3 years ago

Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography

of Macrophage Endogenous S-Nitrosoproteome
Using a Cysteine-Specific Phosphonate
Adaptable Tag in Combination with TiO2 Chromatography
David Escors, Martin R. Larsen, Estela Pérez, Grazyna Kochan, Miren Zuazo, Enrique Santamaría, Xabier Martínez de Morentin, Hugo Arasanz, María Ibáñez-Vea, Gonzalo Fernandez-Hinojal, Honggang Huang, Maria Gato, Joaquin Fernández-Irigoyen
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. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO2) chromatography enrichment prior to nLC–MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.

Publisher URL: http://dx.doi.org/10.1021/acs.jproteome.7b00812

DOI: 10.1021/acs.jproteome.7b00812

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