title: Evaluation of Different N-Glycopeptide Enrichment Methods for N-Glycosylation Sites Mapping in Mouse Brain.
abstract: 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.
authors.affiliation: 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.
authors.forename: Chengqian
authors.initials: C
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Zilu
authors.initials: Z
authors.lastname: Ye
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences , Beijing 100101, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Xue
authors.affiliation: 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.
authors.forename: Qingbo
authors.initials: Q
authors.lastname: Shu
authors.affiliation: 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.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: 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.
authors.forename: Yanlong
authors.initials: Y
authors.lastname: Ji
authors.affiliation: 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.
authors.forename: Ying
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences , Beijing 100101, China.
authors.forename: Jifeng
authors.initials: J
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Fuquan
authors.initials: F
authors.lastname: Yang
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D006020:Glycopeptides; D006031:Glycosylation; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D010103:Oxygen Isotopes; D012680:Sensitivity and Specificity
chemical_list: D006020:Glycopeptides; D010103:Oxygen Isotopes
keywords: N-glycosylation sites; TiO2; ZIC-HILIC; hydrazide; lectins; mouse brain