蛋白质的磁性微球快速酶解与高效富集新方法研究
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摘要
本论文针对蛋白质组学研究中面临的快速酶解以及磷酸化蛋白高效选择性富集方面的热点难点问题,将功能化磁性微球与蛋白质分析结合起来,开展了一系列研究工作,发展了相关的新技术新方法并进行了实际的应用研究,取得了一些创新性研究结果。主要研究内容和取得的主要研究成果摘要如下:
第一章概述了蛋白质组学研究的现状,基于生物质谱的蛋白质组学研究技术以及新兴的翻译后修饰蛋白组学的重要意义;就目前固定化酶反应器及其在蛋白质组学研究中的应用,以及磷酸化蛋白质组学研究中的富集技术研究进展进行了综述;概述了功能化磁性微球及其在生物分析中的应用;最后提出了本论文选题的目的和意义。
第二章分别运用金属离子螯合和共价键合两条技术路线,合成了表面固定酶的磁性硅球材料并将其应用于毛细管/微流控芯片酶解反应器。首先,采用水热法制备了具有超顺磁性的四氧化三铁磁性微球;然后采用溶胶-凝胶法在其表面包覆二氧化硅层,合成了具有核壳结构的Fe_3O_4@SiO_2磁性硅球。在此基础上,发展了两条不同的固定酶的方法,即金属离子螯合法和共价键合法,将胰蛋白酶固定在磁性硅球表面。
利用外加磁场的作用,在不需要制作塞子的情况下,将用两种不同方法合成的固定酶的磁性硅球分别填充到毛细管或者微流控芯片通道内部。蛋白质溶液在泵的推动下,流过毛细管或者微流控芯片通道,从而得到快速的酶解。使用MALDI质谱对收集到的酶解产物进行分析鉴定。结果表明,用两种不同的固定酶方法制得的毛细管/微流控芯片酶反应器均能在5分钟的酶解时间内达到与12小时传统溶液酶解相同或更好的酶解效果。该方法易于操作、成本低;由于材料的磁性,毛细管/芯片通道内的磁性微球还可以进行方便快速地替换,很好地解决了以前文献报道中毛细管/芯片酶反应器只能一次性使用的缺点。对用金属离子螯合方法固定酶的磁性微球而言,还可以用EDTA将磁性微球表面的铜离子除去,再重新引入新的铜离子和蛋白酶,从而实现毛细管/微流控芯片酶反应器的再生。
第三章发展了更为简便的方法,运用一步水热法合成了氨基四氧化三铁磁性微球,从而将固定酶的磁性微球的合成步骤由第二章中的6步简化到3步,大大缩短了合成的时间。
在上述工作的基础上,将固定酶的磁性微球进一步应用于蛋白质的靶上酶解。将磁性微球分散液滴加到已经预先滴加好蛋白溶液的靶板上,酶解一段时间后,用磁化的钢针把固定酶的磁性微球从靶板上分离出来,从而终止酶解过程。这样,我们既利用了固定化酶技术提高了酶解效率,又避免了对质谱离子源光栅的污染。最后,为了证实本方法在复杂生物样品检测中的实际应用性,我们对大鼠肝脏提取蛋白的液相色谱流分就行了酶解鉴定。
第四章针对翻译后修饰蛋白质组研究中亟待解决的磷酸化蛋白及肽段的分离富集问题,用固定金属亲和色谱(IMAC)原理,在Fe_3O_4@SiO_2磁性硅球表面键合上可以螯合金属离子的官能基团,再将对磷酸化蛋白/肽段有特异性吸附作用的铈离子固定在磁球表面。由于材料的磁性,当磷酸化肽段富集到磁性微球上以后,只需将磁铁放置在盛放溶液的离心管外壁,就可以轻松实现富集了磷酸化肽段的磁性微球与非磷酸化肽段溶液的分离。文献调研显示,Fe~(3+)和Ga~(3+)是IMAC富集中最常使用的金属离子,而目前尚未有将Ce~(4+)成功地应用于磷酸化肽富集的文献见诸报道。因此,在本论文中,我们还进一步比较了螯合Ce~(4+)的磁性微球与螯合Fe~(3+)的磁性微球对磷酸化肽段的富集效率。结果表明,螯合Ce~(4+)的磁性微球对复杂混合物中磷酸化肽段的选择性富集效果优于螯合Fe~(3+)的磁性微球。即使在酶解肽段溶液中BSA的量是β-casein的量的50倍的情况下,螯合Ce~(4+)的磁性微球仍可在30 s内将β-casein中的痕量磷酸化肽段有效地富集出来。在此基础上,我们还首次成功地将螯合Ce~(4+)的磁性微球用于选择性富集人血清中(不需要其它的预处理步骤)的磷酸化肽段,为磷酸化蛋白质组学的研究开辟了新的道路。
第五章创新性地探索了在四氧化三铁磁性微球表面均匀包覆过渡金属氧化物的新方法。采用磁球外部包覆碳层为模板包覆过渡金属氧化物、随后灼烧去除碳层的方法,制备了结构紧密的具有核壳结构的Fe_3O_4@M_xO_y超顺磁性微球。该磁性微球具有与IMAC材料不同的富集机理,具有更高的化学惰性和稳定性。利用该方法将不同的过渡金属氧化物,如Al_2O_3,ZrO_2,Ga_2O_3等分别包覆到四氧化三铁磁性微球表面。采用振动样品磁场计、傅立叶变换红外光谱仪、透射及扫描电子显微镜等对产物进行了表征与确认。我们进而优化了这些材料富集磷酸化肽段的实验条件,包括富集体系、富集时间、样品洗脱时间等,验证了表面包覆不同金属氧化物的Fe_3O_4@M_xO_y磁性微球对于复杂体系中的磷酸化肽段均有很好的选择性富集效果,并进一步将其应用于人血清及脱脂牛奶、鼠肝蛋白酶解产物中磷酸化肽的富集鉴定。
总之,本论文针对蛋白质组学研究中面临的快速酶解与高效富集方面的热点难点问题,以功能化磁性微球为基础,以发展相关的蛋白质组学研究新技术新方法并进行实际的应用研究为目标,合成了多种表面带有不同功能基团的磁性微球并建立了相应的分析方法,为解决蛋白质快速酶解、磷酸化肽段高效分离富集及鉴定等问题提供了新颖有效的研究手段和方法。
The advent of genomics and proteomics has increased considerably the need for fast,low-cost,and automated tools from protein analysis.Proteolysis is the key step for positive protein sequencing in proteomics research integrated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS). Moreover,concentration of phosphopeptides or phosphoproteins is also very crucial in phosphoproteomics research.Therefore,new technologies for rapid and high throughput protein digestion and selective enrichment of phosphopeptides are in great demand.On the other hand,during the past decade,magnetic microspheres with micro-and nanometer size are gaining increasing attention due to their ease of manipulation and recovery.Functionalized magnetic microspheres are extensively applied in cell separation,magnetically assisted drug delivery,and enzyme immobilization.Additionally,magnetic materials are the most commonly selected substrates as affinity probes because of the ease of isolation of the magnetic-material-target conjugate from the sample solution based on their magnetic properties.In this study,we focused on preparing several kinds of novel functionalized magnetic polymer microspheres and developing a series of techniques and methods to resolve current problems in proteolysis digestion and concentration of phosphorylated peptides.This dissertation is divided into five parts.
In Chapter 1,advances in proteomics research,current immobilized enzymatic reactor and phosphopeptides enrichment techniques,applications of functionalized magnetic polymer microspheres techniques in proteome research were summarized in details.The intention and meaning of this dissertation were explained.
In Chapter 2,we developed two synthesis routes based on mechanisms of metal-ion chelation and covalent bonding,respectively.With these two synthesis routes,we successfully prepared trypsin-immobilized magnetic silica microspheres and applied them for fabrication of on-column/on-chip enzymatic reactor.Fe_3O_4 magnetic microspheres with superparamagnetic property were first synthesized through sovalthermal reaction.After that,Fe_3O_4@SiO_2 magnetic microspheres with narrow size distribution and uniform thickness of silica were synthesized via sol-gel approach.Thereafter,two different strategies for enzyme immobilization were developed:
First,immobilization method based on metal-ion chelation.The metal chelating agent of iminodiacetic acid(IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the surface of Fe_3O_4@SiO_2 microspheres. The metal ion of copper and enzyme were subsequently adsorbed onto the surface.
Second,immobilization method based on covalent bonding. Aminopropyltriethoxysilane(APTES) and glutaraldehyde(GA) were introduced to functionalize the Fe_3O_4@SiO_2 microspheres in turn for enzyme immobilization. Trypsin was then stably immobilized onto the Fe_3O_4@SiO_2 microspheres through the reaction of primary amines of enzyme with aldehyde groups on the Fe_3O_4@SiO_2 microspheres.
The trypsin-immobilized Fe_3O_4@SiO_2 microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-column or on-chip enzymatic microreactor.The digestion efficiency and reproducibility of the microreactors were demonstrated by using model proteins. When compared with an incubation time of 12 h by free trypsin in the conventional digestion approach,proteins can be digested by the on-column or on-chip microreactor in several minutes.The microreactors were also successfully applied to the analysis of an RPLC fraction of the rat liver extract.The advantage of the prepared microreactors is that the Fe_3O_4@SiO_2 microspheres(which are retained by a magnet) can be fast and efficiently removed,which makes the microreactors replaceable.Moreover,for microreactors prepared using metal-ion chelation method, the Fe_3O_4@SiO_2 microspheres can be easily regenerated by removing copper ions with EDTA and introducing new copper ions and enzyme,and good reproducibility before and after regeneration was obtained.The results revealed their potential for significant simplification of top-down proteomic analysis protocols,without compromising the quality and specificity of analysis.
In Chapter 3,a more convenient method for preparation of trypsin-immobilized magnetic nanospheres was initially developed.At first,amine-functionalized magnetic particles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy.Then,magnetic nanospheres were functionalized with numerous aldehyde(-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanospheres with glutaraldehyde.Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanospheres was achieved through reaction of the aldehyde groups with amine groups of trypsin.The obtained trypsin-immobilized magnetic nanospheres were conveniently applied for protein digestion.The digestion efficiency was demonstrated with peptide mapping analysis of three model proteins.The process of digestion is very facile due to the easy manipulation of magnetic nanospheres.Complete protein digestion was achieved in a short time(5 min),without any complicated reduction and alkylation procedures.
Based on the above results,we further applied these trypsin-immobilized magnetic nanospheres for on-plate digestion.After digestion,the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS.The effects of the temperature and incubation time on the digestion efficiency were characterized.Furthermore,RPLC fractions of rat liver extract were also successfully processed using this novel method.These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.
In Chapter 4,we employed,for the first time,the Ce~(4+)-chelated magnetic silica microspheres to selectively concentrate phosphopeptides from protein digest products. Cerium ions were chelated onto magnetic silica microspheres using the strategy we established before.After enrichment,the phosphopeptide-conjugated magnetic microspheres were separated from the sample solution just by using a magnet.With the optimized enrichment conditions,the performance of the Ce~(4+)-chelated magnetic microspheres was compared with the Fe~(3+)-chelated microspheres using tryptic digested peptides originating from ovalbumin,a five protein mixture containing phosphoproteins and nonphosphoproteins,as well as a mixture ofβ-casein and BSA with a molar ratio of 1:50.Compared to Fe~(3+),Ce~(4+)-chelated magnetic microspheres exhibited more selective isolation ability for concentrating phosphopeptides from complex mixtures.Even when the amount of the tryptic digest product of BSA is 50 times higher than that ofβ-casein in the sample solution,the trace phosphopeptides derived fromβ-casein can still be concentrated effectively by the Ce~(4+)-chelated magnetic microspheres in only 30 s.Furthermore,we initially utilized the Ce~(4+)-chelated magnetic microspheres to directly enrich phosphopeptides from human serum without extra purification steps or tedious treatment,which opens up a possibility for their further application in phosphoproteomics.
In Chapter 5,a novel strategy for preparation of Fe_3O_4@M_xO_y magnetic microspheres with well-defined core-shell structure was initially developed and the as-prepared microspheres were applied for highly selective enrichment of phosphopeptides from tryptic digest product of proteins.To successfully coat iron oxide microspheres with uniform metal oxide shell,magnetic Fe_3O_4 microspheres were first synthesized via a solvothermal reaction,followed by being coated with a thin layer of carbon by polymerization and carbonization of glucose through hydrothermal reaction.Finally,with the use of the Fe_3O_4@C microspheres as templates,metal isopropoxide was prehydrolyzed and absorbed onto the microspheres and eventually converted into metal oxide by calcinations.By using this strategy,we successfully prepared a series of Fe_3O_4@M_xO_y magnetic microspheres,including Fe_3O_4@ZrO_2,Fe_3O_4@Al_2O_3,and Fe_3O_4@Ga_2O_3 magnetic microspheres.The as-prepared Fe_3O_4@M_xO_y core-shell microspheres were used as affinity probes to selectively concentrate phosphopeptides from tryptic digest of model proteins to exemplify their selective enrichment ability of phosphopeptides from complex protein samples.In only 0.5 min,phosphopeptides sufficient for characterization by MALDI-MS could be enriched by the Fe_3O_4@M_xO_y microspheres.The performances of these Fe_3O_4@M_xO_y microspheres were further compared with commonly used commercial IMAC resin and TiO_2 beads,and the results proved stronger selective abilities of Fe_3O_4@M_xO_y microspheres over IMAC resin and TiO_2 beads.Finally,the Fe_3O_4@M_xO_y microspheres were successfully utilized for enrichment of phosphopeptides from human blood serum,digests of non-fat milk and rat liver tissue extracts.The results open up a possibility for their further application in phosphoproteome analysis.
In summary,the main contributes of this dissertation is that we initially synthesized several functional magnetic materials and successfully utilized them for rapid protein digestion and phosphorylated peptides.We aimed at exploring and finding out new techniques in proteolysis and selective concentration of proteome research fields,so that more breakthroughs can be obtained in the proteome research study.
第一章概述了蛋白质组学研究的现状,基于生物质谱的蛋白质组学研究技术以及新兴的翻译后修饰蛋白组学的重要意义;就目前固定化酶反应器及其在蛋白质组学研究中的应用,以及磷酸化蛋白质组学研究中的富集技术研究进展进行了综述;概述了功能化磁性微球及其在生物分析中的应用;最后提出了本论文选题的目的和意义。
第二章分别运用金属离子螯合和共价键合两条技术路线,合成了表面固定酶的磁性硅球材料并将其应用于毛细管/微流控芯片酶解反应器。首先,采用水热法制备了具有超顺磁性的四氧化三铁磁性微球;然后采用溶胶-凝胶法在其表面包覆二氧化硅层,合成了具有核壳结构的Fe_3O_4@SiO_2磁性硅球。在此基础上,发展了两条不同的固定酶的方法,即金属离子螯合法和共价键合法,将胰蛋白酶固定在磁性硅球表面。
利用外加磁场的作用,在不需要制作塞子的情况下,将用两种不同方法合成的固定酶的磁性硅球分别填充到毛细管或者微流控芯片通道内部。蛋白质溶液在泵的推动下,流过毛细管或者微流控芯片通道,从而得到快速的酶解。使用MALDI质谱对收集到的酶解产物进行分析鉴定。结果表明,用两种不同的固定酶方法制得的毛细管/微流控芯片酶反应器均能在5分钟的酶解时间内达到与12小时传统溶液酶解相同或更好的酶解效果。该方法易于操作、成本低;由于材料的磁性,毛细管/芯片通道内的磁性微球还可以进行方便快速地替换,很好地解决了以前文献报道中毛细管/芯片酶反应器只能一次性使用的缺点。对用金属离子螯合方法固定酶的磁性微球而言,还可以用EDTA将磁性微球表面的铜离子除去,再重新引入新的铜离子和蛋白酶,从而实现毛细管/微流控芯片酶反应器的再生。
第三章发展了更为简便的方法,运用一步水热法合成了氨基四氧化三铁磁性微球,从而将固定酶的磁性微球的合成步骤由第二章中的6步简化到3步,大大缩短了合成的时间。
在上述工作的基础上,将固定酶的磁性微球进一步应用于蛋白质的靶上酶解。将磁性微球分散液滴加到已经预先滴加好蛋白溶液的靶板上,酶解一段时间后,用磁化的钢针把固定酶的磁性微球从靶板上分离出来,从而终止酶解过程。这样,我们既利用了固定化酶技术提高了酶解效率,又避免了对质谱离子源光栅的污染。最后,为了证实本方法在复杂生物样品检测中的实际应用性,我们对大鼠肝脏提取蛋白的液相色谱流分就行了酶解鉴定。
第四章针对翻译后修饰蛋白质组研究中亟待解决的磷酸化蛋白及肽段的分离富集问题,用固定金属亲和色谱(IMAC)原理,在Fe_3O_4@SiO_2磁性硅球表面键合上可以螯合金属离子的官能基团,再将对磷酸化蛋白/肽段有特异性吸附作用的铈离子固定在磁球表面。由于材料的磁性,当磷酸化肽段富集到磁性微球上以后,只需将磁铁放置在盛放溶液的离心管外壁,就可以轻松实现富集了磷酸化肽段的磁性微球与非磷酸化肽段溶液的分离。文献调研显示,Fe~(3+)和Ga~(3+)是IMAC富集中最常使用的金属离子,而目前尚未有将Ce~(4+)成功地应用于磷酸化肽富集的文献见诸报道。因此,在本论文中,我们还进一步比较了螯合Ce~(4+)的磁性微球与螯合Fe~(3+)的磁性微球对磷酸化肽段的富集效率。结果表明,螯合Ce~(4+)的磁性微球对复杂混合物中磷酸化肽段的选择性富集效果优于螯合Fe~(3+)的磁性微球。即使在酶解肽段溶液中BSA的量是β-casein的量的50倍的情况下,螯合Ce~(4+)的磁性微球仍可在30 s内将β-casein中的痕量磷酸化肽段有效地富集出来。在此基础上,我们还首次成功地将螯合Ce~(4+)的磁性微球用于选择性富集人血清中(不需要其它的预处理步骤)的磷酸化肽段,为磷酸化蛋白质组学的研究开辟了新的道路。
第五章创新性地探索了在四氧化三铁磁性微球表面均匀包覆过渡金属氧化物的新方法。采用磁球外部包覆碳层为模板包覆过渡金属氧化物、随后灼烧去除碳层的方法,制备了结构紧密的具有核壳结构的Fe_3O_4@M_xO_y超顺磁性微球。该磁性微球具有与IMAC材料不同的富集机理,具有更高的化学惰性和稳定性。利用该方法将不同的过渡金属氧化物,如Al_2O_3,ZrO_2,Ga_2O_3等分别包覆到四氧化三铁磁性微球表面。采用振动样品磁场计、傅立叶变换红外光谱仪、透射及扫描电子显微镜等对产物进行了表征与确认。我们进而优化了这些材料富集磷酸化肽段的实验条件,包括富集体系、富集时间、样品洗脱时间等,验证了表面包覆不同金属氧化物的Fe_3O_4@M_xO_y磁性微球对于复杂体系中的磷酸化肽段均有很好的选择性富集效果,并进一步将其应用于人血清及脱脂牛奶、鼠肝蛋白酶解产物中磷酸化肽的富集鉴定。
总之,本论文针对蛋白质组学研究中面临的快速酶解与高效富集方面的热点难点问题,以功能化磁性微球为基础,以发展相关的蛋白质组学研究新技术新方法并进行实际的应用研究为目标,合成了多种表面带有不同功能基团的磁性微球并建立了相应的分析方法,为解决蛋白质快速酶解、磷酸化肽段高效分离富集及鉴定等问题提供了新颖有效的研究手段和方法。
The advent of genomics and proteomics has increased considerably the need for fast,low-cost,and automated tools from protein analysis.Proteolysis is the key step for positive protein sequencing in proteomics research integrated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS). Moreover,concentration of phosphopeptides or phosphoproteins is also very crucial in phosphoproteomics research.Therefore,new technologies for rapid and high throughput protein digestion and selective enrichment of phosphopeptides are in great demand.On the other hand,during the past decade,magnetic microspheres with micro-and nanometer size are gaining increasing attention due to their ease of manipulation and recovery.Functionalized magnetic microspheres are extensively applied in cell separation,magnetically assisted drug delivery,and enzyme immobilization.Additionally,magnetic materials are the most commonly selected substrates as affinity probes because of the ease of isolation of the magnetic-material-target conjugate from the sample solution based on their magnetic properties.In this study,we focused on preparing several kinds of novel functionalized magnetic polymer microspheres and developing a series of techniques and methods to resolve current problems in proteolysis digestion and concentration of phosphorylated peptides.This dissertation is divided into five parts.
In Chapter 1,advances in proteomics research,current immobilized enzymatic reactor and phosphopeptides enrichment techniques,applications of functionalized magnetic polymer microspheres techniques in proteome research were summarized in details.The intention and meaning of this dissertation were explained.
In Chapter 2,we developed two synthesis routes based on mechanisms of metal-ion chelation and covalent bonding,respectively.With these two synthesis routes,we successfully prepared trypsin-immobilized magnetic silica microspheres and applied them for fabrication of on-column/on-chip enzymatic reactor.Fe_3O_4 magnetic microspheres with superparamagnetic property were first synthesized through sovalthermal reaction.After that,Fe_3O_4@SiO_2 magnetic microspheres with narrow size distribution and uniform thickness of silica were synthesized via sol-gel approach.Thereafter,two different strategies for enzyme immobilization were developed:
First,immobilization method based on metal-ion chelation.The metal chelating agent of iminodiacetic acid(IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the surface of Fe_3O_4@SiO_2 microspheres. The metal ion of copper and enzyme were subsequently adsorbed onto the surface.
Second,immobilization method based on covalent bonding. Aminopropyltriethoxysilane(APTES) and glutaraldehyde(GA) were introduced to functionalize the Fe_3O_4@SiO_2 microspheres in turn for enzyme immobilization. Trypsin was then stably immobilized onto the Fe_3O_4@SiO_2 microspheres through the reaction of primary amines of enzyme with aldehyde groups on the Fe_3O_4@SiO_2 microspheres.
The trypsin-immobilized Fe_3O_4@SiO_2 microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-column or on-chip enzymatic microreactor.The digestion efficiency and reproducibility of the microreactors were demonstrated by using model proteins. When compared with an incubation time of 12 h by free trypsin in the conventional digestion approach,proteins can be digested by the on-column or on-chip microreactor in several minutes.The microreactors were also successfully applied to the analysis of an RPLC fraction of the rat liver extract.The advantage of the prepared microreactors is that the Fe_3O_4@SiO_2 microspheres(which are retained by a magnet) can be fast and efficiently removed,which makes the microreactors replaceable.Moreover,for microreactors prepared using metal-ion chelation method, the Fe_3O_4@SiO_2 microspheres can be easily regenerated by removing copper ions with EDTA and introducing new copper ions and enzyme,and good reproducibility before and after regeneration was obtained.The results revealed their potential for significant simplification of top-down proteomic analysis protocols,without compromising the quality and specificity of analysis.
In Chapter 3,a more convenient method for preparation of trypsin-immobilized magnetic nanospheres was initially developed.At first,amine-functionalized magnetic particles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy.Then,magnetic nanospheres were functionalized with numerous aldehyde(-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanospheres with glutaraldehyde.Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanospheres was achieved through reaction of the aldehyde groups with amine groups of trypsin.The obtained trypsin-immobilized magnetic nanospheres were conveniently applied for protein digestion.The digestion efficiency was demonstrated with peptide mapping analysis of three model proteins.The process of digestion is very facile due to the easy manipulation of magnetic nanospheres.Complete protein digestion was achieved in a short time(5 min),without any complicated reduction and alkylation procedures.
Based on the above results,we further applied these trypsin-immobilized magnetic nanospheres for on-plate digestion.After digestion,the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS.The effects of the temperature and incubation time on the digestion efficiency were characterized.Furthermore,RPLC fractions of rat liver extract were also successfully processed using this novel method.These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.
In Chapter 4,we employed,for the first time,the Ce~(4+)-chelated magnetic silica microspheres to selectively concentrate phosphopeptides from protein digest products. Cerium ions were chelated onto magnetic silica microspheres using the strategy we established before.After enrichment,the phosphopeptide-conjugated magnetic microspheres were separated from the sample solution just by using a magnet.With the optimized enrichment conditions,the performance of the Ce~(4+)-chelated magnetic microspheres was compared with the Fe~(3+)-chelated microspheres using tryptic digested peptides originating from ovalbumin,a five protein mixture containing phosphoproteins and nonphosphoproteins,as well as a mixture ofβ-casein and BSA with a molar ratio of 1:50.Compared to Fe~(3+),Ce~(4+)-chelated magnetic microspheres exhibited more selective isolation ability for concentrating phosphopeptides from complex mixtures.Even when the amount of the tryptic digest product of BSA is 50 times higher than that ofβ-casein in the sample solution,the trace phosphopeptides derived fromβ-casein can still be concentrated effectively by the Ce~(4+)-chelated magnetic microspheres in only 30 s.Furthermore,we initially utilized the Ce~(4+)-chelated magnetic microspheres to directly enrich phosphopeptides from human serum without extra purification steps or tedious treatment,which opens up a possibility for their further application in phosphoproteomics.
In Chapter 5,a novel strategy for preparation of Fe_3O_4@M_xO_y magnetic microspheres with well-defined core-shell structure was initially developed and the as-prepared microspheres were applied for highly selective enrichment of phosphopeptides from tryptic digest product of proteins.To successfully coat iron oxide microspheres with uniform metal oxide shell,magnetic Fe_3O_4 microspheres were first synthesized via a solvothermal reaction,followed by being coated with a thin layer of carbon by polymerization and carbonization of glucose through hydrothermal reaction.Finally,with the use of the Fe_3O_4@C microspheres as templates,metal isopropoxide was prehydrolyzed and absorbed onto the microspheres and eventually converted into metal oxide by calcinations.By using this strategy,we successfully prepared a series of Fe_3O_4@M_xO_y magnetic microspheres,including Fe_3O_4@ZrO_2,Fe_3O_4@Al_2O_3,and Fe_3O_4@Ga_2O_3 magnetic microspheres.The as-prepared Fe_3O_4@M_xO_y core-shell microspheres were used as affinity probes to selectively concentrate phosphopeptides from tryptic digest of model proteins to exemplify their selective enrichment ability of phosphopeptides from complex protein samples.In only 0.5 min,phosphopeptides sufficient for characterization by MALDI-MS could be enriched by the Fe_3O_4@M_xO_y microspheres.The performances of these Fe_3O_4@M_xO_y microspheres were further compared with commonly used commercial IMAC resin and TiO_2 beads,and the results proved stronger selective abilities of Fe_3O_4@M_xO_y microspheres over IMAC resin and TiO_2 beads.Finally,the Fe_3O_4@M_xO_y microspheres were successfully utilized for enrichment of phosphopeptides from human blood serum,digests of non-fat milk and rat liver tissue extracts.The results open up a possibility for their further application in phosphoproteome analysis.
In summary,the main contributes of this dissertation is that we initially synthesized several functional magnetic materials and successfully utilized them for rapid protein digestion and phosphorylated peptides.We aimed at exploring and finding out new techniques in proteolysis and selective concentration of proteome research fields,so that more breakthroughs can be obtained in the proteome research study.
引文
[1]A.Pandey,M.Mann,Nature,2000,405(6788),837-846.
[2]L.Rosamond,A.Alsop,Science,2000,287(5460),1973-1976.
[3]F.Regnier,A.Amini,A.Chakraborty,M.Geng,J.Ji,L.Riggs,C.Sioma,S.Wang,X.Zhang,LC-GC,2001,19,200-213.
[4]S.Cordwell,et al.Electrophoresis,1997,18:1393-1398.
[5]W.P.Blackstock,M.P.Weir,Trends in Biotechnol.1999,17,121-127.
[6]吴世容,李志良,李根容,等,重庆大学学报,2004,27(1),123-127.
[7]P.J.Mintz,J.Kim,K.A.Do,et al.Nat.Biotechnol.,2003,21,57-63.
[8]梁文平,庄乾坤.北京,科学出版社,2003,229-239.
[9]M.Mann,O.N.Jensen,Nat Biotechnol,2003,21(3):255-261.
[10]O.N.Jensen,Proteomics:A Trends Guide,2000,36-42.
[11]J.Bunkenborg,B.J.Pilch,A.V.Podtelejnikov,et al.Proteomics,2004,4(2),454.
[12]肖海军,贺筱蓉.生物学通报,2001,36(7),9-10.
[13]T.N.Krogh T.,Berg,P.Hojrup,Anal.Biochem.,1999,274,153-162.
[14]X.R Mao,G.J.Guo,J.F.Huang,Z.Y.Du,Z.S.Huang,L.Ma,R L i,L.Q.Gu,J.Chem.Tech.B iotech.,2006,8(2),189-195.
[15]M.E.Marin-Zamora,F.Rojas-Melgarejo,F.Garcia-Canovas,P.A.Garcia-Ruiz,J.Chem.Tech.Biotech.,2005,80(12),1356-1364.
[16]L.Gomez,H.L.Ramirez,M.L.Villalonga,HernAdez,R.Villalonga,Enzyme and Microbial Tech.,2006,38(122),22-27.
[17]钱军民,张兴,吕飞,李旭祥,化工新型材料,2002,10,21-24.
[18]彭志英,食品酶学导论.北京:中国轻工业出版社,2002,182-189.
[19]S.J,Bencovic,Ann.Rev.Biochem,1992,6,29-54.
[20]朱祥瑞,徐俊良,浙江大学学报(农业生命科学版),2002,28(1),64-69.
[21]杨昌英,潘家荣,钟珩,湖北化工,2002,6,20-21.
[22]G.Massolini,E.J.Calleri,Sep.Sci.,2005,28(1),7-21.
[23]F.Svec,Electrophoresis,2006,27,947-961.
[24]S.Thelohan,P.Jadaud,I.W.Wainer,Chromatographia,1989,28,551-555.
[25]H.Minakuchi,K.Nakanishi,N.Soga,N.Ishizuka,N.Tanaka,J.Chromatogr.A,1998,797,121-131
[26]N.Tanaka,K.Kobayashi,N.Ishizuka,H.Minakuchi,K.Nakanishi,K.Hosoya,T.Ikegami,J.Chromatogr.A,2002,965,35-49.
[27]H.Minakuchi,K.Nakanishi,N.Soga,N.Ishizuka,N.Tanaka,Anal.Chem.,1996,68,3498-3501.
[28]E.Calleri,C.Temporini,E.Perani,C.Stella,S.Rudaz,D.Lubda,G.Mellerio,J.L.Veuthey,G.Caccialanza,G.Massolini,J.Chromatogr.A,2004,1045,99-109.
[29]郭忠,张清春,雷政登,孔亮,毛希琴,邹汉法,高等学校化学学报,2002,23(7),1277-1280.
[30]N.Amankawa,W.G..Kuhr,Anal.Chem.,1992,64,1610-1613.
[31]J.Kim,J.W.Grate,P.Wang,Chemical Engineering Science,2006,61,1017-1026.
[32]J.P.Chen,D.R.Su,Biotechnology Progress,2001,17,369-375.
[33]B.C.Kim,S.Nair,J.Kim,J.H.Kwak,J.W.Grate,S.H.Kim,M.B.Gu,Nanotechnolgy,2005,16,382-388.
[34]J.F.Diaz,K.J.J.Balkus,Journal of Molecular Catalysis B:Enzymatic,1996,2,115-126.
[35]D.Y.Zhao,Q.S.Huo,J.L.Feng,B.F.Chmelka,G.D.Stucky,Journal of the American Chemical Society,1998,120,6024-6036.
[36]P.Schmidt-Winkel,W.W.Lukens,D.Y.Zhao,P.D.Yang,B.F.Chmelka,G.D.Stucky,Journal of the American Chemical Society,1999,121,254-255.
[37]Y.J.Wang,F.Caruso,Chem.Commun.,2004,13,1528-1529.
[38]Y.L.F.Hsieh,H.Wang,C.Elicone Anal.Chem.,1996,68.455-462.
[39]L.Riggs,C.Sioma,F.E.J.Regnier,Chromatogr.A,2001,924.359-368.
[40]姜泓海,邹汉法,汪海林,张强,郭忠,张清春,陈小明,中国科学,B辑,2000,21(5),385-391.
[41]姜泓海,邹汉法,汪海林,倪坚毅,张强,高等学校化学学报,2000,21(5),702-706.
[42]H.H.Jiang,H.F.Zou,H.L.Wang,J.Y.Ni,Q.Zhang,Y.K.Zhang,J.Chromatogr.A,2000,903.77-84.
[43]S.Hjerten,J.L.Liao,R.Zhang,J.Chromatogr.,1989,473,273-275.
[44]M.Petro,F.Svec,J.M.J.Frechet,Biotech.Bioeng.,1996,48,355-363.
[45]D.S.Peterson,T.Rohr,F.Svec,J.M.J.Frechet,Anal.Chem.,2002,74,4081-4088.
[46]A.K.Palm,M.V.Novotny,Rapid Commun.Mass Spectrom.,2004,18,1374-1382.
[47]J.C.Duan,L.L.Sun,Z.Liang,J.Zhang,H.Wang,L.H.Zhang,W.B.Zhang,Y.K.Zhang,J.Chromatogr.A.2006,1106.165-174.
[48]J.C.Duan,L.Zhen,C.Yang,J.Zhang,L.H.Zhang,W.B.Zhang,Y.K.Zhang Proteomics,2006,6,412-419.
[49]黄家贤,霍岩丽,李涵,晏苏学,孙江晓,袁直,中国生物化学与分子生物学报,2001,17(3),376-3801.
[50]田玉平,王虎,查月珍,杨芃原,化学世界,2005,10,590-592.
[51]P.Cohen,Nat.Cell.Biol.,2002,4(5),127-130.
[52]S.B.Ficarro,M.L.McCleland,P.T.Stukenberg,et al.Nat.Biotechnol.,2002,20(3),301.
[53]E.H.Seeley,L.D.Riggs,F.E.Regnier,J.Chromatogr.B Analyt.Technol.Biomed.Life Sci.,2005,817(1),81.
[54]M.C.Posewitz,R Tempst,Anal.Chem.,1999,71,2883-2892.
[55]A.Stensballe,S.Andersen,O.N.Jensen Proteomics,2001,1,207-222.
[56]K.N.Barnouin,S.R.Hart,A.J.Thompson,M.Okuyama,M.Waterfield,R.Cramer,Proteomics,2005,5,4376-4388.
[57]S.R.Hart,M.D.Waterfield,A.L.Burlingame,R.J.Cramer,J.Am.Soc.Mass Spectrom.,2002,13,1042-1051.
[58]Y.H.Zhang,X.J.Yu,X.Y.Wang,W.Shah,R Y.Yang,Y.Tang,Chem.Commun.,2004,2882-2883.
[59]K.Moser,F.M.White,J.Proteome Res.,2006,5(1),98.
[60]M.O.Collins,L.Yu,M.P.Coba,et al.J.Biol.Chem.,2005,280(18),5972.
[61]P.Cao,J.T.Stults.Rapid Commun.Mass Spectrom.,2000,14(17),1600.
[62]L.Zhang,L.Zhang,W.Zhang,et al.Electrophoresis,2005,26(11),2172.
[63]R.Kange,U.Selditz,M.Granberg,et al.J.Biomol.Tech.,2005,16(2),91.
[64]M.W.Pinkse,P.M.Uitto,M.J.Hilhorst,et al.Anal.Chem.,2004,76(14),3935.
[65]M.R.Larsen,T.E.Thingholm,O.N.Jensen,et al.Mol.Cell.Proteomics,2005,4(7),873
[66]H.K.Kweon,K.Hakansson,Anal.Chem.,2006,8(6),1743.
[67]F.Wolschin,S.Wienkoop,W.Weckwerth,Proteomics,2005,5(17),4389.
[68]J.W.Mandell,Am.J.Pathol.,2003,163(5),1687.
[69]J.Rush,A.Moritz,K.A.Lee,et al.Nat.Biotechnol.,2005,23,94.
[70]M.Gronborg,T.Z.Kristiansen,A.Stensballe,et al.Mol.Cell.Proteomics,2002,1(7),517.
[71]S,Kane.H.Sano.S.C.Liu,et al.J.Biol.Chem.,2002.277(25),22115.
[72]E,Astoul,A,D,Laurence,N,Totty,et al.J.Biol.Chem.,2003,278(11),92675.
[73]A.Laurence,E.Astoul,S.Hanrahan,et al.Eur.J.Immunol.,2004,34,587.
[74]H.Zhou,J.D.Watts,R.Aebersold,Nat.Biotechnol.,2001,19,375-378.
[75]H.V.Jaffe,H.C.Pant,Biochemistry,1998,37,16211-16224.
[76]Y.Oda,T.Nagasu,B.T.Chait,Nat.Biotechnol.,2001,19,379-382.
[77]M.B.Goshe,T.P.Conrads,E.A.Panisko,N.H.Angell,T.D.Veenstra,R.D.Smith,Anal.Chem.,2001,73,2578-2586.
[78]F.Thaler,B.Valsasina,R.Baldi,J.Xie,A.Stewart,A.Isacchi,H.M.Kalisz,L.Rusconi,Anal.Bioanai.Chem.,2003,376,366-373.
[79]S.A.Beausoleil,M.Jedrychowski,D.Schwartz,et al.Proc.Natl.Acad.Sci.USA,2004,101(33),12130.
[80]B.A.Ballif,J.Viilen,S.A.Beausoleil,et al.Mol.Cell.Proteomics,2004,3,1093-1101.
[81]A.Yamagata,D.B.Kristensen,Y.Takeda,et al.Proteomics,2002,2,1267-1276.
[82]E.J.Hayduk,L.H,Choe,K,H.Lee,Electrophoresis,2004,25,2545-2556.
[83]邓新宇,姜颖,贺福初,遗传,2007,29(10),1163-1166.
[84]Q.Hu,R.J.Noll,H.Li,A.Makarov,M.Hardman,C.R.Graham,J.Mass Spectrom.,2005,40(4),430-443.
[85]P.K.Gupta,Int.J.Pharm.,1998,43,167-177.
[86]R.V.Mehta,R.V.Upadhyay,S.W.Charles,et al.Biotechnology Techniques,1997,11(7),493-496.
[87]马宁,谢文磊,现代化工,2007,27,364-369.
[88]邱光明,孙宗华,生物医学工程杂志,1995,12(3),209-213.
[89]L.Han,W.Wang,2004,35(1),15-21.
[90]C.-T.Chen,Y.-C.Chen,Anal.Chem.2005,77,5912-5919.
[91]C.-T.Chen,Y.-C.Chen,P.-J.Tsai,K.-Y.Chien,J.-S.Yu,Y.-C.Chen,J.Proteome Res.,2007,6(1),316-325.
[92]C.-Y.Lo,W.-Y.Chen,C.-T.Chen,Y.-C.Chen.J.Proteome Res.,2007,6(2),887-893.
[93]Z.Bilkova,M.Slovakova,N.Minc,C.Futterer,R.Cecal,D.Horak,M.Bene,I.Potier,J.Kenkova,M.Przybylski,J.-L.Viovy.Electrophoresis,2006,27(9),1811-1824.
[94]J.-M.Kauffmann..Anal.Chem.2004,76,5498-5502.
[95]I.Safarik,M.Safarikova,J.Chromatogr B.1999,722,33-53.
[96]D.Swinbanks,Nature,1995,378,653.
[97]I.Safarik,M.S.Safarikova,.Mon.Chem.,2002,133,737-759.
[98]H.Xu,G.L.Zhang,F.B.Zhang,et al.Chin.J.Chem.Engin.,2003,11(4),456-459.
[1]J.S.Kim,D.R.Knapp,Electrophoresis,2001,22,3993-3999.
[2]D.S.Peterson,T.Rohr,F.Svec,J.M.J.Frechet,Anal.Chem.,2002,74,4081-4088.
[3]K.Sakai-Kato,M.Kato,T.Toyooka,Anal.Chem.,2002,74,2943-2949.
[4]H.Y.Qu,J.L.Kong,P.Y.Yang,B.H.Liu,et al.,Anal.Chem.,2004,76,6426-6433.
[5]H.L.Wu,P.Y.Yang,H.H.Wang,et al.J.Proteome Res.,2004,3,1201-1208.
[6]Y.Liu,H.J.Lu,W.Zhong.P.Y.Song,et al.Anal.Chem.,2006,78,801-812.
[7]C.Wang,R.Oleshuk,F.Ouchen,J.Li,et al.Rapid Commun.Mass Spectrom.,2000,14,1377-1382.
[8]K.Sato,M.Tokeshi,T.Odake,H.Komara,et al.Anal.Chem.,2000,72,1144.
[9]A.B.Jemere,R.D.Oleshuk F.,Ouchen,F.Fajuyigbe,et al.Electrophoresis,2002,23,3537-3546.
[10]D.S.Peterson,T.Rohr,F.Svec,J.M.J.Frechet,J.Proteome Res.2002,1,563-568.
[11]A.Kondo,H.Kamura,K.Higashitani,,Appl.Microbiol.Biotechnol.,1994,41(1),99-105.
[12]严希康,朱留沙等,功能高分子报,1997,10(1),128-129.
[13]W.Choi,K.W.Oh,J.H.Thomas,W.R.Heineman,et al.Lab Chip,2002,2,27-30.
[14]L.G.Rashkovetsky,Y.V.Lyubarskaya,F.Foret,D.E.Hughes,et al.J.Chromatogr.A,1997,781,197-204.
[15]M.Slovakova,N.Minc,Z.Bilkova,C.Smadja,et al.Lab Chip,2005,5,935-943.
[16]Z.Bilkova,M.Slovakova,N.Minc,C.Futterer,et al.Electrophoresis,2006,27,1811-1824.
[17]徐秀青,基于功能磁性材料的蛋白质组学分离鉴定新方法研究,复旦大学博士学位论文,2007。
[18]M.Mary,Scientific and Clinical Applications of Magnetic Carriers,(Eds:U.Hafeli,W.Schutt,M.Zborowski),Plenum Press,New York.1997.p 303.
[19]邓勇辉,功能性磁性聚合物微球的制备、表征及其初步应用。复旦大学博士学位论文,2005。
[20]M.Okubo,H.Minami,T.Komura,J.App.Poly.Sci.,2003,88,428.
[21]马宁,谢文磊,现代化工,2007,27,364-369.
[22]J.Kenkova,F.Foret,Electrophoresis,2004,25,3550-3563.
[23]Z.Guo,S.Y.Xu,Z.D.Lei,H.F.Zou,B.C.Guo,Electrophoresis,2003,24(21),3633-3639.
[24]O.R.Zaborsky,Immobilized Enzymes,CRC Press,Cleveland,OH 1973,pp.61-74.
[25]H.Jiang,H.Zou,H.Wang,J.Ni,et al.J.Chromatogr.A,2000,903,77-86.
[26]K.Okuda,I.Urabe,Y.Yamada,H.Okada,J.Ferment.Bioeng.,1991,71,100-105.
[27]D.R.Walt,V.Agayn,Trends Anal.Chem.,1994,13,425-430.
[28]K.Sakai-Kato,M.Kato,T.Toyo'oka,Anal.Chem.,2002,74,2943.
[29] K. Sakai-Kato, M. Kato, T. Toyo'oka, Anal. Chem., 2003, 75, 388.
[30] Z.-Y. Park, D. H. Russell, Anal. Chem. 2000, 72 (11), 2667-2670.
[31] C. E. Parker, K. B. Tomer, Methods Mol. Biol. 2000, 146, 185- 201.
[32] G. J. Opiteck, K. C. Lewis, J. W. Jorgenson, R. J. Anderegg, Anal. Chem. 1997, 69(8), 1518-1524.
[33] N. C. VerBerkmoes, J. L. Bundy, L. Hauser, K. G. Asano, J. Razumovskaya, F. Larimer, R. L. Hettich, J. L. Stephenson, J. Proteome Res. 2002, 1 (3), 239-252.
[34] H. Deng, X. L. Li, Q. Peng, X. Wang, J. P. Chen, Y. D. Li. Angew. Chem. Int. Ed. 2005, 44, 2782-2785.
[35] Y. Qu, L. Moons, F. Vandesande, J. Chromatogr. B., 1997, 704, 351-358.
[36] S. Huang, X. M. Zhang, J. Chromatogr. A, 2001, 910, 13-18.
[1]A.Stensballe,O.N.Jensen,Proteomics,2001,1,955-966.
[2]D.Ericsson,S.Ekstrom,J.Nillsson,J.Bergquist,G.Marko-Varga,T.Laurell,Proteomics,2001,1,1072-1081.
[3]B.Warscheid,C.A.Fenselau,Proteomics,2004,4,2877-2892.
[4]D.Dogruel,P.Williams,R.W.Nelson,Anal.Chem.,1995,67,4343-4348.
[5]R.W.Nelson,D.Dogruel,J.R.Krone,P.Williams,Rapid Commun.Mass Spectrom.,1995,9,1380-1385.
[6]W.A.Harris,J.P.Reilly,Anal.Chem.,2002,74,4410-4416.
[7]R.J.Arnold,J.P.Reilly,Rapid Commun.Mass Spectrom.,1998,12,630-636.
[8]R.J.Arnold,J.1:'.Reilly,Anal.Biochem.,1999,269,105-112.
[9]S.P.Zheng,C.Yoo,N.Delmotte,F.R.Miller,C.G.Huber,D.M.Lubman,Anal.Chem.,2006,78,5198-5204.
[10]X.Q.Xu,C.H.Deng,M.X.Gao,W.J.Yu,P.Y.Yang,X.M.Zhang,Adv.Mater.,2006,18,3289-3293.
[11]Y.Li,X.Q.Xu,B.Yah,C.H.Deng,W.J.Yu,P.Y.Yang,X.M.Zhang J.Proteome Res.,2007,6,2367-2375.
[12]于文佳,复旦大学,博士学位论文,2005。
[13]A.Fontana,G.Fassina,C.Vita,D.Dalzoppo,M.Zamai,M.Zambonin,Biochemistry,1986,25,1847-1851.
[14]S.J.Hubbard,F.Eisenmenger,J.M.Thornton,Protein Sci.,1994,3,757-768.
[15]Z.-Y.Park,D.H.Russell,Anal.Chem.,2000,72,2667-2670.
[16]Y.Mao,Y.Li,X.M.Zhang,Proteomics 2006,6,420-426.
[17]J.Zhang,X.Q.Xu,M.X.Gao,X.M.Zhang,et al.Proteomics,2007,7,500-512.
[18]L.Wang,J.Bao,L.Wang,F.Zhang,Y.D.Li.Chem.Eur.J.,2006,12,6341-6347.
[1]H.Zhang,X.Zha,Y.Tan,et al.J.Biol.Chem.,2002,277(42),39379-39387.
[2]T.Hunter,Harvey Lect.,1998-99,94,81-119.
[3]J.Chalmersm,W.Kolch,R.Emmettm,et al.J.Chromatogr.B:Analyt.Technol.Biomed.Life Sci.,2004,803(1),111-120.
[4]K.A.Resing,N.G.Ahn,Methods Enzymol.,1997,283,29-44.
[5]R.Aebersold,D.R.Goodlett,Chem.Rev.,2001,101,269-295.
[6]A.Stensballe,S.Andersen,O.N.Jensen,Proteomics,2001,1,207-222.
[7]M.Kokubu,Y.Ishihama,T.Sato;T.Nagasu,Y.Oda,Anal.Chem.,2005,77,5144-5154.
[8]J.D.Dunn,J.T.Watson,M.L.Bruening,Anal.Chem.,2006,78,1574-1580.
[9]S.R.Hart,M.D.Waterfield,A.L.Burlingame,R.Cramer,J.Am Soc.Mass.Spectrom.,2002,13,1042.
[10]Y.Zhang,X.Yu,X.Wang,W.Shah,P.Yang,Y.Tang,Chem.Commun.,2004,2882-2883.
[11]徐秀青,基于功能磁性材料的蛋白质组学分离鉴定新方法研究,复旦大学博士学位论文,2007。
[12]L.Andersson,J.Porath,Anal.Biochem.,1986,154,250-254.
[13]L.Andersson,J.Chromatogr.,1991,539,327-334.
[14]M.C.Posewitz,P.Tempst,Anal.Chem.,1999,71,2883-2892.
[15]S.B.Ficarro,M.L.McCleland,P.T.Stukenberg,D.J.Burke,D.J.Burke,M.M.Ross,J.Shabanowitz,D.F.Hunt,F.M.White,Nat.Biotechnol.,2002,20,301-306.
[16]L.M.Brill,A.R.Salomon,S.B.Ficarro,M.Mukherji,M.Stettler-Gill,E.C.Peters,Anal.Chem.,2004,76,2763-2772.
[17] M. Kokubu, Y. Ishihama, T. Sato, T. Nagasu, Y. Oda, Anal. Chem., 2005, 77, 5144-5154.
[18] M. R. Larsen, T. E. Thingholm, O. N. Jensen, P. Roepstorff, J. D. J(?)rgensen, Mol. Cell. Proteomlcs 2005, 4, 873-886.
[19] M. W. Plnkse, P. M. Uitto, M. J. Hilhorst, B. Ooms, A. J. R. Heck, Anal. Chem. 2004, 76, 3935-3943.
[20] C.-Y. Lo, W.-Y. Chen, C.-T. Chen, Y.-C. Chen, J. Proteome Res., 2007, 6, 887-893.
[21] Y.-C. Chen, W.-Y. Chen, P.-J. Tsai, K.-Y. Chien, J.-S. Yu, Y.-C. Chen, J. Proteome Res., 2007, 6, 316-325.
[22] R. J. Tian, H. Zhang, M. L. Ye, X. G. Jiang, L. H. Hu, X. Li, X. H. Bao, H. F. Zou, Angew. Chem. Int. Ed., 2006, 45, 1-5.
[23] R. M. Valiant, Z. Szabo, L.Trojer, M. Najam-ul-Haq, M. Rainer, C. W. Huck,; R. Bakry, G. K. Bonn, J. Proteome Res. 2007, 6, 44-53.
[24] Mirre E. de Noo, Rob A. E. M. Tollenaar, Aliye O2 zalp, Peter J. K. Kuppen, Marco R. Bladergroen,,Paul H. C. Eilers, Andre' M. Deelder. Anal. Chem., 2005, 77,7232-7241.
[25] Josep Villanueva, John Philip, David Entenberg, Carlos A. Chaparro, Meena K. Tanwar,| Eric C. Holland, Anal. Chem., 2004, 76, 1560-1570.
[26] Matthias P. A. Ebert, Dagmar Niemeyer, Solren O. Deininger, Thomas Wex, Claudia Knippig, Juliane Hoffmann, Jolrg Sauer, Wolfgang Albrecht, Peter Malfertheiner, Christoph Rolcken. J. Proteome Res., 2006, 5, 2152-2158.
[27] S.-Y. Hsieh, R.-K. Chen, Y.-H. Pan, H.-L. Lee, Proteomics, 2006, 6, 3189-3198.
[28] J. N. Adkins, S. M. Varnum, K. J. Auberry, R. J. Moore, N. H. Angell, R. D. Smith, D. L. Springer, J. G. Pounds, Mol. Cell. Proteomics, 2002, 1, 947-955
[29] N. L. Anderson, N. G. Anderson, Mol. Cell. Proteomics, 2002, 1, 845-867.
[30] J. Villanueva, J. Philip, D. Entenbert, C. A. Chaparro, M. K. Tanwar, E. C. Holland, P. Tempst, Anal. Chem., 2004, 76, 1560-1570.
[31] C. A. Burtis, E. R. Ashwood, Eds. Tietz Fundamentals of Clinical Chemistry, 5th ed.; W. B. Saunders Co.: Philadelphia, PA, 2001.
[32] D. Theodorescu, S. Wittke, M. M. Ross, M. Walden, M. Conaway, I. Just, H. Mischak, H. F. Frierson, Lancet Oncol. 2006, 7, 230-240.
[33] H. R. Bergen, G. Vasmatzis, W. A. Cliby, K. L. Johnson, A. L. Oberg, D. C. Muddiman, Dis. Markers 2003, 19, 239-249.
[34] E. Orvisky, S. K. Drake, B. M. Martin, M. Abdel-Hamid, H. W. Ressom, R. S. Varghese, Y. An, D. Saha, G. L. Hortin. C. A. Loffredo, R. Goldman, Proteomics 2006, 6. 2895-2902.
[35]M.P.A.Ebert,D.Niemeyer,S.O.Deininger,T.Wex,C.Knippig,J.Hoffmann,J.Sauer,W.Albrecht,P.Malfertheiner,C.R6cken,J.Proteome Res.,2006,5,2152-2158.
[1]W.Gao,L.Dickinson,C.Grozinger,F.G.Morin,L.Reven,Langmuir,1996,12, 6429.
[2] D. Brovelli, G. Hahner, L. Ruiz, R. Hofer, G. Kraus, A. Waldner, J. Schlosser, P. Oroszlan, M. Ehrat, N. D. Spencer, Langmuir, 1999, 15, 4324.
[3] M. Textor, L. Ruiz, R. Hofer, A. Rossi, K. Feldman, G. Hahner, N. D. Spencer, Langmuir, 2000, 16,3257.
[4] N. D. Spencer, R. H. Textor, Langmuir, 2001, 17, 4014.
[5] M. H. Pinkse, P. M.Uitto, M. J.Hilhorst, B. Ooms, A. J. R. Heck, Anal. Chem., 2004, 76, 3935.
[6] X. L. Lin, Q. Peng, J. X. Yi, X. Wang, Y. D. Li. Chem. Eur. J., 2006, 12, 2383-2391.
[7] C.-T. Chen, Y.-C. Chen. Anal. Chem., 2005(77), 5912-5919.
[8] Y.-C. Chen, W.-Y. Chen, P.-J. Tsai, K.-Y. Chien, J.-S. Yu, Y.-C. Chen, J. Proteome Res., 2007, 6, 316-325.
[9] C.-Y. Lo, W.-Y. Chen, C.-T. Chen, Y.-C. Chen, J. Proteome Res., 2007, 6, 887-893.
[10] H. K. Kweon, K. Hakansson, Anal. Chem., 2006, 78, 1743-1749.
[11] C. B. Amplett, Inorganic Ion Exchangers; Elsevier: Amsterdam, The Netherlands, 1964.
[12] J. A. Blackwell, P. W. Carr, J. Chromatogr., A, 1991, 549, 43-57. [13] J. A. Blackwell, P. W. Carr, J. Chromatogr., A, 1991, 549, 59-75.
[14] D. R. Muller, P. Schindler, M. Coulot, H. Voshol, J. van Oostrum, J. Mass Spectrom., 1999,34,336-345.
[15] H. J. Zhou, S. Y. Xu, M. L. Ye, S. Feng, C. Pan, X. G. Jiang, X. Li, G. H. Han, Y. Fu, H. F. Zou, J. Proteome Res., 2006, 5, 2431-2437.
[16] M. R. Larsen, T. E. Thingholm, O. N. Jensen, P. Roepstorff, T. J. D. J(?)rgensen, Mol. Cell. Proteomics, 2005, 4, 873-886.
[17] D.Theodorescu, S. Wittke, M. M. Ross, M. Walden, M. Conaway, I. Just, H. Mischak, H. F. Frierson, Lancet Oncol., 2006, 7, 230-40.
[18] H. R. Bergen, G. Vasmatzis, W. A. Cliby, K. L. Johnson, A. L.Oberg, D. C. Muddiman, Dis. Markers, 2003, 19, 239-49.
[19] E. Orvisky, S. K. Drake, B. M. Martin, M. Abdel-Hamid, H. W. Ressom, R. S. Varghese, Y. An, D. Saha, H G. Lortin, C. A. Loffredo, R. Goldman, Proteomics, 2006, 6, 2895-2902.
[20] M. P. A. Ebert, D. Niemeyer, S. O. Deininger, T. Wex, C. Knippig, J. Hoffmann, J. Sauer, W. Albrecht. P. Malfertheiner, C. Rocken, J. Proteome Res., 2006, 5, 2152-2158.
[21] C. B. Amplett, Inorganic Ion Exchangers; Elsevier: Amsterdam, The Netherlands, 1964.
[22] D. R. Muller, P. Schindler, M. Coulot, H. Voshol, J. van Oostrum, J. Mass Spectrom. 1999,34,336-345.
[23] M. Katrin, M. W. Forest, J J. Proteome Res., 2006, 5, 98-104.
[2]L.Rosamond,A.Alsop,Science,2000,287(5460),1973-1976.
[3]F.Regnier,A.Amini,A.Chakraborty,M.Geng,J.Ji,L.Riggs,C.Sioma,S.Wang,X.Zhang,LC-GC,2001,19,200-213.
[4]S.Cordwell,et al.Electrophoresis,1997,18:1393-1398.
[5]W.P.Blackstock,M.P.Weir,Trends in Biotechnol.1999,17,121-127.
[6]吴世容,李志良,李根容,等,重庆大学学报,2004,27(1),123-127.
[7]P.J.Mintz,J.Kim,K.A.Do,et al.Nat.Biotechnol.,2003,21,57-63.
[8]梁文平,庄乾坤.北京,科学出版社,2003,229-239.
[9]M.Mann,O.N.Jensen,Nat Biotechnol,2003,21(3):255-261.
[10]O.N.Jensen,Proteomics:A Trends Guide,2000,36-42.
[11]J.Bunkenborg,B.J.Pilch,A.V.Podtelejnikov,et al.Proteomics,2004,4(2),454.
[12]肖海军,贺筱蓉.生物学通报,2001,36(7),9-10.
[13]T.N.Krogh T.,Berg,P.Hojrup,Anal.Biochem.,1999,274,153-162.
[14]X.R Mao,G.J.Guo,J.F.Huang,Z.Y.Du,Z.S.Huang,L.Ma,R L i,L.Q.Gu,J.Chem.Tech.B iotech.,2006,8(2),189-195.
[15]M.E.Marin-Zamora,F.Rojas-Melgarejo,F.Garcia-Canovas,P.A.Garcia-Ruiz,J.Chem.Tech.Biotech.,2005,80(12),1356-1364.
[16]L.Gomez,H.L.Ramirez,M.L.Villalonga,HernAdez,R.Villalonga,Enzyme and Microbial Tech.,2006,38(122),22-27.
[17]钱军民,张兴,吕飞,李旭祥,化工新型材料,2002,10,21-24.
[18]彭志英,食品酶学导论.北京:中国轻工业出版社,2002,182-189.
[19]S.J,Bencovic,Ann.Rev.Biochem,1992,6,29-54.
[20]朱祥瑞,徐俊良,浙江大学学报(农业生命科学版),2002,28(1),64-69.
[21]杨昌英,潘家荣,钟珩,湖北化工,2002,6,20-21.
[22]G.Massolini,E.J.Calleri,Sep.Sci.,2005,28(1),7-21.
[23]F.Svec,Electrophoresis,2006,27,947-961.
[24]S.Thelohan,P.Jadaud,I.W.Wainer,Chromatographia,1989,28,551-555.
[25]H.Minakuchi,K.Nakanishi,N.Soga,N.Ishizuka,N.Tanaka,J.Chromatogr.A,1998,797,121-131
[26]N.Tanaka,K.Kobayashi,N.Ishizuka,H.Minakuchi,K.Nakanishi,K.Hosoya,T.Ikegami,J.Chromatogr.A,2002,965,35-49.
[27]H.Minakuchi,K.Nakanishi,N.Soga,N.Ishizuka,N.Tanaka,Anal.Chem.,1996,68,3498-3501.
[28]E.Calleri,C.Temporini,E.Perani,C.Stella,S.Rudaz,D.Lubda,G.Mellerio,J.L.Veuthey,G.Caccialanza,G.Massolini,J.Chromatogr.A,2004,1045,99-109.
[29]郭忠,张清春,雷政登,孔亮,毛希琴,邹汉法,高等学校化学学报,2002,23(7),1277-1280.
[30]N.Amankawa,W.G..Kuhr,Anal.Chem.,1992,64,1610-1613.
[31]J.Kim,J.W.Grate,P.Wang,Chemical Engineering Science,2006,61,1017-1026.
[32]J.P.Chen,D.R.Su,Biotechnology Progress,2001,17,369-375.
[33]B.C.Kim,S.Nair,J.Kim,J.H.Kwak,J.W.Grate,S.H.Kim,M.B.Gu,Nanotechnolgy,2005,16,382-388.
[34]J.F.Diaz,K.J.J.Balkus,Journal of Molecular Catalysis B:Enzymatic,1996,2,115-126.
[35]D.Y.Zhao,Q.S.Huo,J.L.Feng,B.F.Chmelka,G.D.Stucky,Journal of the American Chemical Society,1998,120,6024-6036.
[36]P.Schmidt-Winkel,W.W.Lukens,D.Y.Zhao,P.D.Yang,B.F.Chmelka,G.D.Stucky,Journal of the American Chemical Society,1999,121,254-255.
[37]Y.J.Wang,F.Caruso,Chem.Commun.,2004,13,1528-1529.
[38]Y.L.F.Hsieh,H.Wang,C.Elicone Anal.Chem.,1996,68.455-462.
[39]L.Riggs,C.Sioma,F.E.J.Regnier,Chromatogr.A,2001,924.359-368.
[40]姜泓海,邹汉法,汪海林,张强,郭忠,张清春,陈小明,中国科学,B辑,2000,21(5),385-391.
[41]姜泓海,邹汉法,汪海林,倪坚毅,张强,高等学校化学学报,2000,21(5),702-706.
[42]H.H.Jiang,H.F.Zou,H.L.Wang,J.Y.Ni,Q.Zhang,Y.K.Zhang,J.Chromatogr.A,2000,903.77-84.
[43]S.Hjerten,J.L.Liao,R.Zhang,J.Chromatogr.,1989,473,273-275.
[44]M.Petro,F.Svec,J.M.J.Frechet,Biotech.Bioeng.,1996,48,355-363.
[45]D.S.Peterson,T.Rohr,F.Svec,J.M.J.Frechet,Anal.Chem.,2002,74,4081-4088.
[46]A.K.Palm,M.V.Novotny,Rapid Commun.Mass Spectrom.,2004,18,1374-1382.
[47]J.C.Duan,L.L.Sun,Z.Liang,J.Zhang,H.Wang,L.H.Zhang,W.B.Zhang,Y.K.Zhang,J.Chromatogr.A.2006,1106.165-174.
[48]J.C.Duan,L.Zhen,C.Yang,J.Zhang,L.H.Zhang,W.B.Zhang,Y.K.Zhang Proteomics,2006,6,412-419.
[49]黄家贤,霍岩丽,李涵,晏苏学,孙江晓,袁直,中国生物化学与分子生物学报,2001,17(3),376-3801.
[50]田玉平,王虎,查月珍,杨芃原,化学世界,2005,10,590-592.
[51]P.Cohen,Nat.Cell.Biol.,2002,4(5),127-130.
[52]S.B.Ficarro,M.L.McCleland,P.T.Stukenberg,et al.Nat.Biotechnol.,2002,20(3),301.
[53]E.H.Seeley,L.D.Riggs,F.E.Regnier,J.Chromatogr.B Analyt.Technol.Biomed.Life Sci.,2005,817(1),81.
[54]M.C.Posewitz,R Tempst,Anal.Chem.,1999,71,2883-2892.
[55]A.Stensballe,S.Andersen,O.N.Jensen Proteomics,2001,1,207-222.
[56]K.N.Barnouin,S.R.Hart,A.J.Thompson,M.Okuyama,M.Waterfield,R.Cramer,Proteomics,2005,5,4376-4388.
[57]S.R.Hart,M.D.Waterfield,A.L.Burlingame,R.J.Cramer,J.Am.Soc.Mass Spectrom.,2002,13,1042-1051.
[58]Y.H.Zhang,X.J.Yu,X.Y.Wang,W.Shah,R Y.Yang,Y.Tang,Chem.Commun.,2004,2882-2883.
[59]K.Moser,F.M.White,J.Proteome Res.,2006,5(1),98.
[60]M.O.Collins,L.Yu,M.P.Coba,et al.J.Biol.Chem.,2005,280(18),5972.
[61]P.Cao,J.T.Stults.Rapid Commun.Mass Spectrom.,2000,14(17),1600.
[62]L.Zhang,L.Zhang,W.Zhang,et al.Electrophoresis,2005,26(11),2172.
[63]R.Kange,U.Selditz,M.Granberg,et al.J.Biomol.Tech.,2005,16(2),91.
[64]M.W.Pinkse,P.M.Uitto,M.J.Hilhorst,et al.Anal.Chem.,2004,76(14),3935.
[65]M.R.Larsen,T.E.Thingholm,O.N.Jensen,et al.Mol.Cell.Proteomics,2005,4(7),873
[66]H.K.Kweon,K.Hakansson,Anal.Chem.,2006,8(6),1743.
[67]F.Wolschin,S.Wienkoop,W.Weckwerth,Proteomics,2005,5(17),4389.
[68]J.W.Mandell,Am.J.Pathol.,2003,163(5),1687.
[69]J.Rush,A.Moritz,K.A.Lee,et al.Nat.Biotechnol.,2005,23,94.
[70]M.Gronborg,T.Z.Kristiansen,A.Stensballe,et al.Mol.Cell.Proteomics,2002,1(7),517.
[71]S,Kane.H.Sano.S.C.Liu,et al.J.Biol.Chem.,2002.277(25),22115.
[72]E,Astoul,A,D,Laurence,N,Totty,et al.J.Biol.Chem.,2003,278(11),92675.
[73]A.Laurence,E.Astoul,S.Hanrahan,et al.Eur.J.Immunol.,2004,34,587.
[74]H.Zhou,J.D.Watts,R.Aebersold,Nat.Biotechnol.,2001,19,375-378.
[75]H.V.Jaffe,H.C.Pant,Biochemistry,1998,37,16211-16224.
[76]Y.Oda,T.Nagasu,B.T.Chait,Nat.Biotechnol.,2001,19,379-382.
[77]M.B.Goshe,T.P.Conrads,E.A.Panisko,N.H.Angell,T.D.Veenstra,R.D.Smith,Anal.Chem.,2001,73,2578-2586.
[78]F.Thaler,B.Valsasina,R.Baldi,J.Xie,A.Stewart,A.Isacchi,H.M.Kalisz,L.Rusconi,Anal.Bioanai.Chem.,2003,376,366-373.
[79]S.A.Beausoleil,M.Jedrychowski,D.Schwartz,et al.Proc.Natl.Acad.Sci.USA,2004,101(33),12130.
[80]B.A.Ballif,J.Viilen,S.A.Beausoleil,et al.Mol.Cell.Proteomics,2004,3,1093-1101.
[81]A.Yamagata,D.B.Kristensen,Y.Takeda,et al.Proteomics,2002,2,1267-1276.
[82]E.J.Hayduk,L.H,Choe,K,H.Lee,Electrophoresis,2004,25,2545-2556.
[83]邓新宇,姜颖,贺福初,遗传,2007,29(10),1163-1166.
[84]Q.Hu,R.J.Noll,H.Li,A.Makarov,M.Hardman,C.R.Graham,J.Mass Spectrom.,2005,40(4),430-443.
[85]P.K.Gupta,Int.J.Pharm.,1998,43,167-177.
[86]R.V.Mehta,R.V.Upadhyay,S.W.Charles,et al.Biotechnology Techniques,1997,11(7),493-496.
[87]马宁,谢文磊,现代化工,2007,27,364-369.
[88]邱光明,孙宗华,生物医学工程杂志,1995,12(3),209-213.
[89]L.Han,W.Wang,2004,35(1),15-21.
[90]C.-T.Chen,Y.-C.Chen,Anal.Chem.2005,77,5912-5919.
[91]C.-T.Chen,Y.-C.Chen,P.-J.Tsai,K.-Y.Chien,J.-S.Yu,Y.-C.Chen,J.Proteome Res.,2007,6(1),316-325.
[92]C.-Y.Lo,W.-Y.Chen,C.-T.Chen,Y.-C.Chen.J.Proteome Res.,2007,6(2),887-893.
[93]Z.Bilkova,M.Slovakova,N.Minc,C.Futterer,R.Cecal,D.Horak,M.Bene,I.Potier,J.Kenkova,M.Przybylski,J.-L.Viovy.Electrophoresis,2006,27(9),1811-1824.
[94]J.-M.Kauffmann..Anal.Chem.2004,76,5498-5502.
[95]I.Safarik,M.Safarikova,J.Chromatogr B.1999,722,33-53.
[96]D.Swinbanks,Nature,1995,378,653.
[97]I.Safarik,M.S.Safarikova,.Mon.Chem.,2002,133,737-759.
[98]H.Xu,G.L.Zhang,F.B.Zhang,et al.Chin.J.Chem.Engin.,2003,11(4),456-459.
[1]J.S.Kim,D.R.Knapp,Electrophoresis,2001,22,3993-3999.
[2]D.S.Peterson,T.Rohr,F.Svec,J.M.J.Frechet,Anal.Chem.,2002,74,4081-4088.
[3]K.Sakai-Kato,M.Kato,T.Toyooka,Anal.Chem.,2002,74,2943-2949.
[4]H.Y.Qu,J.L.Kong,P.Y.Yang,B.H.Liu,et al.,Anal.Chem.,2004,76,6426-6433.
[5]H.L.Wu,P.Y.Yang,H.H.Wang,et al.J.Proteome Res.,2004,3,1201-1208.
[6]Y.Liu,H.J.Lu,W.Zhong.P.Y.Song,et al.Anal.Chem.,2006,78,801-812.
[7]C.Wang,R.Oleshuk,F.Ouchen,J.Li,et al.Rapid Commun.Mass Spectrom.,2000,14,1377-1382.
[8]K.Sato,M.Tokeshi,T.Odake,H.Komara,et al.Anal.Chem.,2000,72,1144.
[9]A.B.Jemere,R.D.Oleshuk F.,Ouchen,F.Fajuyigbe,et al.Electrophoresis,2002,23,3537-3546.
[10]D.S.Peterson,T.Rohr,F.Svec,J.M.J.Frechet,J.Proteome Res.2002,1,563-568.
[11]A.Kondo,H.Kamura,K.Higashitani,,Appl.Microbiol.Biotechnol.,1994,41(1),99-105.
[12]严希康,朱留沙等,功能高分子报,1997,10(1),128-129.
[13]W.Choi,K.W.Oh,J.H.Thomas,W.R.Heineman,et al.Lab Chip,2002,2,27-30.
[14]L.G.Rashkovetsky,Y.V.Lyubarskaya,F.Foret,D.E.Hughes,et al.J.Chromatogr.A,1997,781,197-204.
[15]M.Slovakova,N.Minc,Z.Bilkova,C.Smadja,et al.Lab Chip,2005,5,935-943.
[16]Z.Bilkova,M.Slovakova,N.Minc,C.Futterer,et al.Electrophoresis,2006,27,1811-1824.
[17]徐秀青,基于功能磁性材料的蛋白质组学分离鉴定新方法研究,复旦大学博士学位论文,2007。
[18]M.Mary,Scientific and Clinical Applications of Magnetic Carriers,(Eds:U.Hafeli,W.Schutt,M.Zborowski),Plenum Press,New York.1997.p 303.
[19]邓勇辉,功能性磁性聚合物微球的制备、表征及其初步应用。复旦大学博士学位论文,2005。
[20]M.Okubo,H.Minami,T.Komura,J.App.Poly.Sci.,2003,88,428.
[21]马宁,谢文磊,现代化工,2007,27,364-369.
[22]J.Kenkova,F.Foret,Electrophoresis,2004,25,3550-3563.
[23]Z.Guo,S.Y.Xu,Z.D.Lei,H.F.Zou,B.C.Guo,Electrophoresis,2003,24(21),3633-3639.
[24]O.R.Zaborsky,Immobilized Enzymes,CRC Press,Cleveland,OH 1973,pp.61-74.
[25]H.Jiang,H.Zou,H.Wang,J.Ni,et al.J.Chromatogr.A,2000,903,77-86.
[26]K.Okuda,I.Urabe,Y.Yamada,H.Okada,J.Ferment.Bioeng.,1991,71,100-105.
[27]D.R.Walt,V.Agayn,Trends Anal.Chem.,1994,13,425-430.
[28]K.Sakai-Kato,M.Kato,T.Toyo'oka,Anal.Chem.,2002,74,2943.
[29] K. Sakai-Kato, M. Kato, T. Toyo'oka, Anal. Chem., 2003, 75, 388.
[30] Z.-Y. Park, D. H. Russell, Anal. Chem. 2000, 72 (11), 2667-2670.
[31] C. E. Parker, K. B. Tomer, Methods Mol. Biol. 2000, 146, 185- 201.
[32] G. J. Opiteck, K. C. Lewis, J. W. Jorgenson, R. J. Anderegg, Anal. Chem. 1997, 69(8), 1518-1524.
[33] N. C. VerBerkmoes, J. L. Bundy, L. Hauser, K. G. Asano, J. Razumovskaya, F. Larimer, R. L. Hettich, J. L. Stephenson, J. Proteome Res. 2002, 1 (3), 239-252.
[34] H. Deng, X. L. Li, Q. Peng, X. Wang, J. P. Chen, Y. D. Li. Angew. Chem. Int. Ed. 2005, 44, 2782-2785.
[35] Y. Qu, L. Moons, F. Vandesande, J. Chromatogr. B., 1997, 704, 351-358.
[36] S. Huang, X. M. Zhang, J. Chromatogr. A, 2001, 910, 13-18.
[1]A.Stensballe,O.N.Jensen,Proteomics,2001,1,955-966.
[2]D.Ericsson,S.Ekstrom,J.Nillsson,J.Bergquist,G.Marko-Varga,T.Laurell,Proteomics,2001,1,1072-1081.
[3]B.Warscheid,C.A.Fenselau,Proteomics,2004,4,2877-2892.
[4]D.Dogruel,P.Williams,R.W.Nelson,Anal.Chem.,1995,67,4343-4348.
[5]R.W.Nelson,D.Dogruel,J.R.Krone,P.Williams,Rapid Commun.Mass Spectrom.,1995,9,1380-1385.
[6]W.A.Harris,J.P.Reilly,Anal.Chem.,2002,74,4410-4416.
[7]R.J.Arnold,J.P.Reilly,Rapid Commun.Mass Spectrom.,1998,12,630-636.
[8]R.J.Arnold,J.1:'.Reilly,Anal.Biochem.,1999,269,105-112.
[9]S.P.Zheng,C.Yoo,N.Delmotte,F.R.Miller,C.G.Huber,D.M.Lubman,Anal.Chem.,2006,78,5198-5204.
[10]X.Q.Xu,C.H.Deng,M.X.Gao,W.J.Yu,P.Y.Yang,X.M.Zhang,Adv.Mater.,2006,18,3289-3293.
[11]Y.Li,X.Q.Xu,B.Yah,C.H.Deng,W.J.Yu,P.Y.Yang,X.M.Zhang J.Proteome Res.,2007,6,2367-2375.
[12]于文佳,复旦大学,博士学位论文,2005。
[13]A.Fontana,G.Fassina,C.Vita,D.Dalzoppo,M.Zamai,M.Zambonin,Biochemistry,1986,25,1847-1851.
[14]S.J.Hubbard,F.Eisenmenger,J.M.Thornton,Protein Sci.,1994,3,757-768.
[15]Z.-Y.Park,D.H.Russell,Anal.Chem.,2000,72,2667-2670.
[16]Y.Mao,Y.Li,X.M.Zhang,Proteomics 2006,6,420-426.
[17]J.Zhang,X.Q.Xu,M.X.Gao,X.M.Zhang,et al.Proteomics,2007,7,500-512.
[18]L.Wang,J.Bao,L.Wang,F.Zhang,Y.D.Li.Chem.Eur.J.,2006,12,6341-6347.
[1]H.Zhang,X.Zha,Y.Tan,et al.J.Biol.Chem.,2002,277(42),39379-39387.
[2]T.Hunter,Harvey Lect.,1998-99,94,81-119.
[3]J.Chalmersm,W.Kolch,R.Emmettm,et al.J.Chromatogr.B:Analyt.Technol.Biomed.Life Sci.,2004,803(1),111-120.
[4]K.A.Resing,N.G.Ahn,Methods Enzymol.,1997,283,29-44.
[5]R.Aebersold,D.R.Goodlett,Chem.Rev.,2001,101,269-295.
[6]A.Stensballe,S.Andersen,O.N.Jensen,Proteomics,2001,1,207-222.
[7]M.Kokubu,Y.Ishihama,T.Sato;T.Nagasu,Y.Oda,Anal.Chem.,2005,77,5144-5154.
[8]J.D.Dunn,J.T.Watson,M.L.Bruening,Anal.Chem.,2006,78,1574-1580.
[9]S.R.Hart,M.D.Waterfield,A.L.Burlingame,R.Cramer,J.Am Soc.Mass.Spectrom.,2002,13,1042.
[10]Y.Zhang,X.Yu,X.Wang,W.Shah,P.Yang,Y.Tang,Chem.Commun.,2004,2882-2883.
[11]徐秀青,基于功能磁性材料的蛋白质组学分离鉴定新方法研究,复旦大学博士学位论文,2007。
[12]L.Andersson,J.Porath,Anal.Biochem.,1986,154,250-254.
[13]L.Andersson,J.Chromatogr.,1991,539,327-334.
[14]M.C.Posewitz,P.Tempst,Anal.Chem.,1999,71,2883-2892.
[15]S.B.Ficarro,M.L.McCleland,P.T.Stukenberg,D.J.Burke,D.J.Burke,M.M.Ross,J.Shabanowitz,D.F.Hunt,F.M.White,Nat.Biotechnol.,2002,20,301-306.
[16]L.M.Brill,A.R.Salomon,S.B.Ficarro,M.Mukherji,M.Stettler-Gill,E.C.Peters,Anal.Chem.,2004,76,2763-2772.
[17] M. Kokubu, Y. Ishihama, T. Sato, T. Nagasu, Y. Oda, Anal. Chem., 2005, 77, 5144-5154.
[18] M. R. Larsen, T. E. Thingholm, O. N. Jensen, P. Roepstorff, J. D. J(?)rgensen, Mol. Cell. Proteomlcs 2005, 4, 873-886.
[19] M. W. Plnkse, P. M. Uitto, M. J. Hilhorst, B. Ooms, A. J. R. Heck, Anal. Chem. 2004, 76, 3935-3943.
[20] C.-Y. Lo, W.-Y. Chen, C.-T. Chen, Y.-C. Chen, J. Proteome Res., 2007, 6, 887-893.
[21] Y.-C. Chen, W.-Y. Chen, P.-J. Tsai, K.-Y. Chien, J.-S. Yu, Y.-C. Chen, J. Proteome Res., 2007, 6, 316-325.
[22] R. J. Tian, H. Zhang, M. L. Ye, X. G. Jiang, L. H. Hu, X. Li, X. H. Bao, H. F. Zou, Angew. Chem. Int. Ed., 2006, 45, 1-5.
[23] R. M. Valiant, Z. Szabo, L.Trojer, M. Najam-ul-Haq, M. Rainer, C. W. Huck,; R. Bakry, G. K. Bonn, J. Proteome Res. 2007, 6, 44-53.
[24] Mirre E. de Noo, Rob A. E. M. Tollenaar, Aliye O2 zalp, Peter J. K. Kuppen, Marco R. Bladergroen,,Paul H. C. Eilers, Andre' M. Deelder. Anal. Chem., 2005, 77,7232-7241.
[25] Josep Villanueva, John Philip, David Entenberg, Carlos A. Chaparro, Meena K. Tanwar,| Eric C. Holland, Anal. Chem., 2004, 76, 1560-1570.
[26] Matthias P. A. Ebert, Dagmar Niemeyer, Solren O. Deininger, Thomas Wex, Claudia Knippig, Juliane Hoffmann, Jolrg Sauer, Wolfgang Albrecht, Peter Malfertheiner, Christoph Rolcken. J. Proteome Res., 2006, 5, 2152-2158.
[27] S.-Y. Hsieh, R.-K. Chen, Y.-H. Pan, H.-L. Lee, Proteomics, 2006, 6, 3189-3198.
[28] J. N. Adkins, S. M. Varnum, K. J. Auberry, R. J. Moore, N. H. Angell, R. D. Smith, D. L. Springer, J. G. Pounds, Mol. Cell. Proteomics, 2002, 1, 947-955
[29] N. L. Anderson, N. G. Anderson, Mol. Cell. Proteomics, 2002, 1, 845-867.
[30] J. Villanueva, J. Philip, D. Entenbert, C. A. Chaparro, M. K. Tanwar, E. C. Holland, P. Tempst, Anal. Chem., 2004, 76, 1560-1570.
[31] C. A. Burtis, E. R. Ashwood, Eds. Tietz Fundamentals of Clinical Chemistry, 5th ed.; W. B. Saunders Co.: Philadelphia, PA, 2001.
[32] D. Theodorescu, S. Wittke, M. M. Ross, M. Walden, M. Conaway, I. Just, H. Mischak, H. F. Frierson, Lancet Oncol. 2006, 7, 230-240.
[33] H. R. Bergen, G. Vasmatzis, W. A. Cliby, K. L. Johnson, A. L. Oberg, D. C. Muddiman, Dis. Markers 2003, 19, 239-249.
[34] E. Orvisky, S. K. Drake, B. M. Martin, M. Abdel-Hamid, H. W. Ressom, R. S. Varghese, Y. An, D. Saha, G. L. Hortin. C. A. Loffredo, R. Goldman, Proteomics 2006, 6. 2895-2902.
[35]M.P.A.Ebert,D.Niemeyer,S.O.Deininger,T.Wex,C.Knippig,J.Hoffmann,J.Sauer,W.Albrecht,P.Malfertheiner,C.R6cken,J.Proteome Res.,2006,5,2152-2158.
[1]W.Gao,L.Dickinson,C.Grozinger,F.G.Morin,L.Reven,Langmuir,1996,12, 6429.
[2] D. Brovelli, G. Hahner, L. Ruiz, R. Hofer, G. Kraus, A. Waldner, J. Schlosser, P. Oroszlan, M. Ehrat, N. D. Spencer, Langmuir, 1999, 15, 4324.
[3] M. Textor, L. Ruiz, R. Hofer, A. Rossi, K. Feldman, G. Hahner, N. D. Spencer, Langmuir, 2000, 16,3257.
[4] N. D. Spencer, R. H. Textor, Langmuir, 2001, 17, 4014.
[5] M. H. Pinkse, P. M.Uitto, M. J.Hilhorst, B. Ooms, A. J. R. Heck, Anal. Chem., 2004, 76, 3935.
[6] X. L. Lin, Q. Peng, J. X. Yi, X. Wang, Y. D. Li. Chem. Eur. J., 2006, 12, 2383-2391.
[7] C.-T. Chen, Y.-C. Chen. Anal. Chem., 2005(77), 5912-5919.
[8] Y.-C. Chen, W.-Y. Chen, P.-J. Tsai, K.-Y. Chien, J.-S. Yu, Y.-C. Chen, J. Proteome Res., 2007, 6, 316-325.
[9] C.-Y. Lo, W.-Y. Chen, C.-T. Chen, Y.-C. Chen, J. Proteome Res., 2007, 6, 887-893.
[10] H. K. Kweon, K. Hakansson, Anal. Chem., 2006, 78, 1743-1749.
[11] C. B. Amplett, Inorganic Ion Exchangers; Elsevier: Amsterdam, The Netherlands, 1964.
[12] J. A. Blackwell, P. W. Carr, J. Chromatogr., A, 1991, 549, 43-57. [13] J. A. Blackwell, P. W. Carr, J. Chromatogr., A, 1991, 549, 59-75.
[14] D. R. Muller, P. Schindler, M. Coulot, H. Voshol, J. van Oostrum, J. Mass Spectrom., 1999,34,336-345.
[15] H. J. Zhou, S. Y. Xu, M. L. Ye, S. Feng, C. Pan, X. G. Jiang, X. Li, G. H. Han, Y. Fu, H. F. Zou, J. Proteome Res., 2006, 5, 2431-2437.
[16] M. R. Larsen, T. E. Thingholm, O. N. Jensen, P. Roepstorff, T. J. D. J(?)rgensen, Mol. Cell. Proteomics, 2005, 4, 873-886.
[17] D.Theodorescu, S. Wittke, M. M. Ross, M. Walden, M. Conaway, I. Just, H. Mischak, H. F. Frierson, Lancet Oncol., 2006, 7, 230-40.
[18] H. R. Bergen, G. Vasmatzis, W. A. Cliby, K. L. Johnson, A. L.Oberg, D. C. Muddiman, Dis. Markers, 2003, 19, 239-49.
[19] E. Orvisky, S. K. Drake, B. M. Martin, M. Abdel-Hamid, H. W. Ressom, R. S. Varghese, Y. An, D. Saha, H G. Lortin, C. A. Loffredo, R. Goldman, Proteomics, 2006, 6, 2895-2902.
[20] M. P. A. Ebert, D. Niemeyer, S. O. Deininger, T. Wex, C. Knippig, J. Hoffmann, J. Sauer, W. Albrecht. P. Malfertheiner, C. Rocken, J. Proteome Res., 2006, 5, 2152-2158.
[21] C. B. Amplett, Inorganic Ion Exchangers; Elsevier: Amsterdam, The Netherlands, 1964.
[22] D. R. Muller, P. Schindler, M. Coulot, H. Voshol, J. van Oostrum, J. Mass Spectrom. 1999,34,336-345.
[23] M. Katrin, M. W. Forest, J J. Proteome Res., 2006, 5, 98-104.