Department of Chemistry, Fudan University, Shanghai, 200433, China.
Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China.
Anal Bioanal Chem. 2018 Jan;410(3):989-998. doi: 10.1007/s00216-017-0602-5. Epub 2017 Sep 20.
Protein glycosylation is one of the most important post-translational modifications. Also, efficient enrichment and separation of glycopeptides from complex samples are crucial for the thorough analysis of glycosylation. Developing novel hydrophilic materials with facile and easily popularized synthesis is an urgent need in large-scale glycoproteomics research. Herein, for the first time, a one-step functionalization strategy based on metal-organic coordination was proposed and FeO nanoparticles were directly functionalized with zwitterionic hydrophilic L-cysteine (L-Cys), greatly simplifying the synthetic procedure. The easily synthesized FeO/L-Cys possessed excellent hydrophilicity and brief composition, contributing to affinity for glycopeptides and reduction in nonspecific interaction. Thus, FeO/L-Cys nanoparticles showed outstanding sensitivity (25 amol/μL), high selectivity (mixture of bovine serum albumin and horseradish peroxidase tryptic digests at a mass ratio of 100:1), good reusability (five repeated times), and stability (room temperature storage of 1 month). Encouragingly, in the glycosylation analysis of human serum, a total of 376 glycopeptides with 393 N-glycosylation sites corresponding to 118 glycoproteins were identified after enrichment with FeO/L-Cys, which was superior to ever reported L-Cys modified magnetic materials. Furthermore, applying the one-step functionalization strategy, cysteamine and glutathione respectively direct-functionalized FeO nanoparticles were successfully synthesized and also achieved efficient glycopeptide enrichment in human serum. The results indicated that we have presented an efficient and easily popularized strategy in glycoproteomics as well as in the synthesis of novel materials. Graphical abstract FeO/L-Cys nanoparticles based on one-step functionalization for highly efficient enrichment of glycopeptides.
蛋白质糖基化是最重要的翻译后修饰之一。此外，从复杂样品中高效富集和分离糖肽对于糖基化的深入分析至关重要。开发具有简便易行且易于推广合成的新型亲水材料是大规模糖蛋白质组学研究的迫切需要。在此，首次提出了一种基于金属有机配位的一步功能化策略，并直接将两性离子亲水 L-半胱氨酸 (L-Cys) 功能化到 FeO 纳米颗粒上，极大地简化了合成步骤。合成的 FeO/L-Cys 具有出色的亲水性和简单的组成，有利于与糖肽结合并减少非特异性相互作用。因此，FeO/L-Cys 纳米颗粒表现出出色的灵敏度（25 amol/μL）、高选择性（牛血清白蛋白和辣根过氧化物酶酶解物的混合物，质量比为 100:1）、良好的可重复使用性（重复 5 次）和稳定性（室温储存 1 个月）。令人鼓舞的是，在用 FeO/L-Cys 进行富集后，在人血清的糖基化分析中，共鉴定出 376 种糖肽，对应 118 种糖蛋白，共 393 个 N-糖基化位点，优于以往报道的 L-Cys 修饰的磁性材料。此外，应用一步功能化策略，半胱胺和谷胱甘肽分别成功地直接功能化了 FeO 纳米颗粒，并在人血清中实现了高效的糖肽富集。结果表明，我们在糖蛋白质组学以及新型材料的合成中提出了一种高效且易于推广的策略。
