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

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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

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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.

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Dataset Information

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

Publications

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

Similar Datasets

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