogenous nucleophiles. In addition, adduct formation with human serum albumin-peptide 3 (HSA-T3) at the Cys34 position was demonstrated. 4. In conclusion, BI-94 was unstable at physiological conditions due to chemical instability and irreversible binding to plasma proteins.

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“…Therefore, assays, workflows, instrument methods, and softwareaided approaches have been published and have proven to be useful tools to assess reactive metabolites. [14][15][16][17][18][19][20][21][22] One recent publication by Zhu et al 19 described the use of high-resolution mass spectrometry (HRMS), LTQ Orbitrap Velos, to monitor m/z 272.0888 (deprotonated γ-glutamyl dehydroalanyl-glycine) through extraction of product ion (XoPI) under in-source collision-induced dissociation (SCID) in negative mode. This method was able to achieve high sensitivity by extraction of product ion at exact mass 272.0888 ± 5 ppm, which was generated from select ion monitoring (SIM) at narrow mass range (m/z 269.5 to 274.5 Da), as negligible interference were observed at the same mass range with non-selective CID in the negative mode.…”

Section: Introductionmentioning

confidence: 99%

A high‐throughput glutathione trapping assay with combined high sensitivity and specificity in high‐resolution mass spectrometry by applying product ion extraction and data‐dependent neutral loss

et al. 2019

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Reactive metabolites are thought to play a pivotal role in the pathogenesis of some drug‐induced liver injury (DILI) and idiosyncratic adverse drug reactions (IADRs), which is of concern to patient safety and has been a cause of drugs being withdrawn from the market place. To identify drugs with a lower propensity for causing DILI and/or IADRs, high‐throughput assays to capture reactive metabolites are required in pharmaceutical industry for early drug discovery risk assessment. We describe the development of an assay to detect glutathione adducts with combined high sensitivity, enhanced specificity, and rapid data analysis. In this assay, compounds were incubated with human liver microsomes and a mixture of 1:1 of GSH (γ‐GluCysGly): GSX(γ‐GluCysGly‐13C215N) in a 96‐well plate format. UPLC‐UV and LTQ Orbitrap XL were employed to detect GSH‐adducts using the following mass spectrometry setups: (a) selected ion monitoring (SIM) at m/z of 274 ± 3 Da in negative mode with in‐source fragmentation (SCID), which enables simultaneously monitoring two characteristic product ions of m/z 272.0888 (γ‐glutamyl‐dehydroalanyl‐glycine) and 275.0926 (γ‐glutamyl‐dehydroalanyl‐glycine‐13C215N); (b) full scan mode for acquisition of exact mass of glutathione adducts; (c) data‐dependent MS2 scan through isotopic matching (M:M + 3.00375 = 1:1) for monitoring neutral loss fragments (144 Da from dehydroalanyl‐glycine) and for structural information of glutathione adducts. This approach was qualified using eight compounds known to form GSH conjugates as reported in the literature. The high sensitivity and specificity were demonstrated in identifying unique CysGly adducts in the case of clozapine, diclofenac, and raloxifene and in identifying GSH‐adducts of fragmented parent molecules in the case of amodiaquine and troglitazone. In addition, LC‐UV chromatograms in the presence or absence of GSH/GSX allowed for identification of the rearranged glutathione adducts without aforementioned characteristic fragment ions. Implement of this assay in drug discovery small molecule programs has successfully guided drug design.

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High‐throughput screening for glutathione conjugates using stable‐isotope labeling and negative electrospray ionization precursor‐ion mass spectrometry

Cited by 12 publications

References 23 publications

Exploring the Arabidopsis sulfur metabolome

Exploring the Arabidopsis sulfur metabolome

Irreversible binding of an anticancer compound (BI-94) to plasma proteins

A high‐throughput glutathione trapping assay with combined high sensitivity and specificity in high‐resolution mass spectrometry by applying product ion extraction and data‐dependent neutral loss

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