Northeast Asia Institute of Chinese Medicine, Changchun University of Chinese Medicine, Changchun 130017, China.
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
J Chromatogr A. 2023 Aug 30;1705:464162. doi: 10.1016/j.chroma.2023.464162. Epub 2023 Jun 15.
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.
由于内源性 S-亚硝基化水平低且其化学稳定性差，全面覆盖复杂生物系统中的硫亚硝基化（SNO）蛋白质组仍然具有挑战性。在此，我们优化了 SNOTRAP（通过三芳基膦捕获 SNO）探针的合成路线和蛋白质组学工作流程（包括防止过度烷基化、样品洗涤、胰蛋白酶消化）。通过评估各种实验条件，发现防止过度烷基化是导致鉴定出更多 SNO 蛋白的关键因素。通过改进的基于 SNOTRAP 的蛋白质组学工作流程，我们将鉴定效率提高了约 10 倍，在小鼠脑中鉴定出 1181 种 SNO 蛋白（1714 个 SNO 位点），这代表了最大的内源性 S-亚硝基化蛋白库。此外，我们还确定了 SNO 位点的共识基序，这表明与局部疏水性、酸碱催化以及修饰特定半胱氨酸的周围二级结构有关。总的来说，我们提供了一种通用的工作流程，用于高覆盖鉴定低丰度 SNO 蛋白，具有高富集效率、高特异性（98%）、良好的重现性和易于实施性，有助于阐明多种疾病中硝化应激的机制。
