Zhang Chengqian, Ye Zilu, Xue Peng, Shu Qingbo, Zhou Yue, Ji Yanlong, Fu Ying, Wang Jifeng, Yang Fuquan
Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences , Beijing 100101, China.
University of Chinese Academy of Sciences , Beijing100049, China.
J Proteome Res. 2016 Sep 2;15(9):2960-8. doi: 10.1021/acs.jproteome.6b00098. Epub 2016 Aug 11.
N-Glycosylation of proteins plays a critical role in many biological pathways. Because highly heterogeneous N-glycopeptides are present in biological sources, the enrichment procedure is a crucial step for mass spectrometry analysis. Five enrichment methods, including IP-ZIC-HILIC, hydrazide chemistry, lectin affinity, ZIC-HILIC-FA, and TiO2 affinity were evaluated and compared in the study of mapping N-glycosylation sites in mouse brain. On the basis of our results, the identified N-glycosylation sites were 1891, 1241, 891, 869, and 710 and the FDR values were 3.29, 5.62, 9.54, 9.54, and 20.02%, respectively. Therefore, IP-ZIC-HILIC enrichment method displayed the highest sensitivity and specificity. In this work, we identified a total of 3446 unique glycosylation sites conforming to the N-glycosylation consensus motif (N-X-T/S/C; X ≠ P) with (18)O labeling in 1597 N-glycoproteins. N-glycosylation site information was used to confirm or correct the transmembrane topology of the 57 novel transmembrane N-glycoproteins.
蛋白质的N-糖基化在许多生物途径中起着关键作用。由于生物来源中存在高度异质的N-糖肽，富集过程是质谱分析的关键步骤。在小鼠大脑N-糖基化位点图谱研究中，评估并比较了包括IP-ZIC-HILIC、酰肼化学、凝集素亲和、ZIC-HILIC-FA和TiO2亲和在内的五种富集方法。根据我们的结果，鉴定出的N-糖基化位点分别为1891、1241、891、869和710个，FDR值分别为3.29%、5.62%、9.54%、9.54%和20.02%。因此，IP-ZIC-HILIC富集方法显示出最高的灵敏度和特异性。在这项工作中，我们通过(18)O标记在1597种N-糖蛋白中总共鉴定出3446个符合N-糖基化共有基序（N-X-T/S/C；X≠P）的独特糖基化位点。N-糖基化位点信息用于确认或校正57种新型跨膜N-糖蛋白的跨膜拓扑结构。
