2. Academic Validation
  3. An improved sulfur-nitroso-proteome strategy for global profiling of sulfur-nitrosylated proteins and sulfur-nitrosylation sites in mice

An improved sulfur-nitroso-proteome strategy for global profiling of sulfur-nitrosylated proteins and sulfur-nitrosylation sites in mice

  • J Chromatogr A. 2023 Aug 30:1705:464162. doi: 10.1016/j.chroma.2023.464162.
Hongmei Yang 1,  Linxu Wang 2,  Zhaoyang Xie 3,  Simeng Shao 3,  Yi Wu 3,  Weiyin Xu 3,  Bin Gu 4,  Bo Wang 5
Affiliations

Affiliations

  • 1 Northeast Asia Institute of Chinese Medicine, Changchun University of Chinese Medicine, Changchun 130017, China. Electronic address: [email protected].
  • 2 State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
  • 3 Northeast Asia Institute of Chinese Medicine, Changchun University of Chinese Medicine, Changchun 130017, China.
  • 4 Department of Stomatology, the first medical center, General Hospital of the Chinese people's Liberation Army, Beijing 100036, China. Electronic address: [email protected].
  • 5 State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China. Electronic address: [email protected].
PMID: 37336129 DOI: 10.1016/j.chroma.2023.464162
Abstract

Comprehensive sulfur-nitrosylation (SNO) proteome coverage in complex biological systems remains challenging as a result of the low level of endogenous S-nitrosylation and its chemical instability. Herein, we optimized the synthesis route of SNOTRAP (SNO trapping by triaryl phosphine) probe and the proteomics pipeline (including preventing over-alkylation, sample washing, trypsin digestion). Preventing overalkylation was found to be the key factor resulting in a higher number of identified SNO proteins by evaluating various experimental conditions. With the improved SNOTRAP-based proteomic pipeline, we achieved an improvement of ∼10-fold on identification efficiency, and identified 1181 SNO proteins (1714 SNO sites) in mouse brain, representing the largest repository of endogenous S-nitrosylation. Moreover, we identified the consensus motif of SNO sites, suggesting the correlation with local hydrophobicity, acid-base catalysis, and the surrounding secondary structures for modification of specific cysteines by NO. Collectively, we provide a universal pipeline for the high-coverage identification of low-abundance SNO proteins with high enrichment efficiency, high specificity (98%), good reproducibility, and easy implementation, contributing to the elucidation of the mechanism(s) of nitrosative stress in multiple diseases.

Keywords

Improved SNOTRAP method; Mass spectrometry; Mouse; Overalkylation; S-nitrosylation.

Figures
Products