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.
Highlights
Plant 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
Selection and synthesis of signature peptide standard and isotope‐labeled signature peptide for OsGSTF14 and OsGSTU6 The most critical step of MS-based protein identification and quantitation using tryptic peptides is the selection of suitable tryptic signature peptides
The signature peptides VFGSPTSAEVAR and TPLLAAWAER were confirmed to be absent in the undigested rice sample matrices by mass spectrometry analysis, suggesting that they could be used to quantify OsGSTF14 and OsGSTU6 proteins
Summary
Plant 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. 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. Jain et al [17] showed that OsGSTF5 was AsV stress-induced and differentially expressed in AsV-sensitive and AsV-resistant rice roots during AsV stress, indicating that OsGSTF5 possibly regulates rice tolerance to AsV stress These studies significantly improved our understanding of diversity and functions of individual rice GSTs in response to heavy metal stress at the mRNA transcript level. Direct determination of rice GSTs protein concentrations during Cd exposure is a more effective and reliable approach to explore the possible mechanisms of rice Cd translocation and accumulation
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