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学科主题: 分析化学
题名: Combining mPEG-Hz Derivatization and Filter-Based Size Exclusion for Selective Enrichment of N-glycopeptides
作者: Zhang LH(张丽华) ;  Hou CY(侯春彦) ;  Liang Z(梁振) ;  Zhang YK(张玉奎)
会议文集: 26th International Symposium on Microscale Bioseparations
会议名称: 26th International Symposium on Microscale Bioseparations
会议日期: 2011-5-1
出版日期: 2011
会议地点: 圣地亚哥
通讯作者: 张丽华
出版者: 待补充
出版地: 待补充
合作性质: 墙报
部门归属: 1810
主办者: CASSS
摘要: Although hydrazide (Hz) is widely used for the selective enrichment of N-glycopeptides, it takes a long time to wash off the non-specifically adsorbed peptides on the Hz beads. To solve this problem, in our recent work, a strategy with the combination of methoxy-polyethylene glycol (mPEG)-Hz based derivatization and filter-based size exclusion, was developed, and successfully applied for the facile enrichment of N-glycopeptides. Herein, glycoproteins were first digested, followed by the oxidization of the cis-diol groups of carbohydrates on glycopeptides to aldehydes, which could further form covalent hydrazone bonds with mPEG-Hz. With the increased molecular weights, the derivatized glycopeptides could be separated from other peptides by a centrifugal filter device. After that, PNGase F was added to cut off mPEG-conjugated carbohydrates, which could be kept in the retentate, while the released peptides could be collected in filtrate after centrifuge. HRP, a standard glycoprotein, was used to evaluate this protocol. With 10-kDa mPEG-Hz for N-glycopeptide enrichment, and filter devices with nominal molecular weight limit of 10 kDa to removal non-glycopeptides, 5 peptides with N-glycosite were detected, with the specificity consistent to that obtained with the commercial hydrazide beads. Most importantly, the removal of non-glycopeptides became easy by filter-based size exclusion, beneficial to shorten the sample preparation time. With the further optimization of the size of mPEG-Hz and the cut off molecular weight of filter, improved selectivity of glycopeptide enrichment is expected to achieve, which might be promising in N-glycoproteome analysis.
英文摘要: Although hydrazide (Hz) is widely used for the selective enrichment of N-glycopeptides, it takes a long time to wash off the non-specifically adsorbed peptides on the Hz beads. To solve this problem, in our recent work, a strategy with the combination of methoxy-polyethylene glycol (mPEG)-Hz based derivatization and filter-based size exclusion, was developed, and successfully applied for the facile enrichment of N-glycopeptides. Herein, glycoproteins were first digested, followed by the oxidization of the cis-diol groups of carbohydrates on glycopeptides to aldehydes, which could further form covalent hydrazone bonds with mPEG-Hz. With the increased molecular weights, the derivatized glycopeptides could be separated from other peptides by a centrifugal filter device. After that, PNGase F was added to cut off mPEG-conjugated carbohydrates, which could be kept in the retentate, while the released peptides could be collected in filtrate after centrifuge. HRP, a standard glycoprotein, was used to evaluate this protocol. With 10-kDa mPEG-Hz for N-glycopeptide enrichment, and filter devices with nominal molecular weight limit of 10 kDa to removal non-glycopeptides, 5 peptides with N-glycosite were detected, with the specificity consistent to that obtained with the commercial hydrazide beads. Most importantly, the removal of non-glycopeptides became easy by filter-based size exclusion, beneficial to shorten the sample preparation time. With the further optimization of the size of mPEG-Hz and the cut off molecular weight of filter, improved selectivity of glycopeptide enrichment is expected to achieve, which might be promising in N-glycoproteome analysis.
语种: 英语
内容类型: 会议论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/116022
Appears in Collections:中国科学院大连化学物理研究所_会议论文

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Recommended Citation:
Zhang LH,Hou CY,Liang Z,et al. Combining mPEG-Hz Derivatization and Filter-Based Size Exclusion for Selective Enrichment of N-glycopeptides[C]. 见:26th International Symposium on Microscale Bioseparations. 圣地亚哥. 2011-5-1.
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