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学科主题分析化学
Integration of Protein Digestion Microreactors in a Comprehensive Two-Dimensional Liquid Chromatorgraphy System
Fei LC(费立诚); Yuan HM(袁辉明); Zhang LH(张丽华); Zhang YK(张玉奎)
会议文集36th International Symposium on High-Performance Liquid Phase Separations and Related Techniques
会议名称36th International Symposium on High-Performance Liquid Phase Separations and Related Techniques
会议日期2011-6-19
2011
会议地点布达佩斯
页码103-0
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American Chemical Society
英文摘要Proteins are an extremely important class of (bio)macromolecules as virtually no process within cells occurs without their direct involvement. As a consequence, their analysis is of paramount relevance in fields, such as proteomics, food chemistry, pharmaceutical and life-sciences. One of the most common approaches to determine protein structure, as well as to identify them, consists in (1) digesting them and (2) analyzing the resulting peptides. This idea is at the heart of the “bottom-up” approach to proteomics. Protocols include proteolysis (digestion) in a solution containing both a proteolytic enzyme, e.g. trypsin, and the protein(s) of interest. When done in solution, the proteolysis must generally be carried out with only a small amount of the soluble enzyme to avoid autodigestion of the exogenous enzyme. Therefore, the process is slow, requiring several hours to complete. Some of the problems typical of protein digestion in solution can be overcome with use of the enzyme immobilized on a solid support. This approach enables a high localized concentration of the enzyme and a significant acceleration of the digestion without any appreciable autodigestion due to the site isolation effect [1]. One of the most appealing aspects of on-line two-dimensional liquid chromatography (2D-LC) systems is the possibility of automating the whole analysis. When this is done, operator involvement is greatly reduced and the process is speeded-up. Typically, protein digestion occurs as a preliminary step followed by peptide analysis. As an alternative, the whole protein analysis – including digestion – can be automated. For example, Yuan et al. [2] have recently developed a 2D-LC system for high-throughput analysis of intact proteins, in which on-line tryptic digestion and peptide analysis are combined. In this work, we propose to implement 2D-LC systems which integrate digestion microreactors immobilizing different enzymes. The microreactors are set in parallel after the first dimension protein separation. The advantage of this strategy is that separated proteins are subjected to different proteolytic digestion, hence improving sequence coverage. A second separation conducted on the peptides results in valuable information which can be used to more reliably identify protein sequences. The proposed integrated platform will provide a promising tool for high-throughput, high accuracy and high sequence coverage protein analysis.; Proteins are an extremely important class of (bio)macromolecules as virtually no process within cells occurs without their direct involvement. As a consequence, their analysis is of paramount relevance in fields, such as proteomics, food chemistry, pharmaceutical and life-sciences. One of the most common approaches to determine protein structure, as well as to identify them, consists in (1) digesting them and (2) analyzing the resulting peptides. This idea is at the heart of the “bottom-up” approach to proteomics. Protocols include proteolysis (digestion) in a solution containing both a proteolytic enzyme, e.g. trypsin, and the protein(s) of interest. When done in solution, the proteolysis must generally be carried out with only a small amount of the soluble enzyme to avoid autodigestion of the exogenous enzyme. Therefore, the process is slow, requiring several hours to complete. Some of the problems typical of protein digestion in solution can be overcome with use of the enzyme immobilized on a solid support. This approach enables a high localized concentration of the enzyme and a significant acceleration of the digestion without any appreciable autodigestion due to the site isolation effect [1]. One of the most appealing aspects of on-line two-dimensional liquid chromatography (2D-LC) systems is the possibility of automating the whole analysis. When this is done, operator involvement is greatly reduced and the process is speeded-up. Typically, protein digestion occurs as a preliminary step followed by peptide analysis. As an alternative, the whole protein analysis – including digestion – can be automated. For example, Yuan et al. [2] have recently developed a 2D-LC system for high-throughput analysis of intact proteins, in which on-line tryptic digestion and peptide analysis are combined. In this work, we propose to implement 2D-LC systems which integrate digestion microreactors immobilizing different enzymes. The microreactors are set in parallel after the first dimension protein separation. The advantage of this strategy is that separated proteins are subjected to different proteolytic digestion, hence improving sequence coverage. A second separation conducted on the peptides results in valuable information which can be used to more reliably identify protein sequences. The proposed integrated platform will provide a promising tool for high-throughput, high accuracy and high sequence coverage protein 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/321008/116027
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
Fei LC,Yuan HM,Zhang LH,et al. Integration of Protein Digestion Microreactors in a Comprehensive Two-Dimensional Liquid Chromatorgraphy System[C]. 待补充:待补充,2011:103-0.
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