Citation: ZHANG Ying, LIU Ying, LIU Yang, QIANG Shan, YANG Bing-Ying, HUANG Lin-Juan, WANG Zhong-Fu. Pretreatment Method for Detecting of N-linked Glycans of Glycoprotein in Micro-scale Complex Biological Sample based on Electrospray Ionization Mass Spectrometry[J]. Chinese Journal of Analytical Chemistry, ;2016, 44(2): 265-272. doi: 10.11895/j.issn.0253-3820.150119 shu

Pretreatment Method for Detecting of N-linked Glycans of Glycoprotein in Micro-scale Complex Biological Sample based on Electrospray Ionization Mass Spectrometry

1.
Life Science College, Northwest University, Xi'an 710069, China

Corresponding author: WANG Zhong-Fu,
Received Date: 11 April 2015
Available Online: 7 July 2015
Fund Project: 本文系国家自然科学基金(Nos.31170773, 31370804, 31300678)资助 (Nos.31170773, 31370804, 31300678)

A reliable, efficient and simple method was developed for releasing, enrichment and purification of N-glycan from glycoprotein based on electrospray ionization mass spectrometric (ESI-MS) detection technology. The bovine ribonuclease B(Rib B) and ovalbumin were used as model glycoprotein samples and subjected to direct enzymolysis to investigate the purification effects of four different purification methods. Then the enzymatic efficiency of micro-scale complex fetal bovine serum sample subjected to direct and indirect enzymolysis after polyvinylidene fluoride (PVDF) membrane enrichment was fully investigated. Finally, a pretreatment method of N-linked glycans from glycoprotein in micro-scale complex biological sample analyzed by ESI-MS was established. The PNGase F was exploited for directly releasing of glycans from glycoprotein enriched on PVDF membrane (37℃, 24 h). Then microcrystalline cellulose chromatography column combined with graphite carbon chromatography column was used to enrich and purify glycans from enzymolysis products. The assay was eventually applied to pretreatment of N-glycan in micro-scale biological samples (μg level) of fetal bovine serum and healthy human serum for MS analysis. The method with universality showed good prospects in pretreatment of N-glycan in micro-scale biological sample for MS analysis.
- Micro-scale analysis,
- Glycoprotein,
- N-Linked glycans,
- Electrospray ionization mass spectrometry,
- Pretreatment
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沈阳化工大学材料科学与工程学院沈阳 110142