<br>棉花 Ti-IMAC：开发磷酸化棉花作为磷酸肽富集的新平台,ACS Applied Materials & Interfaces

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棉花 Ti-IMAC：开发磷酸化棉花作为磷酸肽富集的新平台
ACS Applied Materials & Interfaces ( IF 8.3 ) Pub Date : 2023-10-09 , DOI: 10.1021/acsami.3c08697
Danqing Wang ₁ , Junfeng Huang _{2,

3,

4} , Haoran Zhang ₁ , Ting-Jia Gu ₂ , Lingjun Li _{1,

2,

5,

6}

Affiliation

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。在分子水平上了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强大工具。然而，复杂生物样品中的低电离效率和低丰度的磷酸肽使其质谱分析具有挑战性；在 MS 分析之前，始终需要具有高效率和选择性的富集策略。在这项研究中，我们开发了一种磷酸化棉纤维基 Ti(IV)-IMAC 材料（称为 Cotton Ti-IMAC），它可以作为磷酸肽富集的新平台。棉纤维可以在一步中有效地与磷酸基团共价接枝，然后可以固定钛离子以捕获磷酸肽。使用经济高效的试剂只需 4 小时即可制备该材料。得益于棉纤维的柔韧性和过滤性，该材料可以轻松地包装为旋转吸头，使浓缩过程变得方便。 Cotton Ti-IMAC 成功地从蛋白质标准消化物中富集了磷酸肽，并表现出高选择性 (BSA/β-酪蛋白 = 1000:1) 和出色的灵敏度 (0.1 fmol/μL)。此外，仅从 100 μg HeLa 细胞消化物中富集后，即可在一次 LC-MS/MS 进样中对 2354 种磷酸肽进行分析，富集特异性高达 97.51%。总而言之，我们相信 Cotton Ti-IMAC 可以作为一个广泛适用且强大的平台，用于实现大规模磷酸肽富集并扩展我们对复杂生物系统中磷酸蛋白质组学的了解。

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Cotton Ti-IMAC: Developing Phosphorylated Cotton as a Novel Platform for Phosphopeptide Enrichment

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.

更新日期：2023-10-09

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