1 One-pot synthesis of magnetic colloidal nanocrystal clusters coated 2 with chitosan for selective enrichment of glycopeptides 3 Chunli Fang a Q1 , Zhichao Xiong b , Hongqiang Qin c , Guang Huang c , Jing Liu c , 4 Mingliang Ye c , Shun Feng a, *, Hanfa Zou c, ** 5 a Key Laboratory of Oil Gas Fine Chemicals, Ministry of Education Xinjiang Uyghur Autonomous Region, College of Chemistry and Chemical Engineering, 6 Xinjiang University, Urumqi 830046, China 7 b Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, China 8 c Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China H I G H L I G H T S G R A P H I C A L A B S T R A C T  Chitosan coated MCNC materials were prepared by a simple one-pot method.  The Fe 3 O 4 @CS MCNC materials showed super hydrophilicity.  The material had high selectivity and efficiency for enrichment of glyco- peptides. A R T I C L E I N F O Article history: Received 26 March 2014 Received in revised form 9 May 2014 Accepted 25 May 2014 Available online xxx Keywords: Glycopeptides Magnetic colloidal nanocrystal clusters Chitosan Hydrophilic interaction liquid chromatography A B S T R A C T Selective enrichment of glycopeptides prior to the mass spectrometry (MS) analysis is essential due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among the enrichment approaches, hydrophilic interaction liquid chromatography (HILIC) based on magnetic separation has become a popular method in recent years. As the conventional synthesis procedures of these materials are tedious and time-consuming with at least four steps. Herein, magnetic colloidal nanocrystal clusters coated with chitosan (Fe 3 O 4 @CS MCNCs) have been successfully prepared by a simple one-pot method. The resulting Fe 3 O 4 @CS MCNCs demonstrated an excellent ability for glycopeptide enrichment with high selectivity, low detection limit and high binding capacity. Furthermore, in the analysis of real complicated biological sample, 283 unique N-glycosylation sites corresponding to 175 glycosylated proteins were identified in three replicate analyses of 45 mg protein sample extracted from HeLa cells, indicating the great potential in detection and identification of low abundant glycopeptides in glycoproteome analysis. ã 2014 Published by Elsevier B.V. 9 1. Introduction 10 Protein glycosylation is one of the most important post- 11 translational modifications (PTMs) and plays important structural 12 and functional roles in multiple biological processes, including cell 13 –cell interaction, immune defence, cell growth and differentiation 14 [1,2]. And approximately 50% of the mammalian proteome is 15 composed of glycoproteins [3]. Aberrant glycosylation on some * Corresponding author at: No. 14 Shengli Road, Urumqi, China. Tel.: +86 991 8582087; fax: +86 991 8582087. ** Corresponding author at: No. 457 Zhongshan Road, Dalian, China. Tel.: +86 411 84379610; fax: +86 411 84379620. E-mail addresses: fengshun@xju.edu.cn (S. Feng), hanfazou@dicp.ac.cn (H. Zou). http://dx.doi.org/10.1016/j.aca.2014.05.037 0003-2670/ ã 2014 Published by Elsevier B.V. Analytica Chimica Acta xxx (2014) xxx–xxx G Model ACA 233281 1–7 Please cite this article in press as: C. Fang, et al., One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.037 Contents lists available at ScienceDirect Analytica Chimica Acta journa l home page : www.e lsevier.com/loca te/aca