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学科主题: 分析化学
题名: Technology and Method Development for Proteome Analysis
作者: Zou HF(邹汉法) ;  Ye ML(叶明亮) ;  Wu RA(吴仁安)
会议文集: The Croucher ASI for the functional phosphoproteomics in study of plant cell signaling and molecular systems biology
会议名称: The Croucher ASI for the functional phosphoproteomics in study of plant cell signaling and molecular systems biology
会议日期: 2011-1-3
出版日期: 2011
会议地点: 香港
其他题名: 蛋白质分析新技术新方法
通讯作者: 邹汉法
出版者: 待补充
出版地: 待补充
合作性质: 特邀报告
部门归属: 1809
主办者: 香港裘槎基金会高级研究所
摘要: Monolithic columns have been widely applied in capillary liquid chromatography (μLC) and capillary electrochromatography (CEC) due to their easy preparations, low back pressure, convective mass transfer process and high separation performances comparing to the particle packed columns or the open tubular columns. By taking advantages of polymeric monoliths, we have prepared a SCX monolithic column based on the functional monomer ethylene glycol methacrylate phosphate (EGMP) to trap and fractionate peptides in shotgun proteome analysis. A biphasic monolithic column was synthesized with 10 cm segment of SCX monolithic and 65 cm segment of RP monolith in a single 100 μm i.d. capillary in this study. This biphasic monolithic column was applied in online multidimensional separation for shotgun proteome analysis under relatively low operating pressure of ~900 psi, and good separation performance of this biphasic column was demonstrated. Furthermore, the inorganic silica-based monolithic columns demonstrate good solvent resistance C18 stationary phase for highly efficient separation of tryptic digest of yeast proteins, and more than 5000 unique peptides were identified by μLC-MS/MS analysis under 500 min gradient elution. Low-molecular weight (MW) proteome has drawn great attention in understanding biological systems for its adequate information of probable biomarkers and signal molecules in biology and pathology. An MPS-based “one-step” size-selective enzymatic digestion mode for achieving the highly efficient proteome analysis of low-MW proteins from a complex protein mixture was established by combination of size-exclusive separation and enzyme immobilization on a thiol-modified SBA-15 material (SBA-15-SH). Furthermore, highly ordered mesoporous silica particles were successfully applied for enrichment of the peptidome from human plasma with their highly ordered pores and nearly 95% in-pore surface area to ensure the highly selective enrichment based on a combination of adsorption and a size-exclusion mechanism. In silico digestion of proteins in human proteome database by trypsin indicates that a significant percentage of tryptic N-glycopeptides is not in the preferred detection mass range of shotgun proteomics approach, that is, from 800 to 3500 Da. And the quite big size of glycan groups may block trypsin to access the K, R residues near N-glycosites for digestion, which will result in generation of big glycopeptides. Thus many N-glycosites could not be localized if only trypsin was used to digest proteins. We developed a comprehensive way to analyze the N-glycoproteome of human liver tissue by combination of hydrazide chemistry method and multiple enzyme digestion, which leads to great improvement in coverage of N glycosites identified. Application of glycoproteome analysis in biomarker discovery was demonstrated.
英文摘要: Monolithic columns have been widely applied in capillary liquid chromatography (μLC) and capillary electrochromatography (CEC) due to their easy preparations, low back pressure, convective mass transfer process and high separation performances comparing to the particle packed columns or the open tubular columns. By taking advantages of polymeric monoliths, we have prepared a SCX monolithic column based on the functional monomer ethylene glycol methacrylate phosphate (EGMP) to trap and fractionate peptides in shotgun proteome analysis. A biphasic monolithic column was synthesized with 10 cm segment of SCX monolithic and 65 cm segment of RP monolith in a single 100 μm i.d. capillary in this study. This biphasic monolithic column was applied in online multidimensional separation for shotgun proteome analysis under relatively low operating pressure of ~900 psi, and good separation performance of this biphasic column was demonstrated. Furthermore, the inorganic silica-based monolithic columns demonstrate good solvent resistance C18 stationary phase for highly efficient separation of tryptic digest of yeast proteins, and more than 5000 unique peptides were identified by μLC-MS/MS analysis under 500 min gradient elution. Low-molecular weight (MW) proteome has drawn great attention in understanding biological systems for its adequate information of probable biomarkers and signal molecules in biology and pathology. An MPS-based “one-step” size-selective enzymatic digestion mode for achieving the highly efficient proteome analysis of low-MW proteins from a complex protein mixture was established by combination of size-exclusive separation and enzyme immobilization on a thiol-modified SBA-15 material (SBA-15-SH). Furthermore, highly ordered mesoporous silica particles were successfully applied for enrichment of the peptidome from human plasma with their highly ordered pores and nearly 95% in-pore surface area to ensure the highly selective enrichment based on a combination of adsorption and a size-exclusion mechanism. In silico digestion of proteins in human proteome database by trypsin indicates that a significant percentage of tryptic N-glycopeptides is not in the preferred detection mass range of shotgun proteomics approach, that is, from 800 to 3500 Da. And the quite big size of glycan groups may block trypsin to access the K, R residues near N-glycosites for digestion, which will result in generation of big glycopeptides. Thus many N-glycosites could not be localized if only trypsin was used to digest proteins. We developed a comprehensive way to analyze the N-glycoproteome of human liver tissue by combination of hydrazide chemistry method and multiple enzyme digestion, which leads to great improvement in coverage of N glycosites identified. Application of glycoproteome analysis in biomarker discovery was demonstrated.
语种: 英语
内容类型: 会议论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/115929
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:
Zou HF,Ye ML,Wu RA. Technology and Method Development for Proteome Analysis[C]. 见:The Croucher ASI for the functional phosphoproteomics in study of plant cell signaling and molecular systems biology. 香港. 2011-1-3.
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