In Situ Synthesis of Magnetic Mesoporous Phenolic Resin for the Selective Enrichment of Glycopeptides
In Situ Synthesis of Magnetic Mesoporous Phenolic Resin for the Selective Enrichment of Glycopeptides
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原位合成磁性介孔酚醛树脂选择性富集糖肽
DOI：
10.1021/acs.analchem.8b00708
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发表时间：
2018
影响因子：
7.4
通讯作者：
Yang Pengyuan
中科院分区：
文献类型：
--
作者：
Zhang Quanqing;Huang Yuanyu;Jiang Biyun;Hu Yajun;Xie Juanjuan;Gao Xing;Jia Bin;Shen Huali;Zhang Weijia;Yang Pengyuan
蛋白质糖基化是许多生物过程的重要参与者，是蛋白质翻译后修饰的关键。由于糖肽与非糖肽相比含量较低，糖基化位点的微观异质性，需要高选择性的糖肽纯化方法来进行糖蛋白组学的全面表征。本工作以支化聚乙烯亚胺(PEI)为交联剂，在间苯二酚/甲醛单体水溶液体系中，采用原位乳液聚合法，从磁性Fe3O4@SiO2溶液中制备了磁性介孔酚醛树脂。多种表征方法表明，基质金属氧化物具有良好的生物相容性、介孔结构、含氮官能度、良好的亲水性和耐溶剂性。利用基质金属蛋白酶表面亲水基团与糖肽上的糖组分相互作用，选择性地捕获N-糖肽(人免疫球蛋白或辣根过氧化物酶消化/牛血清白蛋白=1：50)，回收率高(70.18-97.23%)，结合能力强(400 mg g-1)，重复性好。基于基质金属蛋白酶在标准糖蛋白胰酶解液浓缩中的优异性能，我们进一步将其用于从人血清胰酶解液中捕获N-糖肽。从超低样本量(0.025μL)的人血清中共鉴定出15个独特的N-糖肽。总体而言，在三次重复分析的重叠部分中，我们从人血清(0.35μL)中鉴定出与119种糖蛋白相对应的356个独特的N-糖肽。这些结果表明，基质金属蛋白酶在大规模N-糖蛋白组学研究中具有很大的潜力。
Protein glycosylation is a significant participant in a mass of biological processes, which is a pivotal protein post-translational modification. Due to the low contents of glycopeptides compared with nonglycopeptides and the microheterogeneity of glycosylation sites, highly selective enrichment methods for the purification of glycopeptides are required for the comprehensive characterization of glycoproteomics. In this work, a type of magnetic mesoporous phenolic resin (MMP) was prepared using branched polyethylenimine (PEI) as a cross-linker from a homogeneous magnetic Fe3O4@SiO2solution in a resorcinol/formaldehyde monomer aqueous system via an in situ emulsion polymerization procedure. The results showed that MMP exhibited good biocompatibility, a mesoporous structure, nitrogen-containing functionality, excellent hydrophilicity, and solvent resistance by using multiple characterization methods. By taking advantage of the interaction between hydrophilic groups on the MMP and glycan components on the glycopeptides, the acquired MMP was utilized to the selective capture of N-glycopeptides (human IgG or HRP tryptic digests/BSA proteins = 1:50), good recovery yield (70.18–97.23%), superior binding capacity (400 mg g–1), and excellent reproducibility. Based on the outstanding performance in standard glycoproteins tryptic digests enrichment, MMP was further used to capture N-glycopeptides from tryptic digests of human serum. A total of 15 unique N-glycopeptides were identified from an ultralow sample volume (0.025 μL) of human serum. Overall, we identified 356 unique N-glycopeptides corresponding to 119 glycoproteins from human serum (0.35 μL) in the overlap of three replicate analyses. All the results have demonstrated that MMP has great potential in large-scale N-glycoproteomics research.