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Redox proteomics DIALRP to study MND response in DU145 cells

ABSTRACT: In this study, we developed a label-free redox proteomics method using data-independent acquisition mass spectrometry (DIA-MS), termed DIALRP, to comprehensively analyze cysteine oxidative modifications in the prostate cancer cell line DU145 under oxidative stress induced by menadione (MND).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Masaki Matsumoto

PROVIDER: PXD057921 | JPOST Repository | Thu Jul 31 00:00:00 BST 2025

REPOSITORIES: jPOST

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			Action	DRS
	20240607_DU145_1_DIC0_Mena0_All.raw	Raw
	20240607_DU145_1_DIC0_Mena0_Oxi.raw	Raw
	20240607_DU145_1_DIC0_Mena50_All.raw	Raw
	20240607_DU145_1_DIC0_Mena50_Oxi.raw	Raw
	20240607_DU145_2_DIC0_Mena0_All.raw	Raw

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Publications

Data-Independent Acquisition (DIA)-Based Label-Free Redox Proteomics (DIALRP) Identifies Prominent Cysteine Oxidations in Translation Machinery in Prostate Cancer Cells Under Oxidative Stress.

Kobayashi Daiki D Takami Tomoyo T Matsumoto Masaki M

Journal of proteome research 20250731 9

Oxidative stress is a key factor in numerous physiological and pathological processes, including aging, cancer, and neurodegenerative diseases. Protein cysteine residues are particularly susceptible to oxidative stress-induced modifications that can alter their structure and function, thereby affecting intracellular signaling pathways. In this study, we performed a data-independent acquisition mass spectrometry (DIA-MS)-based label-free redox proteomics method, termed DIALRP, to comprehensively ...[more]

PMID: 40740030

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SP3-enabled rapid and high coverage chemoproteomic identification of redox-sensitive cysteines

Project description:Cysteine residues undergo various oxidative modifications, acting as key sensors of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Given that ROS and RNS have known roles in many pathophysiological processes, numerous proteome-wide strategies to profile cysteine oxidation state have emerged in the last decade. Recent advancements to traditional redox profiling methods include incorporation of costly isotopic labeling reagents to allow for more quantitative assessment of oxidation states. These methods are typically carried out by using sequential thiol capping and reduction steps in order to label redox-sensitive cysteines, often found in di-cysteine motifs (‘CXXC’ or ‘CXXXC’). Tailored, pricy algorithms are commonly used to analyze redox-profiling datasets, the majority of which cannot accurately quantify site-of-labeling in redox-motifs; moreover, accurate quantification is confounded by excess labeling reagents during sample preparation. Here, we present a low-cost redox-profiling workflow using newly synthesized isotopic reagents compatible with SP3-bead technology, termed SP3-ROx, that allows for high throughput, rapid identification of redox-sensitive cysteines. We optimize cysteine labeling quantification using the FragPipe suite, an open source GUI for MSfragger-based search algorithm. Application of SP3-ROx to naive and activated T cells identifies redox-senstive cysteines, showcasing the utility of this workflow to study biological processes.

2022-03-02 | PXD029500 | Pride

SP3-enabled rapid and high coverage chemoproteomic identification of redox-sensitive cysteines

2022-03-02 | PXD031647 | Pride

Oct4 redox sensitivity potentiates reprogramming and differentiation

Project description:The transcription factor Oct4/Pou5f1 is a key component of the regulatory circuitry governing pluripotency. Oct4 is also widely used to generate induced pluripotent stem cells (iPSCs) from somatic cells. These observations provide compelling rationale to understand Oct4’s functions. Here we used domain swapping and mutagenesis to compare Oct4’s reprogramming activity with the paralog Oct1/Pou2f1, identifying a redox-sensitive DNA binding domain cysteine residue (Cys48) as a key determinant of both reprogramming and differentiation. In combination with the Oct4 N-terminus, mutating Oct1’s serine at this position to cysteine is sufficient to confer strong reprogramming activity. Conversely, Oct4C48S strongly reduces reprogramming potential. Oct4C48S renders the protein insensitive to oxidative inhibition of DNA binding. The same mutation prevents oxidation-mediated protein ubiquitylation. An engineered Pou5f1C48S point mutation has little effect on undifferentiated cells embryonic stem cells (ESCs), but upon retinoic acid (RA)-mediated differentiation causes retention of Oct4 expression, decreased proliferation, and increased apoptosis. Transcriptional profiling reveals that the retention of pluripotency also manifests at the RNA level. Pou5f1C48S ESCs also contribute poorly to adult somatic tissues and form less differentiated teratomas. Collectively, the data support a model in which Oct4 redox sensing is a positive reprogramming determinant during one or more reprogramming steps, and mediates Oct4 degradation during differentiation.

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Beyond Glucose: The Crucial Role of Redox Signaling in β-Cell Metabolic Adaptation

Project description:Objective: Redox signaling mediated by reversible oxidative cysteine thiol modifications is crucial for driving cellular adaptation to dynamic environmental changes, maintaining homeostasis, and ensuring proper function. This is particularly critical in pancreatic β-cells, which are highly metabolically active and play a specialized role in whole organism glucose homeostasis. Glucose stimulation in β-cells triggers signals leading to insulin secretion, including changes in ATP/ADP ratio and intracellular calcium levels. Additionally, lipid metabolism and reactive oxygen species (ROS) signaling are essential for β-cell function and health. Methods: We employed IodoTMT isobaric labeling combined with tandem mass spectrometry to elucidate redox signaling pathways in pancreatic β-cells. Results: Glucose stimulation significantly increases ROS levels in β-cells, leading to targeted reversible oxidation of proteins involved in key metabolic pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, pyruvate metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum (ER), and insulin secretion. Furthermore, the glucose-induced increase in reversible cysteine oxidation correlates with the presence of other post-translational modifications, including acetylation and phosphorylation. Conclusions: Proper functioning of pancreatic β-cell metabolism relies on fine-tuned regulation, achieved through a sophisticated system of diverse post-translational modifications that modulate protein functions. Our findings demonstrate that glucose induces the production of ROS in pancreatic β-cells, leading to targeted reversible oxidative modifications of proteins. Furthermore, protein activity is modulated by acetylation and phosphorylation, highlighting the complexity of the regulatory mechanisms in β-cell function.

2024-09-15 | GSE274980 | GEO

S-bacillithiolation protects against hypochlorite stress in Bacillus subtilis as revealed by transcriptomics and redox proteomics

Project description:Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In Bacillus subtilis the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate, BSH), termed as S-bacillithiolation. Here, we have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid in B. subtilis. The expression profile of NaOCl stress is indiciative of disulfide stess as shown by the induction of the thiol- and oxidative stress-specific Spx, CtsR and PerR regulons. Thiol redox proteomics identified only few cytoplasmic proteins with reversible thiol-oxidations in response to NaOCl stress that include GapA and MetE. Shotgun-LC-MS/MS analyses revealed that GapA, Spx and PerR are oxidized to intramolecular disulfides by NaOCl stress. Furthermore, we identified six S-bacillithiolated proteins in NaOCl-treated cells, including the OhrR repressor, two methionine synthases MetE and YxjG, the inorganic pyrophosphatase PpaC, the 3-D-phophoglycerate dehydrogenase SerA and the putative bacilliredoxin YphP. S-bacillithiolation of the OhrR repressor leads to up-regulation of the OhrA peroxiredoxin that confers together with BSH specific protection against NaOCl. S-bacillithiolation of MetE, YxjG, PpaC and SerA causes hypochlorite-induced methionine starvation as supported by the induction of the S-box regulon. The mechanism of S-glutathionylation of MetE has been described in Escherichia coli also leading to enzyme inactivation and methionine auxotrophy. In summary, our studies discover an important role of the BSH redox buffer in protection against hypochloric acid by S-bacillithiolation of the redox-sensing regulator OhrR and of four enzymes of the methionine biosynthesis pathway.

2011-07-04 | GSE27637 | GEO

Long-term exposure to cold plasma-generated ROS âan in vitro model for redox-related diseases of the skin

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