蛋白质的 N-糖基化修饰与多种重要的生理、病理进程密切相关，是多种重大疾病诊断标志物研究的热点。由于糖蛋白本身多是低丰度表达的蛋白质，且糖链结构具有高度微不均一性，这使得蛋白质糖基化修饰的分析具有一定的挑战。本研究利用表面引发-原子转移自由基聚合（ SI-ATRP ）法，以带有双键的氨基葡萄糖为单体（GMAG），成功制备了新型核壳型亲水聚合物-硅胶杂化填料（pGMAG-SiO2）。由于在硅胶表面引入致密的亲水聚合物层，该填料不仅保持了硅胶良好的机械强度，而且显著提高了其亲水性，因此非常适合作为亲水填料用于蛋白质的N-糖链富集。以麦芽七糖和鸡卵清蛋白的N-糖链为研究对象，考察了该填料对N-糖链的富集效果，并将该杂化填料成功用于人血浆中糖蛋白 N-糖链的富集检测，共鉴定了47种糖型。以上结果表明，pGMAG-SiO2填料对N-糖链具有较高的亲和性，可以用于 N-糖链的高覆盖率鉴定。
Protein N-glycosylation is one of the most important post-translational modifications closely correlated with many important biological and pathological processes. The structural alterations of N-linked glycans in glycoproteins are always associated with many diseases,such as diabetes,heart failure and malignant tumors. Therefore,it is very important to establish sen-sitive methods for high-throughput N-glycan profiling. However,the low abundance of the N-glycoproteins and the heterogeneity of the N-glycans make it a challenge to analyse the pro-tein glycosylation sensitively. In this work,we had synthesized core-shell hydrophilic polymer-silica hybrid materials(pGMAG-SiO2)for the efficient enrichment of protein N-glycans. Firstly, pGMAG-SiO2 was prepared by in situ growth of glucose polymer on the surface of silica microp-articles using surface-initiated atom transfer radical polymerization( SI-ATRP)technique. The strong hydrophilicity of the material makes it suitable for the enrichment of N-glycans released from complex samples. Secondly,maltoheptaose and the N-glycans from chicken egg albumin were used as standard samples to optimize the enrichment conditions and evaluate the enrich-ment efficiency of pGMAG-SiO2. Finally,pGMAG-SiO2 was applied to the enrichment of N-linked glycans from human plasma proteins and 47 glycoforms were successfully identified after enrichment. These results demonstrated the high enrichment efficiency and significant applica-tion value of pGMAG-SiO2 in the analysis of N-glycans.
参考文献
| [1] | Zaia, J..At last, functional glycomics[J].Nature methods,20111(1):55-57. |
| [2] | Lau K S;Partridge E A;Grigorian A.[J].CELL,2007129(1):123. |
| [3] | Balog,C.I.A.;Stavenhagen,K.;Fung,W.L.J.;Koeleman,C.A.;McDonnell,L.A.;Verhoeven,A.;Mesker,W.E.;Tollenaar,R.A.E.M.;Deelder,A.M.;Wuhrer,M..N-glycosylation of colorectal cancer tissues: A liquid chromatography and mass spectrometry-based investigation[J].Molecular & cellular proteomics: MCP,20129(9):571-585. |
| [4] | Robajac D;Masnikosa R;Filimonovic D.[J].J Med Bio-chem,201231(3):205. |
| [5] | Anderson N L;Anderson N G.[J].Molecular & Cellular Proteomics,20021(11):845. |
| [6] | Ruiz-May,E.;Hucko,S.;Howe,K.J.;Zhang,S.;Sherwood,R.W.;Thannhauser,T.W.;Rose,J.K.C..A comparative study of lectin affinity based plant n-glycoproteome profiling using tomato fruit as a model[J].Molecular & cellular proteomics: MCP,20142(2):566-579. |
| [7] | Ruiz-May E;Catalá C;Rose J K.[J].Methods in Molecular Biology,20141072:633. |
| [8] | Xu Y;Bailey U M;Punyadeera C.[J].Rapid Communications in Mass Spectrometry,201428(5):471. |
| [9] | Zhang Y T;Ma W F;Li D.[J].SMALL,201410(7):1379. |
| [10] | Wan H H;Yan J Y;Yu L.[J].ANALYST,2011136(21):4422. |
| [11] | 马成;潘一廷;张琪;王继峰;钱小红;应万涛.利用亲水富集策略分析人血浆中N-糖基化蛋白及N-糖类型[J].色谱,2013(11):1057-1063. |
| [12] | Regnier F E;Noel R.[J].Journal of Chromatography Science,197614:316. |
| [13] | Jackowska M;Bocian S;Buszewski B.[J].ANALYST,2012137(19):4610. |
| [14] | Tissot B;Ceroni A;Powell A K.[J].Analytical Chemistry,200880(23):9204. |
| [15] | 宋子凤;张庆林;张养军;秦伟捷;钱小红.新型鳞片状聚合物修饰硅胶填料固定化酶的制备及其在蛋白质组鉴定中的应用[J].色谱,2012(6):549-554. |
| [16] | 范超;宋子凤;秦伟捷;蔡耘;钱小红.新型亲水聚合物修饰硅胶颗粒固定化酶的制备及在蛋白质组鉴定中的应用[J].色谱,2013(5):423-428. |
| [17] | Lattova E;Perreault HN;Krokhin O.Matrix-assisted laser desorption/ionization tandem mass spectrometry and post-source decay fragmentation study of phenylhydrazones of N-linked oligosaccharides from ovalbumin[J].Journal of the American Society for Mass Spectrometry,20045(5):725-735. |
| [18] | Zhong Y;Shen S;Zhou Y D.[J].MEDICAL ONCOLOGY,201431(3):864. |
| [19] | Vermassen,T.;Speeckaert,M.M.;Lumen,N.;Rottey,S.;Delanghe,J.R..Glycosylation of prostate specific antigen and its potential diagnostic applications[J].Clinica chimica acta: International journal of clinical chemistry and applied molecular biology,201219/20(19/20):1500-1505. |
| [20] | Lai Q;Avolio A W;Graziadei I.[J].Journal of Hepatology,201460(6):1332. |
| [21] | Bereman, MS;Young, DD;Deiters, A;Muddiman, DC.Development of a Robust and High Throughput Method for Profiling N-Linked Glycans Derived from Plasma Glycoproteins by NanoLC-FTICR Mass Spectrometry[J].Journal of proteome research,20097(7):3764-3770. |
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