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学科主题: 物理化学
题名: Preparation of Monolithic Capillary Columns for Proteome Analysis
作者: Zou HF(邹汉法) ;  Ye ML(叶明亮) ;  Wang FJ(王方军) ;  Wu MH(吴明火) ;  Wu RA(吴仁安)
会议名称: 17th International Symposium on Electro- and Liquid Phase-separation Techniques
会议日期: 2010-8-29
出版日期: 2010-08-29
会议地点: 美国
其他题名: 用于蛋白质分析的毛细管整体住的制备
通讯作者: 邹汉法
部门归属: 十八室
主办者: Oklahoma State University
摘要: Monolithic columns have been widely applied in capillary liquid chromatography (LC) 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. With dependence on the nature of the precursors used, the monolithic columns can be categorized into two major types: the organic polymer-based and the inorganic silica-based monolithic 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 strong anion-exchange (SAX) capillary monolith was prepared to enrich phosphopeptides from protein digest for MALDI-TOF MS analysis. It was found that phosphopeptides could be specifically retained on the SAX column at high pH around 8 and could be eluted by 5% formic acid. Thus, the protein digests without any pretreatment could be loaded onto the SAX column under basic pH condition; after removing nonphosphopeptides by washing, the bound phosphopeptides could be eluted directly onto a MALDI target and analyzed by MALDI-TOF MS. This approach significantly simplified the analytical procedures and reduced the sample loss, a detection limit as low as 50 amol for the analysis of phosphopeptides from α-casein digest was achieved. 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. A fully automated system with integration of a reversed phase-strong cation exchange (RP-SCX) biphasic trap column into vented sample injection system was developed to realize online sample loading, isotope dimethyl labeling and online multidimensional separation of the proteome samples. However, both the polymeric and silica monoliths have their own disadvantages. Recently, the organic-inorganic hybrid monolithic columns have been attracting great attention since the organic functional moieties can be incorporated into the inorganic silica monolithic matrices via the sol-gel process to give various properties, such as good mechanical stability and solvent resistance. A “one-pot” process for the preparation of organic-silica hybrid capillary monolithic columns by concurrently using organic monomers and alkoxysilanes was developed.
语种: 中文
内容类型: 会议论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/114152
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,Wang FJ,et al. Preparation of Monolithic Capillary Columns for Proteome Analysis[C]. 见:17th International Symposium on Electro- and Liquid Phase-separation Techniques. 美国. 2010-8-29.
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