In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins

// Pan Fang 1 , Xin-jian Wang 2 , Yu Xue 3 , Ming-qi Liu 1 , Wen-feng Zeng 4 , Yang Zhang 1 , Lei Zhang 1 , Xing Gao 1 , Guo-quan Yan 3 , Jun Yao 1 , Hua-li Shen 1, 5 , Peng-yuan Yang 1, 3, 5 1 Minhang Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai, China 2 State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China 3 Department of Chemistry, Fudan University, Shanghai, China 4 Key Laboratory of Intelligent Information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS, Beijing, China 5 Department of Systems Biology for Medicine and School of Basic Medical Sciences, Fudan University, Shanghai, China Correspondence to: Hua-li Shen, email: shenhuali@fudan.edu.cn Peng-yuan Yang, email: pyyang@fudan.edu.cn Keywords: N-glycoproteomics, mouse brain, mass spectrometry, brain physiological activities, disease biomarker Received: January 25, 2016      Accepted: April 26, 2016      Published: May 31, 2016 ABSTRACT N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation strategies. Altogether, 13492 N-glycopeptides containing 8386 N-glycosylation sites on 3982 proteins were identified. After evaluating the performance of the above methods, we proposed a simple and efficient workflow for large-scale N-glycosylation site mapping. The optimized workflow yielded 80% of the initially identified N-glycosylation sites with considerably less effort. Analysis of the identified N-glycoproteins revealed that many of the mouse brain proteins are N-glycosylated, including those proteins in critical pathways for nervous system development and neurological disease. Additionally, several important biomarkers of various diseases were found to be N-glycosylated. These data confirm that N-glycosylation is important in both physiological and pathological processes in the brain, and provide useful details about numerous N-glycosylation sites in brain proteins.