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J Proteome Res. 2016 Sep 2;15(9):2960-8. doi: 10.1021/acs.jproteome.6b00098. Epub 2016 Aug 11.

Evaluation of Different N-Glycopeptide Enrichment Methods for N-Glycosylation Sites Mapping in Mouse Brain.

Zhang C1,2, Ye Z1,2, Xue P1, Shu Q1,2, Zhou Y1,2, Ji Y1,2, Fu Y1,2, Wang J1, Yang F1,2.

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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.


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.


N-glycosylation sites; TiO2; ZIC-HILIC; hydrazide; lectins; mouse brain

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