Dataset Information

Adaptation of CysPAT technique to characterize endogenous S-nitrosylation in RAW264.7 cells

ABSTRACT: The aim of the study was to adapt the CysPAT technique to efficiently identify and characterize endogenous S-nitrosylation in cells under physiological conditions of an experiment from a small amount of sample. Using a macrophage cell line RAW 264.7 we compared the efficacy of the method in the identification of total endogenous S-nitrosylation and the corresponding modification of the samples treated with exogenous SNO donor (GSNO) or with DTT. We identified 999 unique formerly S-nitrosylated peptides (SIA peptides) and 965 unique SNO sites from untreated RAW cells which belong to 569 endogenous nitrosylated proteins. We discovered 579 novel S-nitrosylation sites and identified 232 proteins as new S-nitrosylation targets. A total of 1450 S-nitrosylated peptides belonging to 795 proteins were identified in samples treated with GSNO (n = 3). Interestingly, we found a large overlap (>53%) of S-nitrosylated peptides in both subsets of samples, demonstrating a high sensitivity of the method to analyze endogenous S-nitrosylation. The large number of identified endogenous S-nitrosylated peptides allowed the identification of two nitrosylation consensus sites, and to highlight protein translation and redox processes as key S-nitrosylation targets in macrophages.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Macrophage

SUBMITTER: Maria Ibañez-Vea

LAB HEAD: David Escors

PROVIDER: PXD008182 | Pride | 2022-02-22

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Control1_082_Nitro_PhosphoCystag.mgf	Mgf
	Control1_082_Nitro_PhosphoCystag.wiff	Wiff
	Control1_082_Nitro_PhosphoCystag_PSMs.xlsx	Xlsx
	Control2_082_Nitro_PhosphoCystag.mgf	Mgf
	Control2_082_Nitro_PhosphoCystag.wiff	Wiff

Items per page:

1 - 5 of 24

Publications

Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO<sub>2</sub> Chromatography.

Ibáñez-Vea María M Huang Honggang H Martínez de Morentin Xabier X Pérez Estela E Gato Maria M Zuazo Miren M Arasanz Hugo H Fernández-Irigoyen Joaquin J Santamaría Enrique E Fernandez-Hinojal Gonzalo G Larsen Martin R MR Escors David D Kochan Grazyna G

Journal of proteome research 20180125 3

Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile ...[more]

PMID: 29338241

Similar Datasets

Project description:Trypomastigotes from Trypanosoma cruzi adhere to the extracellular matrix (ECM) in order to invade mammalian host cells. Incubation of trypomastigotes with ECM leads to a decrease of nitric oxide synthase (NOS) activity and associated post-translational modifications (PTMs). Herein, resin-assisted enrichment of thiols combined with mass spectrometry were employed to map site-specific S-nitrosylated (SNO) proteins from T. cruzi trypomastigotes incubated (MTy) or not (Ty) with ECM. We confirmed the reduction of S-nitrosylation upon incubation with ECM, associated to a rewiring of the subcellular distribution and intracellular signaling pathways of the SNO proteins between MTy and Ty conditions. Forty (~10.7%) and 248 (~66.4%) SNO-peptides were uniquely identified in MTy and Ty, respectively, in addition to 85 peptides (~22.7%) quantified in both conditions. S-nitrosylated proteins were enriched in a wide range of cellular components, including surface membranes and vesicles, and are involved in ribosome, transport, carbohydrate and lipid metabolisms. Of note, we described for the first time nytrosylation of histones H2B and H3 on Cys64 and Cys126, respectively, which is was more abundant in MTy. Additionally, sequence alignment shows that H3 (Cys126) histone is also present in T.b.brucei, but not in L. major, while H2B (Cys64) is absent in both parasites and other higher eukaryotes. Protein-protein interaction networks analyzed by STRING revealed ribosomal proteins, proteins involved in carbon and fatty acid metabolism to be among the enriched protein complexes. Among the SNO ribosomal proteins, 90% were identified and quantified uniquely or upregulated in the Ty fraction. Enzymes involved in oxidoreductase, kinases and phosphatases were identified as nitrosylated and phosphorylated in the same conditions suggesting a crosstalk between these modifications linked to regulation of energy metabolism of T. cruzi in the presence of ECM. Although enzymatic activity of NOS was described in T. cruzi, its identification is elusive. In silico mapping of putative nitric oxide synthase (NOS) gene(s) based on domain architecture identified four putative T. cruzi proteins expressing a putative C-terminal NOS domain: two putative P450 reductases, a putative FMN/FAD containing oxidoreductase and a putative oxidoreductase-protein. Our results provide the first site-specific characterization of S-nitrosylated proteins in T. cruzi linked to the roles of NO in reprogramming parasite cells to invade mammalian hosts.

Project description:Hypermethylation of helicase-like transcription factor (HLTF) in colorectal cancer (CRC) cells occurs more frequently in men than women. Progressive epigenetic silencing of HLTF in tumor cells is accompanied by negligible expression in the tumor microenvironment (TME). Cell line-derived xenografts were established in control (Hltf+/+) and Hltf-deleted male Rag2-/-IL2rg-/- mice by direct orthotopic microinjection of HLTF+/+HCT116 cells into the submucosa of the cecum. Combinatorial induction of IL6 and S100A8/A9 in the Hltf-deleted TME with ICAM-1 and IL8 in the primary tumor activated a positive feedback loop. The proinflammatory niche produced a major shift in CDX metastasis to peritoneal dissemination compared to controls. Inducible nitric oxide (iNOS) gene expression and transactivation of the iNOS-S100A8/A9 signaling complex in Hltf-deleted TME reprogrammed the human S-nitroso-proteome. S-nitroso (SNO) proteins POTEE, TRIM52 and UN45B are S-nitrosylated on the conserved I/L-X-C-X2-D/E motif indicative of iNOS-S100A8/A9-mediated S-nitrosylation. 2D-DIGE and protein identification by MALDI-TOF/TOF mass spectrometry authenticated S-nitrosylation of 53 individual cysteines in half-site motifs (I/L-X-C or C-X-X-D/E) in CDX tumors. POTEE-SNO in CDX tumors is both a general S-nitrosylation target and an iNOS-S100A8/A9 site-specific (Cys638) target in the Hltf-deleted TME. REL is an example of convergence of transcriptomic-S-nitroso-proteomic signaling. The gene is transcriptionally activated in CDX tumors with an Hltf-deleted TME, and REL-SNO (Cys143) is found in primary CDX tumors and all metastatic sites. Primary CDX tumors from Hltf-deleted TME shared 60% of their S-nitroso-proteome with all metastatic sites. Forty percent of SNO-proteins from primary CDX tumors were variably expressed at metastatic sites. Global S-nitrosylation of proteins in pathways related to cytoskeleton and motility is strongly implicated in the metastatic dissemination of CDX tumors. Hltf-deletion from the TME plays a major role in the pathogenesis of inflammation and links protein S-nitrosylation in primary CDX tumors with spatiotemporal continuity in metastatic progression even when the tumor cells express HLTF.

Project description:Human prion diseases are fatal neurodegenerative disorders characterized by neuronal damage in brain. Protein S-nitrosylation, the covalent adduction of a NO to cysteine, plays a role in human brain biology, and brain dysfunction is a prominent feature of pPrion disease, yet the direct brain targets of S-nitrosylation are largely unknown. We described the first proteomic analysis of global S-nitrosylation in brain tissues of sporadic Creutzfeldt–Jakob disease (sCJD), fatal familial insomnia (FFI) and genetic CJD with a substitution of valine for glycine at codon 114 of the prion protein gene (G114V gCJD) accompanying with normal control with isobaric tags for relative and absolute quantitation (iTRAQ) combined with a nano-HPLC/Q Exactive Mass spectrometry platform. In parallel, we used several approaches to provide quality control for the experimentally defined S-nitrosylated proteins. Total 1509 S-nitrosylated proteins (SNO-proteins) were identified, and cerebellum tissues appeared to contain more commonly differentially expressed SNO-proteins (DESPs) than cortex of sCJD, FFI and gCJD. Three selected SNO-proteins were verified by Western blots, consistent with proteomics assays. Gene ontology analysis showed that more up-regulated DESPs were involved in metabolism, cell cytoskeleton/structure and immune system both in cortex and cerebellum, while more down-regulated ones in both regions were involved in cell cytoskeleton/structure, cell-cell communication and miscelaneous function protein. Pathway analysis suggested that systemic lupus erythematosus, pathogenic Escherichia coli infection, extracellular matrix-receptor interaction were the most commonly affected pathways, which were identified from at least two different diseases. Using STRING database, the network of immune system and cell cytoskeleton and structure were commonly identified in the context of the up-regulated and down-regulated DESPs, respectively, both in cortex and cerebellum. Our study thus have implications for understanding the molecular mechanisms of human prion diseases related to abnormal protein S-nitrosylation and pave the way for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases.