CysQuant: simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)
TISSUE(S): Plant Cell, Shoot
SUBMITTER: 
Patrick Willems
LAB HEAD: Kris Gevaert
PROVIDER: PXD043895 | Pride | 2023-07-21
REPOSITORIES: Pride
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2018-03-05 | PXD007278 | Pride
Project description:Most of the redox proteomics strategies are focused on the identification and relative quantification of cysteine oxidation changes without considering the variation in the total levels of the proteins. However, the regulation of protein synthesis and protein degradation are also associated with many of the regulatory mechanisms of the cells, being therefore important to consider the changes in total protein levels in the Post Translational Modifications (PTMs) focused analyses, such as cysteine redox characterization. This aspect was only address in the cysTMTRAQ method, which combines two types of isobaric tags in the same experiment leading to a considerable increase in the costs of the analysis. Therefore, a novel integrative approach combining the SWATH-MS method with differential alkylation using non-isotopically labeled alkylating reagents (oxSWATH) is presented, thus integrating the information regarding relative cysteine oxidation with the comparative analysis of the total protein levels in a cost effective highthrouput approach. The proposed method was tested using a redox regulated protein, and further applied to a comparative analysis of secretomes obtained under control or oxidative stress conditions to strengthen the importance of considering the changes in protein total levels. OxSWATH allowed to determine the relative proportion of reduced and reversible oxidized oxoforms of the proteins, and by considering total protein levels, to determine the total levels of each fraction, which are then used for comparative analysis. This approach can be an important tool in redox centered approaches, being able to not only analyze the overall reduce/reversible oxidized state of proteins but able to be further applied in the characterization of the different oxoforms of the individual cysteines. Moreover, since samples are acquired in SWATH-MS mode, besides the redox centered analysis, a generic differential protein expression analysis can be also performed, allowing a truly comprehensive evaluation of proteomics changes upon oxidative stimulus.
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Project description:Nonsteroidal anti-inflammatory drugs (NSAIDs), including salicylic acid (SA), target mammalian cyclooxygenases. In plants, SA is a defense hormone that regulates NON-EXPRESSOR OF PATHOGENESIS RELATED GENES 1 (NPR1), the master transcriptional regulator of immunity-related genes. We identify that the oxicam-type NSAIDs tenoxicam (TNX), meloxicam, and piroxicam, but not other types of NSAIDs, exhibit an inhibitory effect on immunity to bacteria and SA-dependent plant immune response. TNX treatment decreases NPR1 levels, independently from the proposed SA receptors NPR3 and NPR4. Instead, TNX induces oxidation of cytosolic redox status, which is also affected by SA and regulates NPR1 homeostasis. A cysteine labeling assay reveals that cysteine residues in NPR1 can be oxidized in vitro, leading to disulfide-bridged oligomerization of NPR1, but not in vivo regardless of SA or TNX treatment. Therefore, this study indicates that oxicam inhibits NPR1-mediated SA signaling without affecting the redox status of NPR1.
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Project description:Long wavelength Ultraviolet (UVA-1) radiation causes oxidative stress that leads to the formation of noxious substances within the skin. As a defensive mechanism skin cells produce detoxifying enzymes and antioxidants when they detect modified molecules. We have recently shown that UVA-1 irradiation oxidizes the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the synthesis of the stress response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1 and (UV-) oxidized phospholipids on the global gene expression in human dermal fibroblasts. We identified a cluster of genes that were co-induced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit (GCLM), aldo-keto reductases-1-C1 and -C2 (AKR1C1, AKR1C2), and interleukin 8 (IL8). These genes are members of the cellular stress response system termed âantioxidant responseâ or âPhase II detoxificationâ. Accordingly, the regulatory regions of all these genes contain binding sites for NF-E2-related factor 2 (Nrf2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation of Nrf2. Silencing expression of Nrf2 using siRNA or using cells and tissue from Nrf2-deficient mice, we show that the induction of the co-regulated genes was suppressed. Expression of other canonical UVA-1-induced genes, including cyclooxygenase 2 (Cox2) and interleukin 6 (IL6) was unaltered in the absence of Nrf2. Together, our data show that UVA-1-mediated lipid oxidation induces induction of antioxidant response genes, which is dependent on the redox-regulated transcription factor Nrf2. To activate Nrf2 is a major strategy for novel antioxidant drugs, the skin photo-adaptation (SPA) inducers. Our finding that specific uv-oxidized lipids act similar sheds a new (ultraviolet) light on the usually detrimental âimageâ of UV generated lipid mediators. Experiment Overall Design: we profiled global mRNA expression levels in human dermal fibroblasts that had been treated with either UVA-1 or oxidized lipids. To investigate the effect of oxidized phospholipids on gene regulation, we used two preparations, which differed in their degree of oxidation; the minimally oxidized UV-PAPC resulting from UVA-1 irradiation of PAPC, and air-oxidized PAPC (OxPAPC), which represents the full spectrum of oxidation products (Gruber 07) (Reis et al., 2005). We irradiated dermal fibroblasts with UVA-1 (40J/cm²) or treated them with UV-PAPC, OxPAPC or native PAPC (100µg/ml each). We analyzed global gene expression four hours after stimulation with gene arrays (Affymetrix U133A Plus 2.0 Gene Chips).
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Project description:Cysteine oxidative modification of cellular proteins is crucial for many aspects of cardiac hypertrophy development. However, integrated dissection of multiple types of cysteine oxidation proteome in cardiac hypertrophy is currently missing. Here we developed a novel discovery platform encompassing a customized biotin switch-based quantitative proteomics pipeline and an advanced analytic workflow to comprehensively profile the landscape of cysteine oxidation in ISO-induced cardiac hypertrophy mouse model. Specifically, we identified a total of 3,717 proteins containing 6,837 oxidized cysteine sites by at least one of reversible cysteine oxidation, cysteine sulfinylation (CysSO2H), and cysteine sulfonylation (CysSO3H). Analyzing the hypertrophy signatures that are reproducibly discovered from computational workflow highlighted a group of fatty acid beta-oxidation enzymes with a continual decreased temporal pattern and a significant decreased abundance in reversible oxidation with no temporal or abundance change in total cysteine, revealing the oxidative regulatory map of fatty acid metabolism in cardiac hypertrophy, which is featured by an overall reduced oxidative metabolism. Our cysteine oxidation platform depicts a dynamic and integrated landscape of the cysteine oxidative proteome, extracted molecular signature, and provided mechanistic insights in cardiac hypertrophy.
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Project description:To investigate selectivity of mRNA oxidation, total RNA and oxidized RNA isolated from Neuro 2a cells before and after H2O2 treatment were employed for microarray analysis. It was found that selective oxidation of mRNA already occurs under normal culture conditions but was increased by H2O2, especially in a subset of mRNAs related to certain functions. Moreover, mRNA oxidation level is also related to its abundance or stability (half-life time). This shows for the first time that mRNA oxidation is associated with RNA homeostasis including function, stability and abundance depending on cellular redox status in a genome-wide scale. Neuro 2a cells received hydrogen peroxide treatment or no treatment as a control. Samples were applied for RNA extraction and ARP labeling, which could bind with apurinic/apyrimidinic sites, and then a pull-down process to isolate oxidized RNA. Total RNA and oxidized RNA were used for subsequent transcriptomic profiling. 4 types of samples were analyzed: Basal-total: untreated N2a cells labeled with ARP, but not processed for the pull-down assay. Ox-total: hydrogen peroxide-treated N2a cells labeled with ARP, but not processed for the pull-down assay. Basal-ARP: untreated N2a cells labeled with ARP, and processed for the pull-down assay. ARP-derivatized RNA, which is also oxidized RNA, was concentrated and used for the microarray analysis. Ox-ARP: hydrogen peroxide-treated N2a cells labeled with ARP, and processed for the pull-down assay. ARP-derivatized RNA, which is also oxidized RNA, was concentrated and used for the microarray analysis.
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Project description:Redox proteomics identifies additional putative targets of Sod1 redox regulation. The high abundance and broad cellular distribution of Sod1 suggests that it may function as a redox regulator of a broad variety of substrates in addition to GAPDH. To test this, we conducted a high-powered quantitative redox proteomics screen of WT and sod1Δ yeast to identify Sod1-dependent redox substrates. We used a combined SILAC-TMT approach, whereby sod1Δ-dependent changes in protein abundance are quantified through Stable Isotope Labeling with Amino acids in Cell culture (SILAC) and changes in reversible cysteine oxidation are quantified through cysteine-reactive Iodoacetyl Tandem Mass Tags (iodo-TMT) before and after reduction with DTT. Consequently, the study reveals a broad range of proteins that undergo significant changes in abundance, cysteine oxidation, or both. Focusing first on the effects of SOD1 deletion on proteome-wide protein abundance, we independently analyzed the SILAC MS data. A total of 4,409 proteins were confidently detected and quantified, and ~9% of these (373) exhibited significant differences in abundance between the two strains. Of these, 114 (30.6%) exhibit a significant decrease in protein abundance in sod1Δ cells compared to 259 (69.4%) proteins that exhibit a significant increase in abundance. Overall, we revealed a larger network of cysteine-containing proteins that are oxidized in a Sod1- dependent manner using mass spectrometry-based redox proteomics approaches.
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