Department of Chemistry, University of Wisconsin─Madison, Madison, Wisconsin 53706, United States.
School of Pharmacy, University of Wisconsin─Madison, Madison, Wisconsin 53705, United States.
ACS Appl Mater Interfaces. 2023 Oct 18;15(41):47893-47901. doi: 10.1021/acsami.3c08697. Epub 2023 Oct 9.
Protein phosphorylation is an important post-translational modification (PTM), which is involved in many important cellular functions. Understanding protein phosphorylation at the molecular level is critical to deciphering its relevant biological processes and signaling networks. Mass spectrometry (MS) has become a powerful tool for the comprehensive profiling of protein phosphorylation. Yet the low ionization efficiency and low abundance of phosphopeptides among complex biological samples make its MS analysis challenging; an enrichment strategy with high efficiency and selectivity is always necessary prior to MS analysis. In this study, we developed a phosphorylated cotton-fiber-based Ti(IV)-IMAC material (termed as Cotton Ti-IMAC) that can serve as a novel platform for phosphopeptide enrichment. The cotton fiber can be effectively grafted with phosphate groups covalently in a single step, where the titanium ions can then be immobilized to enable capturing phosphopeptides. The material can be prepared using cost-effective reagents within only 4 h. Benefiting from the flexibility and filterability of cotton fibers, the material can be easily packed as a spin-tip and make the enrichment process convenient. Cotton Ti-IMAC successfully enriched phosphopeptides from protein standard digests and exhibited a high selectivity (BSA/β-casein = 1000:1) and excellent sensitivity (0.1 fmol/μL). Moreover, 2354 phosphopeptides were profiled in one LC-MS/MS injection after enriching from only 100 μg of HeLa cell digests with an enrichment specificity of up to 97.51%. Taken together, we believe that Cotton Ti-IMAC can serve as a widely applicable and robust platform for achieving large-scale phosphopeptide enrichment and expanding our knowledge of phosphoproteomics in complex biological systems.
蛋白质磷酸化是一种重要的翻译后修饰（PTM），参与许多重要的细胞功能。在分子水平上理解蛋白质磷酸化对于破译其相关的生物过程和信号网络至关重要。质谱（MS）已成为全面分析蛋白质磷酸化的有力工具。然而，复杂生物样品中磷酸肽的低离子化效率和低丰度使得其 MS 分析具有挑战性；在 MS 分析之前，总是需要一种高效和选择性的富集策略。在这项研究中，我们开发了一种基于磷酸化棉纤维的 Ti(IV)-IMAC 材料（称为 Cotton Ti-IMAC），可作为一种新型的磷酸肽富集平台。棉纤维可以通过一步共价键合有效地接枝磷酸基团，然后将钛离子固定化以捕获磷酸肽。该材料可以在仅 4 小时内使用经济有效的试剂制备。得益于棉纤维的灵活性和过滤性，该材料可以很容易地包装成一个自旋尖，使富集过程变得方便。Cotton Ti-IMAC 成功地从蛋白质标准消化物中富集了磷酸肽，表现出高选择性（BSA/β-酪蛋白=1000:1）和优异的灵敏度（0.1 fmol/μL）。此外，仅从 100μg HeLa 细胞消化物中进行富集，在一次 LC-MS/MS 注射中就可以分析到 2354 种磷酸肽，富集特异性高达 97.51%。总之，我们相信 Cotton Ti-IMAC 可以作为一种广泛适用且强大的平台，用于实现大规模磷酸肽的富集，并扩展我们对复杂生物系统中磷酸蛋白质组学的认识。
