3 years ago

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

Characterization
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

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.