Institute of Drug Discovery Technology, Ningbo University, Ningbo, Zhejiang 315211, China; CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, Liaoning 116023, China.
CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, Liaoning 116023, China.
J Chromatogr A. 2024 Aug 16;1730:465064. doi: 10.1016/j.chroma.2024.465064. Epub 2024 Jun 9.
Small ubiquitin-like modifier (SUMO) modification regulates various eukaryotic cellular processes and plays a pivotal role in interferon (IFN)-mediated antiviral defense. While immunoprecipitation enrichment method is widely used for proteome-wide analysis of endogenous SUMOylation, the inability to target all SUMO forms and high cost of antibodies limited its further application. Herein, we proposed an antibody-free enrichment method based on SUMO-specific protease and strong anion exchange chromatography (SPAX) to globally profile the endogenous SUMOylation. The SUMO1/2/3-modified peptides could be simultaneously enriched by SAX chromatography by utilizing its electrostatic interaction with SUMO1/2/3 remnants, which contained multiple aspartic acids (D) and glutamic acids (E). To remove the co-enriched D/E-containing peptides which might interfere with the detection of low-abundance SUMOylated peptides, SUMO-specific protease was used to cleave the SUMO1/2/3 remnants from enriched SUMOylated peptides. As the deSUMOylated peptides lost SUMO remnants, their interaction with SAX materials became weaker, and the D/E-containing peptides could thus be depleted through the second SAX separation. The SPAX method identified over twice the SUMOylated sites than using SAX method only, greatly improving the identification coverage of endogenous SUMOylated sites. Our strategy was then applied to the site-specific identification and quantification of endogenous SUMOylation in A549 cells stimulated by IFN-γ for the first time. A total of 226 SUMOylated sites on 146 proteins were confidently identified, among which multiple up-regulated sites were involved in IFN-mediated antiviral defense, demonstrating the great promise of SPAX to globally profile and discover endogenous SUMOylation with significant biological functions.
小泛素样修饰物 (SUMO) 修饰调节各种真核细胞过程，并在干扰素 (IFN) 介导的抗病毒防御中发挥关键作用。虽然免疫沉淀富集方法广泛用于内源性 SUMOylation 的蛋白质组学分析，但由于无法靶向所有 SUMO 形式和抗体成本高，限制了其进一步应用。在此，我们提出了一种基于 SUMO 特异性蛋白酶和强阴离子交换色谱 (SPAX) 的无抗体富集方法，用于全局分析内源性 SUMOylation。通过利用静电相互作用与 SUMO1/2/3 残基结合，SAX 色谱可同时富集 SUMO1/2/3 修饰的肽段，而 SUMO1/2/3 残基中含有多个天冬氨酸 (D) 和谷氨酸 (E)。为了去除可能干扰低丰度 SUMO 化肽检测的共富集的含有 D/E 的肽段，使用 SUMO 特异性蛋白酶从富集的 SUMO 化肽中切割 SUMO1/2/3 残基。由于去 SUMO 化肽段失去了 SUMO 残基，它们与 SAX 材料的相互作用变弱，因此含有 D/E 的肽段可以通过第二次 SAX 分离去除。与仅使用 SAX 方法相比，SPAX 方法鉴定的 SUMO 化位点数量增加了一倍以上，极大地提高了内源性 SUMO 化位点的鉴定覆盖率。然后，我们首次将该策略应用于 IFN-γ刺激的 A549 细胞中内源性 SUMOylation 的特异性鉴定和定量。在 146 种蛋白质上鉴定到 226 个具有置信度的 SUMO 化位点，其中多个上调的位点参与 IFN 介导的抗病毒防御，这表明 SPAX 具有很大的潜力用于全局分析和发现具有重要生物学功能的内源性 SUMOylation。
