Quantitation of glutathione S-transferases in rice (Oryza sativa L.) roots exposed to cadmium by liquid chromatography–tandem mass spectrometry using isotope-labeled wing peptides as an internal standard

Abstract

BackgroundPlant glutathione S-transferases (GSTs, EC 2.5.1.18) are multifunctional enzymes involved in heavy metal cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione to heavy metals. Previous studies demonstrated that individual rice GSTs were differentially induced by heavy metal exposure at the mRNA transcript level. However, little information is available concerning changes in protein concentration of rice GSTs under heavy metal stress. Because the correlation between changes in protein concentration and gene expression under abiotic stress is poor, direct determination of rice GSTs protein concentrations during cadmium (Cd) exposure is a more effective and reliable approach to explore possible mechanisms of rice Cd translocation and accumulation.ResultsThis study established an optimized and advanced liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based targeted proteomics assay for quantification of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots. The tryptic signature peptides were chosen as surrogate analytes and winged peptides containing the isotope-labeled signature peptides were used as the internal standards. The signature peptides exhibited good linearity in the range of 0.6–60 and 0.3–30 nM, respectively. The limit of detection and limit of quantification were 4.5 and 14.5 µg/g for OsGSTF14, respectively, and 2.1 and 7.0 µg/g for OsGSTU6. The spiking recoveries rates at low, medium and high levels were in the range of 72.5–93.4%, with intra- and inter-day precisions of 5.5–9.1 and 4.2–10.2%, respectively.ConclusionsThe assay successfully quantified the temporal and dose responses of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots, with good accuracy, precision and high-throughput. This assay will have significant application in developing quantification methods of other proteins in Cd-stressed rice, which may provide more insight into the mechanisms of Cd translocation and accumulation in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-017-0214-2) contains supplementary material, which is available to authorized users.

Mass Spectrometry-Based Quantification of Tau in Human Cerebrospinal Fluid Using a Complementary Tryptic Peptide Standard

“…To this end, we introduced flanking residues (3 residues each) preceding and following the tryptic cleavage site to provide context for Tryp-N and Tryp-C enzymes and to assess digestion efficiency. This CompTryp peptide is analogous to “winged” peptides described for Tryp-C standards − and allows monitoring of both complete and partially digested intermediates following Typ-C and Tryp-N digestion (Figure ). The complete digestion efficiency for Tryp-C and Tryp-N was calculated as 99.8% and 94.1%, respectively.…”

Liquid Chromatography-Tandem Mass Spectrometry Analysis of Acetaminophen Covalent Binding to Glutathione S-Transferases

“…GSTs represent major detoxification enzymes with a protein size of ~25 kDa (Eaton and Bammler, 1999) and multifunctional purposes, including induction of microsomal peroxidation (Strange et al, 2000) and heavy metal resistance (Zhang et al, 2013; Cao et al, 2017), as well as possible roles in diseases, such as pulmonary fibrosis (He et al, 2019). Members of cytosolic GSTs tend to dimerize with subunits of the same class, which results in a larger number of enzyme possibilities than genes (Sheehan et al, 2001).…”

Genome-wide identification of the GST gene family and its expression pattern analysis under cold stress in quinoa (Chenopodium quinoa Willd.)

“…and pepper [7]. We hypothesized that gene duplication events occurred mainly in the Tau and Phi subfamilies, probably as a result of their substantial role in the tolerance to biotic and abiotic stresses and speci c environmental factors, including salt tolerance, drought tolerance, tolerance to metal ions, and anthocyanin accumulation [16,40,43,44]. As shown by both Tajima's D neutrality test and the Ka/Ks ratio, the Ka/Ks of duplicated CqGST gene pairs in quinoa GSTs during prolonged evolution was generally less than 1, indicating that the duplicated genes were under purifying selection pressure, which demonstrates the elimination of deleterious duplications and increases the likelihood of fusion of newly duplicated genes.…”