Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins,Journal of Proteome Research

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Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2021-11-15 , DOI: 10.1021/acs.jproteome.1c00788
Huiyu Wang ₁ , Liqiang Qian ₂ , Zhi Shang ₃ , Zeyuan Wang ₁ , Yan Zhang ₄ , Chengxi Cao ₅ , Hua Xiao ₁

Affiliation

Cellular nucleic acid-binding proteins (NABPs), namely, DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in many biological processes. However, extracting NABPs with high efficiency in living cells is challenging, which greatly limited their proteomics analysis and comprehensive characterization. Here, we discovered that titanium (IV) ion-immobilized metal affinity chromatography (Ti⁴⁺-IMAC) material could enrich DNA and RNA with high efficiency (96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore developed a Ti⁴⁺-IMAC method for the joint extraction of DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking, DBPs and RBPs were covalently linked to nucleic acids (NAs) and further denatured by organic solvents. After Ti⁴⁺-IMAC capture, 2000 proteins were identified in 293T cells, among which 417 DBPs and 999 RBPs were revealed, showing promising selectivity for NABPs. We further applied the Ti⁴⁺-IMAC capture method to lung cancer cell lines 95C and 95D, which have different tumor progression abilities. The DNA- and RNA-binding capabilities of many proteins have been dysregulated in 95D. Under our conditions, Ti⁴⁺-IMAC can be used as a selective and powerful tool for the comprehensive characterization of both DBPs and RBPs, which might be utilized to study their dynamic interactions with nucleic acids.

中文翻译：

固定化钛 (IV) 离子亲和色谱有助于细胞核酸结合蛋白的高效蛋白质组学分析

细胞核酸结合蛋白 (NABPs)，即 DNA 结合蛋白 (DBPs) 和 RNA 结合蛋白 (RBPs)，在许多生物过程中发挥着重要作用。然而，在活细胞中高效提取 NABPs 具有挑战性，这极大地限制了它们的蛋白质组学分析和综合表征。在这里，我们发现钛 (IV) 离子固定金属亲和层析 (Ti ⁴⁺ -IMAC) 材料可以高效富集 DNA 和 RNA（分别为 96.82 ± 2.67 和 85.75 ± 2.99%）。因此，我们开发了一种用于联合提取 DBP 和 RBP 的 Ti ⁴⁺ -IMAC 方法。通过利用甲醛 (FA) 交联，DBPs 和 RBPs 与核酸 (NAs) 共价连接，并通过有机溶剂进一步变性。钛后⁴⁺-IMAC捕获，在293T细胞中鉴定出2000个蛋白质，其中发现了417个DBP和999个RBP，显示出对NABP的良好选择性。我们进一步将Ti ⁴⁺ -IMAC 捕获方法应用于具有不同肿瘤进展能力的肺癌细胞系95C 和95D。许多蛋白质的 DNA 和 RNA 结合能力在 95D 中已经失调。在我们的条件下，Ti ⁴⁺ -IMAC 可用作全面表征 DBPs 和 RBPs 的选择性且强大的工具，可用于研究它们与核酸的动态相互作用。

更新日期：2022-01-07

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