Citation: LI Feng, KANG Jingwu. Enrichment of glycoproteins in human serum using concanavalin A-functionalized magnetic nanoparticles and identification by mass spectrometry[J]. Chinese Journal of Chromatography, ;2014, 32(4): 369-375. doi: 10.3724/SP.J.1123.2013.12018 shu

Enrichment of glycoproteins in human serum using concanavalin A-functionalized magnetic nanoparticles and identification by mass spectrometry

LI Feng ,
KANG Jingwu ^,

1.
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China

Corresponding author: KANG Jingwu,
Received Date: 18 December 2013
Available Online: 8 January 2014
Fund Project: 国家自然科学基金项目(21175146，20975109). (21175146，20975109)

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein novel magnetic nanoparticles with an average size around 135 nm in diameter were prepared for the enrichment of glycoproteins in human serum. The prepared magnetic nanoparticles possessed uniform core/shell/shell structure which was composed of 8 nm magnetite internal core and double layers consisting of silica and poly glycidyl methacrylate (GMA). The latter was constructed by seed polymerization. Modified by a polyethylene hydrophilic linker, it made the surfaces of the magnetic nanoparticles highly hydrophilic so as to reduce the nonspecific adsorption of proteins. We examined affinity purification of glycoprotein in diluted human serum using our prepared magnetic nanoparticles with immobilization of concanavalin A (MNP@ConA). The enriched proteins were reduced, alkylated and digested with trypsin. These peptides then were separated by offline two-dimensional chromatography. Protein identification was realized with nano-high performance liquid chromatography-orbitrap mass spectrometry. A total of 80 proteins were identified, among them 76 proteins were found to be glycoproteins by use of bioinformatic tools. β-2-Glycoprotein 1 present in serum at low mass concentration around 0.00001 g/L was also identified. This demonstrates the capability of magnetic nanoparticle for recovering minute amounts of glycoproteins from a fluid exhibiting a dynamic concentration range more than 12 orders of magnitude. Overall, MNP@ConA has been proven to be an efficient alternative to currently available immobilization supports.
- liquid chromatography (LC),
- mass spectrometry (MS),
- enrichment,
- glycoprotein,
- lectin,
- magnetic nanoparticle
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