TIPP: A Novel Iodoacetamide Reaction-Induced Protein Precipitation Approach for Proteome-Wide Ligand-Target Identification

Ligand-bound proteins have higher stability and thereby greater resistance to denaturing-induced precipitation than free proteins, which forms the basis of several modification-free ligand target protein identification methods. However, due to the vast sequence diversity of cellular proteins, different proteins respond differently to distinct denaturation mechanisms, and a single denaturation mechanism can only cover certain target proteins. Therefore, developing efficient target protein identification methods based on novel denaturation mechanisms is of great significance for improving the coverage of target identification. Herein, we propose a novel target protein identification method relying on a new precipitation mechanism, namely, Target Identification by Iodoacetamide reaction-induced Protein Precipitation (TIPP). The TIPP method uses iodoacetamide (IAA), a thiol-blocking reagent commonly used in shotgun proteomics, to irreversibly alkylate the thiol groups of free cysteine residues, thereby inducing protein denaturation and precipitation. Under IAA treatment, proteins in different conformational states have different exposures of free thiol groups, resulting in different precipitation efficiencies, which is the key theoretical basis for the design of the TIPP method for target protein identifications. The feasibility of the developed TIPP method was evaluated by identifying target proteins of various ligand compounds, including MTX, TG101348, and staurosporine. In addition, this method was also used to monitor the target proteins of metal ions with the metal ion chelator TPEN. The developed method has similar efficiency and good complementarity with the traditional method, and can be combined with the traditional method to increase the coverage of target identification. Therefore, TIPP is expected to become a powerful strategy to achieve comprehensive ligand-target identification by complementing other drug target identification methods.配体结合蛋白（ligand-bound proteins）相较于游离蛋白具备更高稳定性，因此对变性诱导沉淀（denaturing-induced precipitation）的抗性更强，这一特性构成了多种无修饰配体靶蛋白鉴定方法的核心原理。然而，由于细胞蛋白质序列多样性极高，不同蛋白质对不同变性机制的响应存在显著差异，单一变性机制仅能覆盖部分靶蛋白。因此，开发基于新型变性机制的高效靶蛋白鉴定方法，对提升靶标鉴定覆盖范围具有重要意义。 在此，我们提出一种基于全新沉淀机制的靶蛋白鉴定新方法——碘乙酰胺反应诱导蛋白沉淀靶标鉴定法（Target Identification by Iodoacetamide reaction-induced Protein Precipitation，简称TIPP）。该方法采用鸟枪法蛋白质组学（shotgun proteomics）中常用的巯基封闭试剂碘乙酰胺（iodoacetamide, IAA），对游离半胱氨酸残基的巯基进行不可逆烷基化修饰，进而诱导蛋白质变性与沉淀。在碘乙酰胺处理条件下，处于不同构象状态的蛋白质其游离巯基暴露程度存在差异，进而导致沉淀效率不同，这正是TIPP方法用于靶蛋白鉴定的核心理论基础。 我们通过鉴定多种配体化合物（包括MTX、TG101348以及星形孢菌素（staurosporine））的靶蛋白，对所开发的TIPP方法的可行性进行了评估。此外，该方法还结合金属离子螯合剂TPEN，实现了金属离子靶蛋白的鉴定。所开发的TIPP方法与传统方法效率相当，且具备良好的互补性，二者联用可进一步提升靶标鉴定的覆盖范围。因此，通过与其他药物靶标鉴定方法互补联用，TIPP有望成为实现全面配体-靶标鉴定的高效策略。