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学科主题分析化学
Combining mPEG-Hz Derivatization and Filter-Based Size Exclusion for Selective Enrichment of N-glycopeptides
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
2011
会议地点圣地亚哥
页码66-0
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
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.; 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
文献类型会议论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/116022
专题中国科学院大连化学物理研究所
通讯作者Zhang LH(张丽华)
推荐引用方式
GB/T 7714
Zhang LH,Hou CY,Liang Z,et al. Combining mPEG-Hz Derivatization and Filter-Based Size Exclusion for Selective Enrichment of N-glycopeptides[C]. 待补充:待补充,2011:66-0.
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