Liu Liting, Yu Meng, Zhang Ying, Wang Changchun, Lu Haojie
Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, P. R. China.
ACS Appl Mater Interfaces. 2014 May 28;6(10):7823-32. doi: 10.1021/am501110e. Epub 2014 Apr 29.
In view of the biological significance of glycosylation for human health, profiling of glycoproteome from complex biological samples is highly inclined toward the discovery of disease biomarkers and clinical diagnosis. Nevertheless, because of the existence of glycopeptides at relatively low abundances compared with nonglycosylated peptides and glycan microheterogeneity, glycopeptides need to be highly selectively enriched from complex biological samples for mass spectrometry analysis. Herein, a new type of hydrazide functionalized core-shell magnetic nanocomposite has been synthesized for highly specific enrichment of N-glycopeptides. The nanocomposites with both the magnetic core and the polymer shell hanging high density of hydrazide groups were prepared by first functionalization of the magnetic core with polymethacrylic acid by reflux precipitation polymerization to obtain the Fe3O4@poly(methacrylic acid) (Fe3O4@PMAA) and then modification of the surface of Fe3O4@PMAA with adipic acid dihydrazide (ADH) to obtain Fe3O4@poly(methacrylic hydrazide) (Fe3O4@PMAH). The abundant hydrazide groups toward highly specific enrichment of glycopeptides and the magnetic core make it suitable for large-scale, high-throughput, and automated sample processing. In addition, the hydrophilic polymer surface can provide low nonspecific adsorption of other peptides. Compared to commercially available hydrazide resin, Fe3O4@PMAH improved more than 5 times the signal-to-noise ratio of standard glycopeptides. Finally, this nanocomposite was applied in the profiling of N-glycoproteome from the colorectal cancer patient serum. In total, 175 unique glycopeptides and 181 glycosylation sites corresponding to 63 unique glycoproteins were identified in three repeated experiments, with the specificities of the enriched glycopeptides and corresponding glycoproteins of 69.6% and 80.9%, respectively. Because of all these attractive features, we believe that this novel hydrazide functionalized core-shell magnetic nanocomposite will shed new light on the profiling of N-glycoproteome from complex biological samples in high throughput.
鉴于糖基化对人类健康的生物学意义，对复杂生物样品中的糖蛋白质组进行分析对于发现疾病生物标志物和临床诊断具有重要意义。然而，由于与非糖基化肽相比，糖肽的丰度相对较低，且存在聚糖微异质性，因此需要从复杂生物样品中高度选择性地富集糖肽，以便进行质谱分析。在此，合成了一种新型的酰肼功能化核壳磁性纳米复合材料，用于高度特异性富集N-糖肽。通过回流沉淀聚合用聚甲基丙烯酸对磁性核进行首次功能化，得到Fe3O4@聚（甲基丙烯酸）（Fe3O4@PMAA），然后用己二酸二酰肼（ADH）对Fe3O4@PMAA的表面进行修饰，得到Fe3O4@聚（甲基丙烯酸酰肼）（Fe3O4@PMAH），从而制备出具有高密度酰肼基团的磁性核和聚合物壳的纳米复合材料。大量的酰肼基团对糖肽具有高度特异性富集作用，且磁性核使其适用于大规模、高通量和自动化的样品处理。此外，亲水性聚合物表面可降低其他肽的非特异性吸附。与市售酰肼树脂相比，Fe3O4@PMAH将标准糖肽的信噪比提高了5倍以上。最后，将该纳米复合材料应用于结直肠癌患者血清中N-糖蛋白质组的分析。在三次重复实验中，共鉴定出175个独特的糖肽和181个糖基化位点，对应63种独特的糖蛋白，富集的糖肽和相应糖蛋白的特异性分别为69.6%和80.9%。由于所有这些吸引人的特性，我们相信这种新型的酰肼功能化核壳磁性纳米复合材料将为高通量分析复杂生物样品中的N-糖蛋白质组提供新的思路。
