School of Pharmacy, Nanjing Medical University, Nanjing, 211166, China.
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China.
Talanta. 2022 Sep 1;247:123580. doi: 10.1016/j.talanta.2022.123580. Epub 2022 May 26.
N-phosphorylation modifications are crucial for prokaryotic signal transduction and verified as intermediate for several metabolic enzymes, yet the landscape of N-phosphorylation remains an obstacle due to the lack of effective identification strategies. One of the difficulties derives from the labile phosphoramidate bond (P-N) under acidic conditions, making it easily hydrolyze during the routine analysis. Meanwhile, O-phosphopeptides influence the accurate identification of N-phosphorylation sites during mass spectrometry (MS) analysis. Herein, a selective enrichment tandem β-elimination assisted identification (abbreviated as EnaBe) strategy was established to address the difficulties. Firstly, N-phosphoproteins could be captured within 10 min by SiO@DpaZn microspheres under neutral conditions, which was benefited from rapid mass transfer. Secondly, the β-elimination efficiency could reach over 92% within 3 h under the optimized condition, and MS signals of N-phosphopeptides were significantly enhanced by integrating the β-elimination treatment. Finally, N-phosphoproteins from E. coli lysates were analyzed by EnaBe approach and 16 N-phosphoproteins with high confidence were identified. Furthermore, functional analysis showed that the proteins played vital roles in phosphorylation process and bacterial primary metabolic processes including glucose, purine and ADP metabolism. All above results demonstrated the superiority of the EnaBe strategy.
N-磷酸化修饰对于原核信号转导至关重要，并且已被证实为几种代谢酶的中间产物，然而由于缺乏有效的鉴定策略，N-磷酸化的全貌仍然是一个障碍。其中一个困难源于在酸性条件下不稳定的磷酰胺键（P-N），使其在常规分析过程中容易水解。同时，O-磷酸肽会影响质谱（MS）分析中 N-磷酸化位点的准确鉴定。在此，建立了一种选择性富集串联β-消除辅助鉴定（简称 EnaBe）策略来解决这些困难。首先，在中性条件下，SiO@DpaZn 微球可以在 10 分钟内捕获 N-磷酸蛋白，这得益于快速的传质。其次，在优化条件下，β-消除效率可在 3 小时内达到 92%以上，并且通过整合β-消除处理，N-磷酸肽的 MS 信号得到显著增强。最后，通过 EnaBe 方法分析大肠杆菌裂解物中的 N-磷酸蛋白，并鉴定出 16 种具有高可信度的 N-磷酸蛋白。此外，功能分析表明，这些蛋白质在磷酸化过程以及细菌初级代谢过程中发挥着重要作用，包括葡萄糖、嘌呤和 ADP 代谢。所有这些结果都证明了 EnaBe 策略的优越性。
