Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
State Key Laboratory of Proteomics, National Center for Protein Sciences Beijing, Beijing Institute of Lifeomics, Beijing Proteome Research Center, Beijing 102206, China.
ACS Biomater Sci Eng. 2022 Jun 13;8(6):2676-2683. doi: 10.1021/acsbiomaterials.2c00385. Epub 2022 May 26.
Metal oxide affinity chromatography (MOAC) is considered to be one of the most effective methods for phosphopeptide enrichment. However, most of the materials used in the method are powder; frequent centrifugation is necessitated during the enrichment process, and potential risks of loss of peptides and materials and clogging of the column employed for liquid chromatography-mass spectrometry (LC-MS) arise. Moreover, the reusability of these materials to achieve sustainability was hardly investigated. To overcome these limitations, herein, inorganic titanium dioxide (TiO) was coated onto the skeletal surface of the organic cellulose monolith (CM) material with a coral-like structure a sol-gel method. This produced an organic-inorganic hybrid TiO-CM material, which contained a combination of organic and inorganic substances, making it mimic the mollusk shell in terms of composition. The prepared TiO-CM material as monolith exhibited excellent mechanical strength and did not break during the enrichment process; thus, the tedious implementation of multiple centrifugation cycles was prevented, thereby streamlining the experimental procedure and avoiding the loss of peptides and materials. Moreover, a large amount of TiO was introduced onto the surface of the CM material, and thus, the resultant TiO-CM material exhibited a large surface area. As a result, the fabricated TiO-CM material was successfully applied to the enrichment of phosphopeptides obtained from the tryptic digests of a BSA/β-casein (mass ratio, 500/1) mixture. The results were superior to those achieved for commercial TiO beads, confirming that TiO-CM has excellent selectivity for phosphopeptides and reusability. Furthermore, 9287 unique phosphopeptides derived from the 2661 phosphoproteins were successfully identified from two milligrams of tryptic digests of Hela cell exosomes obtained through five independent replications after enriching using the TiO-CM material. The results indicated that the material has good application prospects in the analysis of protein phosphorylation. Furthermore, TiO-CM consists of green and cheap cellulose as the skeleton, and its synthesis process is environment-friendly, simple, and inexpensive.
金属氧化物亲和色谱（MOAC）被认为是最有效的磷酸肽富集方法之一。然而，该方法中使用的大多数材料都是粉末状的；在富集过程中需要频繁离心，并且存在肽和材料损失以及用于液相色谱-质谱（LC-MS）的柱子堵塞的潜在风险。此外，这些材料的可重复使用性以实现可持续性几乎没有被研究过。为了克服这些限制，本文采用溶胶-凝胶法将无机二氧化钛（TiO）涂覆到具有珊瑚状结构的有机纤维素整体材料（CM）的骨架表面上。这产生了一种有机-无机杂化 TiO-CM 材料，它包含有机和无机物质的组合，使其在组成上模仿软体动物壳。制备的 TiO-CM 整体材料表现出优异的机械强度，在富集过程中不会破裂；因此，避免了繁琐的多次离心循环的实施，从而简化了实验程序并避免了肽和材料的损失。此外，大量的 TiO 被引入 CM 材料的表面，因此，所得的 TiO-CM 材料具有较大的表面积。结果，成功地将制备的 TiO-CM 材料应用于从 BSA/β-酪蛋白（质量比，500/1）混合物的胰蛋白酶消化物中富集得到的磷酸肽。结果优于商业 TiO 珠的结果，证实 TiO-CM 对磷酸肽具有优异的选择性和可重复使用性。此外，在使用 TiO-CM 材料富集后，通过五个独立重复实验，从 2 毫克 Hela 细胞外泌体的胰蛋白酶消化物中成功鉴定出 2661 个磷酸蛋白衍生的 9287 个独特磷酸肽。结果表明，该材料在蛋白质磷酸化分析中具有良好的应用前景。此外，TiO-CM 由绿色和廉价的纤维素作为骨架组成，其合成过程环保、简单且廉价。
