毛细管等电聚焦/加压毛细管电色谱多维联用及其在多肽蛋白质分离中的应用
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摘要
随着人类对生命体系探究的不断深入,对分析分离工具和方法的需求日渐迫切。发展二维或者是多维的高效、高分辨率和高峰容量的分析技术已经成为从根本上解决复杂体系分离问题的方法之一。尤其是随着蛋白组学研究的推进,缺乏专门针对蛋白质、多肽等生物样品的高效分离平台已经成为制约其发展的瓶颈。本研究基于毛细管等电聚焦(cIEF)与加压毛细管电色谱(pCEC)正交的分离机理,以搭建cIEF/pCEC二维分离系统为研究方向,分别对cIEF和pCEC单维以及离线和在线cIEF/pCEC二维体系搭建中的关键内容、相关技术进行了系统的研究和讨论。首次实现了cIEF和pCEC的联用分析,该二维体系从样品的等电点、疏水性、分子量和带电荷情况等方面进行多重定位,实现了对复杂蛋白质/多肽样品的高效全多维分析。证明了该二维分离体系在复杂生物样品分离中超强的分离能力和广阔的应用前景,有望成为蛋白质组学研究的重要工具。为今后cIEF-LC、CE-pCEC的联用提供了新的思路,为针对蛋白质/多肽复杂样品的多维分离研究提供了强有力的技术基础。
本论文共分为五章,主要内容包括:
第一章分别从方法、原理,检测方法以及应用等方面综述了cIEF和pCEC相关的研究背景。同时介绍了多维分离方法的理论依据,总结了多维液相色谱(MDLC),液相色谱-毛细管电泳(LC-CE),二维毛细管电泳(2D-CE)等联用技术的进展,并对cIEF及pCEC相关的多维研究现状分别进行详述。
第二章建立了一步法cIEF分离蛋白质和多肽的方法。对毛细管壁涂层方法、两性电解质的浓度、聚焦电压、添加剂等关键因素进行优化,建立了一种高效的分离两性物质的一步法cIEF。本文建立的羟丙基纤维素(HPC)涂层,100次重现性良好。与未涂层的毛细管相比,对酸性蛋白质的分离有明显改善。另外对这种cIEF方法进行重现性的考察,6种标准蛋白质分离的日内精密度均小于4%,日间精密度以及批次重现性的RSD都在4-12%之间。该方法稳定可靠,操作简便,重现性好,具有很强的聚焦能力,在优化的条件下可以实现对复杂多肽和蛋白质样品的高效分离。
第三章则对pCEC分离多肽的条件进行了探讨,本章以牛血清白蛋白(BSA)的酶解多肽为样品,主要对洗脱梯度、工作电压以及分离的重现性进行优化和考察。实验结果表明随着电压的升高,电场强度增强,样品保留时间缩短,分离速度提高。-6 kV的工作电压下,比不加电时对肽段的分离在分辨率、柱效以及峰容量上都有所改善。
第四章在前两章研究的基础上,对cIEF/pCEC二维离线系统构建中的关键问题进行优化。一步法的cIEF方便了样品迁移,使二维过程更易行。第一维毛细管柱末端的电隔离槽装置,方便了样品的收集,保证了实验的安全。以BSA的胰蛋白酶解肽段对该二维平台进行评价,通过全多维分析共检测到大约300个肽段,理论峰容量约为36000。比单维分离的方法,总峰容量和整体分离能力都得到了很大的提升。将cIEF/pCEC二维系统应用在人血红细胞破碎液(HRBCL)的蛋白质和酶解多肽的图谱构建中。建立了可靠的cIEF/pCEC离线分离体系,为搭建在线cIEF/pCEC二维平台奠定了坚实的基础。
第五章在离线分离的基础上,以电隔离槽和六通阀作为接口搭建了cIEF与pCEC在线二维联用平台,并用BSA酶解多肽进行评价。同时将其应用在HRBCL及其胰蛋白酶裂解液的研究中,证明该系统不仅可以用于复杂多肽样品的分离,而且对复杂的生物大分子也可以实现很好的分离。同时与后一维不加电的cIEF/μHPLC相比,发现cIEF/pCEC联用系统对样品的分离柱效更高,分离速度更快,在整个分离效果上有非常明显的改善。
本论文对cIEF/pCEC联用系统进行了研究,分别首次构建了离线和在线联用体系,并实现了对复杂蛋白质和多肽样品的高效分析。cIEF的聚焦能力和pCEC的双重分离机制使该二维系统在分离复杂生物样品中具有独特优势,为复杂生物样品的分离提供了一种新颖有效的分离工具,为今后cIEF及pCEC的多维联用研究打下了坚实的基础。
As the quest in understanding of life science advances, the demand for more powerful analytical tools becomes more and more urgent. Multi-dimensional separation technique becomes an effective solution for exploring complex system. Especially, the advancement of proteomics makes it essential to set up better analytical tools with higher performance, higher selectivity and higher peak capacity for separation and analysis of proteins and peptides. This dissertation focuses on coupling capillary isoelectric focusing (cIEF) with pressurized electrochromatography (pCEC) based on their orthogonal separation mechanisms. Samples can be separated by their isoelectric point, hydrophobic, molecular weight and charge condition in this system. It highlights on the key problems and relevant content in establishing both on-line and off-line cIEF/pCEC systems. The novel cIEF/pCEC two-dimensional (2D) system was realized for the first time and used in comprehensive analysis of complex peptides/proteins, demonstrating outstanding separation ability for complex biological sample and broad application prospects. The current 2D coupling system can be further extended to other multi-dimensional platforms, such as cIEF-LC, CE-pCEC, providing a strong technology fundamention for multi-dimensional separations and may promote the prosperous development of proteomics research.
This dissertation is presented in five chapters, and the main contents are as following.
The first chapter introduced the background of cIEF and pCEC from methodology, theory, detection method and applications respectively. Moreover, the outlook of multi-dimensional separation was summarized including MDLC, LC-CE and 2D-CE, where the development of cIEF or pCEC were outlined in detail.
In chapter two, to establish a robust and high performance one-step cIEF, the major factors which affect the separation in cIEF were discussed, such as the coating method of the capillary, the concentration of carrier ampholytes, the focusing voltage as well as buffer additives. The HPC coated capillary was stable in 100 runs and proteins were separated better than in uncoated capillary. Besides, the reproducibility of the method was assessed by 6 standard proteins. A with-in day precision in RSD was less than 4% (n=5), while the between-day precision and bulk reproducibility were between 4-12%. Overall, this one-step cIEF, which was easy to operate, had a satisfied reproducibility and high focusing ability. It ensured an efficient separation of complex peptides/proteins in utilized condition.
Separation conditions of peptides by pCEC were explored in the third chapter. The BSA tryptic digests were selected as the sample to evaluate the elution gradient, separation voltage, and reproducibility. With the increase of separation voltage, the separation velocity was increased. Compared to 0 kV, the resolution, column efficiency and capacity were all improved in -6 kV. The reproducibility was also satisfactory.
Based on the research in two chapters above, chapter 4 focused on the key problems of establishing the off-line cIEF/pCEC two-dimensional system. The one-step cIEF which transferred samples by EOF made the two-dimensional process easier. It was much simpler and safer to collect the sample in the first dimension by introducing an electrical decoupler in the end of the capillary of cIEF. By a comprehensive separation of off-line cIEF/pCEC, more than 300 peaks were detected in BSA digests and the theoretic capacity was about 36000. Notablely, the capacity and separation ability were both significantly improved in comparison with a single dimensional method. Furthermore, this off-line 2D system was applied to the mapping of human red blood cell lysate (HRBCL) and its digests. A robust and high efficiency off-line cIEF/pCEC was set and laid a solid foundation for the on-line coupling.
In chapter 5, we moved on to the construction of on-line cIEF/pCEC. To couple cIEF with pCEC on-line, the decoupler in the capillary of cIEF and a six port valve utilized as the interface. Meanwhile, HRBCL and its digests were respectively analyzed by this platform. Hundreds of compounds were detected by UV detector and detailed profiles were shown. In addition, cIEF/μHPLC, where no voltage was applied in the second dimension, can not match the column efficiency, separation velocity with on-line cIEF/pCEC. The overall separation ability of on-line cIEF/pCEC was much better, and it costs shorter time in comparison with off-line system.
cIEF/pCEC 2D systems, including off-line and on-line, were established and evaluated for the first time. Both systems can separate complex peptides/proteins samples with high efficiency and capacity. The high focusing ability of cIEF and double separation mechanisms of pCEC play vital role in the successful junction. All these advantages make cIEF/pCEC system an effective and high performance technique in analyzing complex biological samples. The novel two-dimensional combination lays a solid foundation for the future study of multi-dimensional systems and may greatly affect the research in proteomics.
本论文共分为五章,主要内容包括:
第一章分别从方法、原理,检测方法以及应用等方面综述了cIEF和pCEC相关的研究背景。同时介绍了多维分离方法的理论依据,总结了多维液相色谱(MDLC),液相色谱-毛细管电泳(LC-CE),二维毛细管电泳(2D-CE)等联用技术的进展,并对cIEF及pCEC相关的多维研究现状分别进行详述。
第二章建立了一步法cIEF分离蛋白质和多肽的方法。对毛细管壁涂层方法、两性电解质的浓度、聚焦电压、添加剂等关键因素进行优化,建立了一种高效的分离两性物质的一步法cIEF。本文建立的羟丙基纤维素(HPC)涂层,100次重现性良好。与未涂层的毛细管相比,对酸性蛋白质的分离有明显改善。另外对这种cIEF方法进行重现性的考察,6种标准蛋白质分离的日内精密度均小于4%,日间精密度以及批次重现性的RSD都在4-12%之间。该方法稳定可靠,操作简便,重现性好,具有很强的聚焦能力,在优化的条件下可以实现对复杂多肽和蛋白质样品的高效分离。
第三章则对pCEC分离多肽的条件进行了探讨,本章以牛血清白蛋白(BSA)的酶解多肽为样品,主要对洗脱梯度、工作电压以及分离的重现性进行优化和考察。实验结果表明随着电压的升高,电场强度增强,样品保留时间缩短,分离速度提高。-6 kV的工作电压下,比不加电时对肽段的分离在分辨率、柱效以及峰容量上都有所改善。
第四章在前两章研究的基础上,对cIEF/pCEC二维离线系统构建中的关键问题进行优化。一步法的cIEF方便了样品迁移,使二维过程更易行。第一维毛细管柱末端的电隔离槽装置,方便了样品的收集,保证了实验的安全。以BSA的胰蛋白酶解肽段对该二维平台进行评价,通过全多维分析共检测到大约300个肽段,理论峰容量约为36000。比单维分离的方法,总峰容量和整体分离能力都得到了很大的提升。将cIEF/pCEC二维系统应用在人血红细胞破碎液(HRBCL)的蛋白质和酶解多肽的图谱构建中。建立了可靠的cIEF/pCEC离线分离体系,为搭建在线cIEF/pCEC二维平台奠定了坚实的基础。
第五章在离线分离的基础上,以电隔离槽和六通阀作为接口搭建了cIEF与pCEC在线二维联用平台,并用BSA酶解多肽进行评价。同时将其应用在HRBCL及其胰蛋白酶裂解液的研究中,证明该系统不仅可以用于复杂多肽样品的分离,而且对复杂的生物大分子也可以实现很好的分离。同时与后一维不加电的cIEF/μHPLC相比,发现cIEF/pCEC联用系统对样品的分离柱效更高,分离速度更快,在整个分离效果上有非常明显的改善。
本论文对cIEF/pCEC联用系统进行了研究,分别首次构建了离线和在线联用体系,并实现了对复杂蛋白质和多肽样品的高效分析。cIEF的聚焦能力和pCEC的双重分离机制使该二维系统在分离复杂生物样品中具有独特优势,为复杂生物样品的分离提供了一种新颖有效的分离工具,为今后cIEF及pCEC的多维联用研究打下了坚实的基础。
As the quest in understanding of life science advances, the demand for more powerful analytical tools becomes more and more urgent. Multi-dimensional separation technique becomes an effective solution for exploring complex system. Especially, the advancement of proteomics makes it essential to set up better analytical tools with higher performance, higher selectivity and higher peak capacity for separation and analysis of proteins and peptides. This dissertation focuses on coupling capillary isoelectric focusing (cIEF) with pressurized electrochromatography (pCEC) based on their orthogonal separation mechanisms. Samples can be separated by their isoelectric point, hydrophobic, molecular weight and charge condition in this system. It highlights on the key problems and relevant content in establishing both on-line and off-line cIEF/pCEC systems. The novel cIEF/pCEC two-dimensional (2D) system was realized for the first time and used in comprehensive analysis of complex peptides/proteins, demonstrating outstanding separation ability for complex biological sample and broad application prospects. The current 2D coupling system can be further extended to other multi-dimensional platforms, such as cIEF-LC, CE-pCEC, providing a strong technology fundamention for multi-dimensional separations and may promote the prosperous development of proteomics research.
This dissertation is presented in five chapters, and the main contents are as following.
The first chapter introduced the background of cIEF and pCEC from methodology, theory, detection method and applications respectively. Moreover, the outlook of multi-dimensional separation was summarized including MDLC, LC-CE and 2D-CE, where the development of cIEF or pCEC were outlined in detail.
In chapter two, to establish a robust and high performance one-step cIEF, the major factors which affect the separation in cIEF were discussed, such as the coating method of the capillary, the concentration of carrier ampholytes, the focusing voltage as well as buffer additives. The HPC coated capillary was stable in 100 runs and proteins were separated better than in uncoated capillary. Besides, the reproducibility of the method was assessed by 6 standard proteins. A with-in day precision in RSD was less than 4% (n=5), while the between-day precision and bulk reproducibility were between 4-12%. Overall, this one-step cIEF, which was easy to operate, had a satisfied reproducibility and high focusing ability. It ensured an efficient separation of complex peptides/proteins in utilized condition.
Separation conditions of peptides by pCEC were explored in the third chapter. The BSA tryptic digests were selected as the sample to evaluate the elution gradient, separation voltage, and reproducibility. With the increase of separation voltage, the separation velocity was increased. Compared to 0 kV, the resolution, column efficiency and capacity were all improved in -6 kV. The reproducibility was also satisfactory.
Based on the research in two chapters above, chapter 4 focused on the key problems of establishing the off-line cIEF/pCEC two-dimensional system. The one-step cIEF which transferred samples by EOF made the two-dimensional process easier. It was much simpler and safer to collect the sample in the first dimension by introducing an electrical decoupler in the end of the capillary of cIEF. By a comprehensive separation of off-line cIEF/pCEC, more than 300 peaks were detected in BSA digests and the theoretic capacity was about 36000. Notablely, the capacity and separation ability were both significantly improved in comparison with a single dimensional method. Furthermore, this off-line 2D system was applied to the mapping of human red blood cell lysate (HRBCL) and its digests. A robust and high efficiency off-line cIEF/pCEC was set and laid a solid foundation for the on-line coupling.
In chapter 5, we moved on to the construction of on-line cIEF/pCEC. To couple cIEF with pCEC on-line, the decoupler in the capillary of cIEF and a six port valve utilized as the interface. Meanwhile, HRBCL and its digests were respectively analyzed by this platform. Hundreds of compounds were detected by UV detector and detailed profiles were shown. In addition, cIEF/μHPLC, where no voltage was applied in the second dimension, can not match the column efficiency, separation velocity with on-line cIEF/pCEC. The overall separation ability of on-line cIEF/pCEC was much better, and it costs shorter time in comparison with off-line system.
cIEF/pCEC 2D systems, including off-line and on-line, were established and evaluated for the first time. Both systems can separate complex peptides/proteins samples with high efficiency and capacity. The high focusing ability of cIEF and double separation mechanisms of pCEC play vital role in the successful junction. All these advantages make cIEF/pCEC system an effective and high performance technique in analyzing complex biological samples. The novel two-dimensional combination lays a solid foundation for the future study of multi-dimensional systems and may greatly affect the research in proteomics.
引文
[1] Service, R F. Proteomics 2.0: the view ahead. Science, 2001, 294:2076
[2] Chen J, Lee C S, Shen Y, et al.. Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis, 2002, 23: 3143-3148
[3] Tsuda T. Chromatographic behavior in electrochromatography, Anal. Chem., 1988, 60(17):1677-1680
[4] Zhang K, Gao R Y, Jiang Z J,et al.. Pressurized capillary electrochromatography separation of peptides with strong cation exchange and hydrophilic interaction. J. Sep. Sci., 2003, 26: 1389-1394
[5] Eimer T, Unger K, Tsuda T, et al.. Pressurized flow electrochromatography with reversed phase capillary columns. Anal. Chem., 1995, 352(1):649-653
[6] Hjerténa S, Zhu M D. Adaptation of the equipment for high-performance electrophoresis to isoelectric focusing. J Chromatogr. A, 1985, 346: 265-270
[7] Diaz R R, Wehr T, Zhu M. Capillary isoelectric focusing. Electrophoresis, 1997, 18: 2134-2144
[8] Hiraoka A, Tominaga I, Hori K. One-step capillary isoelectric focusing of the proteins in cerebrospinal fluid and serum of patients with neurological disorders. J. Chromatogr. A, 2002, 961:147-153
[9] Yang C, Wang S S, Chang C Y, et al.. Capillary isoelectric focusing with an open tubular immobilized pH gradient. Anal. Chem., 2010, 82, 1580-1583
[10] Horka, M., Kub?cek, O., Ruzicka, F., et al.. Capillary isoelectric focusing of native and inactivated microorganisms. J. Chromatogr. A., 2007, 1155, 164-171
[11] Shen Y F, Smith R D. High-resolution capillary isoelectric focusing of proteins using highly hydrophilic-substituted cellulose-coated capillaries. J. Microcolumn Separations, 2000,12(3): 135-141
[12] Vincenzi J A, Franco M T M. Novel approach for the analysis of glycated hemoglobin using capillary isoelectric focusing with chemical mobilization. J. Chromatogr. B, 2003, 785(2): 285-292
[13]杨春,姬磊,张维冰等.毛细管等电聚焦电泳技术进展.色谱, 2003, 21(2): 121-125
[14] Dou P, Liu Z, He J G, et al. Rapid and high-resolution glycoform profiling of recombinant human erythropoietin by capillary isoelectric focusing with whole column imaging detection. J. Chromatogr. A, 2008, 1190:372-376
[15] Ramsay L M, Dickerson J A, Dada O, et al. Femtomolar concentration detection limit and zeptomole mass detection limit for protein separation by capillary isoelectric focusing and laser-induced fluorescence detection. Anal. Chem., 2009, 81:1741-1746
[16] Shimura K. Recent advances in IEF in capillary tubes and microchips. Electrophoresis, 2009, 30:11-28
[17] Lechner M, Seifner A, RizziA M. Capillary isoelectric focusing hyphenated to single- and multistage matrix-assisted laser desorption/ionization-mass spectrometry using automated sheath-flow-assisted sample deposition. Electrophoresis, 2008, 29:1974-1984
[18]朱贵杰,张维冰,张丽华等.聚丙烯酰胺基质固定化pH梯度毛细管整体柱的制备及其对蛋白质的分离.中国科学B辑:化学, 2007, 37(1):43-47
[19] Weinberger R. Practical capillary electrophoresis. San Die-go: Academic Press Inc, 1993
[20]吴漪,张晓晖,魏娟等.毛细管电色谱和加压毛细管电色谱的进展与应用.色谱, 2009, 27(5): 609-620.
[21] Tsuda T. Electrochromatography using high applied voltage. Ana1. Chem., 1987, 59:521-523
[22] Yan C. US Pa ten t, 6569325. 2003205227
[23] Zhang K, Jiang Z J , Yao C Y, et al. Sepatation of peptides by pressurized capillary electrochromatoraphy. J. Chromatogr A, 2003, 987: 453-459
[24]吴孔弦,谷雪,阎超.加压毛细管电色谱法用于银杏叶的指纹图谱研究,分析化学, 2009, 37(4) :581-568
[25]林子俺,庞纪磊,黄慧等.毛细管电色谱联用技术的研究进展,色谱,2010,28:273-283
[26] Wen E, Rathore A S, Horváth C. Enhancement of electroosmotic flow in capillary electrochromatography. Electrophoresis, 2001, 22: 3720-3728
[27]袁瑞娟,王怀锋,刘丹宁,等.毛细管电色谱技术研究进展.化学进展, 2006, 18(9) : 1181-1190
[28]金慧,范松华,王彦,等.奶粉中三聚氰胺的加压毛细管电色谱法测定.分析测试学报, 2010, 29 (5):519 -522
[29] Wu Y, Zhao Z Z, Wu K X, et al. Am. Lab, 2010, 42:38-41
[30] Cheng X, Wang Q J, Zhang S, et al. Determination of four kinds of carbamate pesticides by capillary zone electrophoresis with amperometric detection at a polyamide-modified carbon paste electrode. Talanta, 2007, 71:1083-1088
[31] Li H J , Liu X P, Niu W X, et al. CEC with tris (2, 2’-bipyridyl) ruthenium (II) electrochemiluminescent detection. Electrophoresis, 2008, 29(22) : 4475-4481
[32] Liu S F, W u X P, Xie Z H, et a1. On-line coupling of pressurized capillary electrochromatography with end-column amperometric detection for analysis of estrogens. Electrophoresis, 2005, 26(12): 2342-2350
[33] Liu S F, Zhang X, Lin X C, et al. Development of a new method for analysis of Sudan dyes by pressurized CEC with amperometric detection. Electrophoresis, 2007,28(11): 1696-1703.
[34] Liang Z, Duan J C, Zhang L H, et al. Microdialysis Sampling Method for Evaluation of Binding Kinetics of Small Molecules to Macromolecules. Anal. Chem., 2004, 76: 6935-6939
[35] Lu M H, Zhang L, Feng Q, et a1. Pressure-assisted capillary electrochromatography with electrospray ionization-mass spectrometry based on silica-based monolithic column for rapid analysis of narcotics. Electrophoresis, 2008, 29:936-943
[36] Lu M H , Zhang L, Lu Q M, et al. Rapid analysis of peptides and amino acids by CE-ESI-MS using chemically modified fused-silica capillaries. Electrophoresis, 2009, 30, 2273-2279
[37] Yang C M, E I Rassi Z. Electrically driven microseparation methods for pesticides and metabolites. II: On-line and offline preconcentration of urea herbicides in capillary electrochromatography. Electrophoresis, 1999, 20: 2337-2342
[38] Gfrbrer P, Schewitz J, Puseeker K, et a1. Gradient elution capillary electrochromatography and hyphenation with nuclear magnetic resonance. Electrophoresis, l999, 20:3-8
[39]张锴,阎超,江正瑾,等.多肽的反相梯度加压毛细管电色谱分离.分析测试学报, 2003, 22 (3) : 32-34
[40]赵良娟,邹娟娟,王秀丽,等.梯度加压毛细管电色谱分离蛋白质.色谱, 2005, 23(6): 669-672
[41] Xie G X, Su M M, Li P, et al. Analysis of urinary metabolites for metabolomic study by pressurized CEC. Electrophoresis, 2007, 28(23): 4459-4468
[42] Xie G X, Qiu M F, Zhao A H, et al. Fingerprint Analysis of Flos Carthami by Pressurized CEC and LC. Chromatographia, 2006, 64: 739-743
[43] Xie G X, Zhao A H, Li P, et al. Fingerprint analysis of Rhizoma chuanxiong by pressurized capillary electrochromatography and high-performance liquid chromatography. Biomedical Chromatography, 2007, 21(8): 867-875
[44] Lin B, Zheng M M, Ng S C, et al. Development of in-tube solid-phase microextraction coupled to pressure-assisted CEC and its application to the analysis of propranolol enantiomers in human urine. Electrophoresis, 2007, 28(15): 2771-2780
[45] Liu S F, Zhang X, Lin X C, et al. High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel. Electrophoresis, 2007, 28 (11) : 3966-3975
[46] LüH X, Wu X P, Xie Z H, et al. Separation and determination of seven fluoroquinolones by pressurized capillary electrochromatography. J. Sep. Sci., 2005, 28(16): 2210-2217
[47]张养军,蔡耘,王京兰等.蛋白质组学研究中的色谱分离技术.色谱. 2003,21(1): 20-26
[48] Giddings J C. Sample dimensionality:a predictor of order-disorder in component peak distribution in multidimensional separation. J. Chromatogr. A, 1995,703:3-15
[49] Giddings J C, Concepts and comparisons in multi-dimensional separation. J. High Resout Chromatogr Commun, 1987, 10: 319-323
[50] Giddings J C. Two-dimensional separations: concept and promise. Anal. Chem., 1984, 56 (12): 1258A-1270A
[51] Giddings J C. Concepts and comparisons in multidimensional separation. Journal of High Resolution Chromatography, 1987, 10: 319-407
[52] Sweeney A P, Shalliker R A. Development of a two-dimensional liquid chromatography system with trapping and sample enrichment capabilities. J. Chromatogr. A, 2002, 968:41-52
[53] Murphy R E, Schure M R, Foley J P. Effect of sampling rate on resolution in comprehensive two-dimensional liquid chromatography. Anal. Chem., 1998, 70(8): 1585-1592
[54]孟兆玲,刘道杰.多维液相色谱-质谱联用技术及其在复杂体系分离分析中的应用.药物分析杂志. 2007, 27(10):1674-1679
[55] Ma S, Chen L X, Luo G A, et al. Offline comprehensive two-dimensional high-performance liquid chromatography system with size exclusion column and reverse phase column for separation of complex traditional Chinese medicine Qingkailing injection. J. Chromatogr. A, 2006, 1127(1-2):207-213
[56] I m K, Park H.W., Kim Y, et al. Comprehensive two-dimensional liquid chromatography analysis of a block copolymer. Anal. Chem., 2007, 79(3): 1067-1072
[57]郭菲,王彦,王刃锋,等.二维液相色谱-质谱联用分离中药复方葛根芩连汤中的有效成分.色谱, 2008, 26(1) : 152-160
[58] Zhou L, Beuerman R, Chew A P, et al. Quantitative analysis of N-Linked glycoproteins in tear fluid of climatic droplet keratopathy by glycopeptide capture and iTRAQ. J. Proteome Res., 2009, 8: 1992-2003
[59]吴剑威,赵润怀,陈波,等.多维液相色谱及其在中药研究中的应用.中草药, 2010, 41, 6: 1019-1023
[60] Stroink T, Wiesea G, Lingemanb H, et al.Develoment of an on-line size exclusion chromatographic-reversed-phase liquid chromatographic two-dimensional system for the quantitative determination of peptides with concentration prior to re-versed-phase liquid chromatographic separation.Anal Chim Acta, 2001, 444(2):193-203
[61] Suzanne A, Odile V T, Herve′C. Heart-cutting 2D-CE with on-line preconcentration for the chiral analysis of native amino acids. Electrophoresis, 2010,31, 1029-1035
[62]徐溢,申纪伟,曹明霞,等.含双T切换接口的胶束电动色谱-毛细管区带电泳二维电泳芯片上混合氨基酸的分离富集.分析化学研究报告, 2009, 10: 1421- 1425
[63] Dickerson J A, Dovichi N J. Capillary sieving electrophoresis and micellar electrokinetic capillary chromatography produce highly correlated separation of tryptic digests. Electrophoresis, 2010, 31, 2461-2464
[64] Lemmon A V, Jorgenson J W. Transverse flow gating interface for the coupling of microcolumn LC with CZE in a comprehensive two-dimensional system. Anal. Chem., 1993, 65 (11):1576-1581
[65] Chambers, Andrew G.; Mellors, J. Scott; Henley, W. Hampton; Ramsey, J. Michael. 238th ACS National Meeting, Washington DC, United States, August 16-20, 2009
[66]黄爽,徐韶瑛,张祥民.毛细管高效液相色谱-毛细管电泳二维分离接口的优化.分析化学, 2000, 28(12): 1467-1471
[67] Wang Y J, Balgley B M, Rudnick P A, et al. Integrated capillary isoelectric focusing/nano-resersed phase liquid chromatography coupled with ESI-MS for characterization of intact Yeast proteins. J. Proteome Res., 2005, 4 (1) :33-42
[68] Chen J Z, Balgley B M , Devoe D L , et al. Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis. Anal. Chem., 2003 , 75(13) : 3145-3152
[69] Sheng L, Pawliyn J. Comprehensive two dimensional separation based on coupling micellar electrokinetic chromatography with capillary isoelectric focusing. Analyst, 2002, 127:1159-1163
[70]刘和春,杨春,杨青,等.毛细管等电聚焦/毛细管无胶筛分电泳二维蛋白质分离平台的构建.分析化学研究报告, 2004, 32(3) : 273-277
[76]刘和春,杨春,张维冰.毛细管等电聚焦/毛细管区带电泳二维蛋白质分离平台的构建及性能考察.高等学校化学学报, 2004, 25(8) :1451-1453
[72] Yang C, Liu H C, Yang Q, et al. On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface. Anal. Chem., 2003, 75: 215-218
[73] Dickerson J A, Ramsay L M, Dada O O, et al. Two-dimensional capillary electrophoresis: Capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection. Electrophoresis, 2010, 31:2650-2654
[74] Mohan D, Lee C S. On-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis: A two-dimensional separation system for proteomics. Electrophoresis, 2002, 23:3160-3167
[75] Zhang M Q, Rassi Z El. Two-dimensional microcolumn separation platform forproteomics consisting of on-line coupled capillary isoelectric focusing and capillary electrochromatography. 1. Evaluation of the capillary-based two-dimensional platform with proteins, peptides, and human serum. J. Proteome Res.. 2006, 5: 2001-2008
[76] Stahl M, Jakob A, Von Brocke A, et al. Comparison of different setups for one- and two-dimensional capillary high-performance liquid chromatography and pressurized capillary electrochromatography coupled on-line with mass spectrometry. Electrophoresis, 2002, 23: 2949-2962
[77]吴漪,王彦,谷雪,等.强阳离子交换毛细管液相色谱-反相加压毛细管电色谱二维系统的构建及其在中药黄柏提取物分离中的应用色谱. 2010, 28(3): 226-230
[78] Righetti, P G, Glanazza, E, Gelfi, C, et al. Isoelectric focusing in immobilized pH gradients. Anal. Chem., 1989, 61:1602-1612
[79] Zhu M D, Rodriguez R, Wehr T. Optimizing separation parameters in capillary iso- electric focusing. J. Chromatogr A, 1991, 559:479-488
[80] Mazzeo J R, Krull J S. Capillary isoelectric focusing of proteins in uncoated fused-silica capillaried using polymeric assitices. Anal. Chem., 1991, 63:2852-2857
[81]Shen Y F, Berger S J, Anderson G A, et al. High-efficiency capillary isoelectric focusing of peptides. Anal. Chem., 2000, 72:2154-2159
[82] Graf M, Watzig H. Capillary isoelectric focusing reproducibility and protein adsorption. Electrophoresis, 2004, 25: 2959-2964
[83]曹枫,张维冰,阎超,等.压力对加压电色谱分离选择性影响的研究.分析化学, 2004, 32 (2) : 143-148
[84] Wu X P, Wang L, Xie Z H, et al. Rapid separation and determination of carbamate insecticides using isocratic elution pressurized capillary electrochromatography. Electrophoresis, 2006, 27: 768-777
[85] Bradford M M. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72: 24-32
[86] Li M J, Zhou J Y, Gu X, et al. Quantitative capillary electrophoresis and its application in analysis of alkaloids in tea, coffee, coca cola and theophylline tablets. J. Sep. Sci., 2009, 32: 267-274
[87] Hu S, Wang Z L, Li P B, et al. Amperometric detection in capillary electrophoresis with an etched joint. Anal. Chem., 1997, 69 (2): 264-267.
[88] Chen J, Lee C S, Shen Y, et al. Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis, 2002, 23: 3143-3148
[2] Chen J, Lee C S, Shen Y, et al.. Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis, 2002, 23: 3143-3148
[3] Tsuda T. Chromatographic behavior in electrochromatography, Anal. Chem., 1988, 60(17):1677-1680
[4] Zhang K, Gao R Y, Jiang Z J,et al.. Pressurized capillary electrochromatography separation of peptides with strong cation exchange and hydrophilic interaction. J. Sep. Sci., 2003, 26: 1389-1394
[5] Eimer T, Unger K, Tsuda T, et al.. Pressurized flow electrochromatography with reversed phase capillary columns. Anal. Chem., 1995, 352(1):649-653
[6] Hjerténa S, Zhu M D. Adaptation of the equipment for high-performance electrophoresis to isoelectric focusing. J Chromatogr. A, 1985, 346: 265-270
[7] Diaz R R, Wehr T, Zhu M. Capillary isoelectric focusing. Electrophoresis, 1997, 18: 2134-2144
[8] Hiraoka A, Tominaga I, Hori K. One-step capillary isoelectric focusing of the proteins in cerebrospinal fluid and serum of patients with neurological disorders. J. Chromatogr. A, 2002, 961:147-153
[9] Yang C, Wang S S, Chang C Y, et al.. Capillary isoelectric focusing with an open tubular immobilized pH gradient. Anal. Chem., 2010, 82, 1580-1583
[10] Horka, M., Kub?cek, O., Ruzicka, F., et al.. Capillary isoelectric focusing of native and inactivated microorganisms. J. Chromatogr. A., 2007, 1155, 164-171
[11] Shen Y F, Smith R D. High-resolution capillary isoelectric focusing of proteins using highly hydrophilic-substituted cellulose-coated capillaries. J. Microcolumn Separations, 2000,12(3): 135-141
[12] Vincenzi J A, Franco M T M. Novel approach for the analysis of glycated hemoglobin using capillary isoelectric focusing with chemical mobilization. J. Chromatogr. B, 2003, 785(2): 285-292
[13]杨春,姬磊,张维冰等.毛细管等电聚焦电泳技术进展.色谱, 2003, 21(2): 121-125
[14] Dou P, Liu Z, He J G, et al. Rapid and high-resolution glycoform profiling of recombinant human erythropoietin by capillary isoelectric focusing with whole column imaging detection. J. Chromatogr. A, 2008, 1190:372-376
[15] Ramsay L M, Dickerson J A, Dada O, et al. Femtomolar concentration detection limit and zeptomole mass detection limit for protein separation by capillary isoelectric focusing and laser-induced fluorescence detection. Anal. Chem., 2009, 81:1741-1746
[16] Shimura K. Recent advances in IEF in capillary tubes and microchips. Electrophoresis, 2009, 30:11-28
[17] Lechner M, Seifner A, RizziA M. Capillary isoelectric focusing hyphenated to single- and multistage matrix-assisted laser desorption/ionization-mass spectrometry using automated sheath-flow-assisted sample deposition. Electrophoresis, 2008, 29:1974-1984
[18]朱贵杰,张维冰,张丽华等.聚丙烯酰胺基质固定化pH梯度毛细管整体柱的制备及其对蛋白质的分离.中国科学B辑:化学, 2007, 37(1):43-47
[19] Weinberger R. Practical capillary electrophoresis. San Die-go: Academic Press Inc, 1993
[20]吴漪,张晓晖,魏娟等.毛细管电色谱和加压毛细管电色谱的进展与应用.色谱, 2009, 27(5): 609-620.
[21] Tsuda T. Electrochromatography using high applied voltage. Ana1. Chem., 1987, 59:521-523
[22] Yan C. US Pa ten t, 6569325. 2003205227
[23] Zhang K, Jiang Z J , Yao C Y, et al. Sepatation of peptides by pressurized capillary electrochromatoraphy. J. Chromatogr A, 2003, 987: 453-459
[24]吴孔弦,谷雪,阎超.加压毛细管电色谱法用于银杏叶的指纹图谱研究,分析化学, 2009, 37(4) :581-568
[25]林子俺,庞纪磊,黄慧等.毛细管电色谱联用技术的研究进展,色谱,2010,28:273-283
[26] Wen E, Rathore A S, Horváth C. Enhancement of electroosmotic flow in capillary electrochromatography. Electrophoresis, 2001, 22: 3720-3728
[27]袁瑞娟,王怀锋,刘丹宁,等.毛细管电色谱技术研究进展.化学进展, 2006, 18(9) : 1181-1190
[28]金慧,范松华,王彦,等.奶粉中三聚氰胺的加压毛细管电色谱法测定.分析测试学报, 2010, 29 (5):519 -522
[29] Wu Y, Zhao Z Z, Wu K X, et al. Am. Lab, 2010, 42:38-41
[30] Cheng X, Wang Q J, Zhang S, et al. Determination of four kinds of carbamate pesticides by capillary zone electrophoresis with amperometric detection at a polyamide-modified carbon paste electrode. Talanta, 2007, 71:1083-1088
[31] Li H J , Liu X P, Niu W X, et al. CEC with tris (2, 2’-bipyridyl) ruthenium (II) electrochemiluminescent detection. Electrophoresis, 2008, 29(22) : 4475-4481
[32] Liu S F, W u X P, Xie Z H, et a1. On-line coupling of pressurized capillary electrochromatography with end-column amperometric detection for analysis of estrogens. Electrophoresis, 2005, 26(12): 2342-2350
[33] Liu S F, Zhang X, Lin X C, et al. Development of a new method for analysis of Sudan dyes by pressurized CEC with amperometric detection. Electrophoresis, 2007,28(11): 1696-1703.
[34] Liang Z, Duan J C, Zhang L H, et al. Microdialysis Sampling Method for Evaluation of Binding Kinetics of Small Molecules to Macromolecules. Anal. Chem., 2004, 76: 6935-6939
[35] Lu M H, Zhang L, Feng Q, et a1. Pressure-assisted capillary electrochromatography with electrospray ionization-mass spectrometry based on silica-based monolithic column for rapid analysis of narcotics. Electrophoresis, 2008, 29:936-943
[36] Lu M H , Zhang L, Lu Q M, et al. Rapid analysis of peptides and amino acids by CE-ESI-MS using chemically modified fused-silica capillaries. Electrophoresis, 2009, 30, 2273-2279
[37] Yang C M, E I Rassi Z. Electrically driven microseparation methods for pesticides and metabolites. II: On-line and offline preconcentration of urea herbicides in capillary electrochromatography. Electrophoresis, 1999, 20: 2337-2342
[38] Gfrbrer P, Schewitz J, Puseeker K, et a1. Gradient elution capillary electrochromatography and hyphenation with nuclear magnetic resonance. Electrophoresis, l999, 20:3-8
[39]张锴,阎超,江正瑾,等.多肽的反相梯度加压毛细管电色谱分离.分析测试学报, 2003, 22 (3) : 32-34
[40]赵良娟,邹娟娟,王秀丽,等.梯度加压毛细管电色谱分离蛋白质.色谱, 2005, 23(6): 669-672
[41] Xie G X, Su M M, Li P, et al. Analysis of urinary metabolites for metabolomic study by pressurized CEC. Electrophoresis, 2007, 28(23): 4459-4468
[42] Xie G X, Qiu M F, Zhao A H, et al. Fingerprint Analysis of Flos Carthami by Pressurized CEC and LC. Chromatographia, 2006, 64: 739-743
[43] Xie G X, Zhao A H, Li P, et al. Fingerprint analysis of Rhizoma chuanxiong by pressurized capillary electrochromatography and high-performance liquid chromatography. Biomedical Chromatography, 2007, 21(8): 867-875
[44] Lin B, Zheng M M, Ng S C, et al. Development of in-tube solid-phase microextraction coupled to pressure-assisted CEC and its application to the analysis of propranolol enantiomers in human urine. Electrophoresis, 2007, 28(15): 2771-2780
[45] Liu S F, Zhang X, Lin X C, et al. High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel. Electrophoresis, 2007, 28 (11) : 3966-3975
[46] LüH X, Wu X P, Xie Z H, et al. Separation and determination of seven fluoroquinolones by pressurized capillary electrochromatography. J. Sep. Sci., 2005, 28(16): 2210-2217
[47]张养军,蔡耘,王京兰等.蛋白质组学研究中的色谱分离技术.色谱. 2003,21(1): 20-26
[48] Giddings J C. Sample dimensionality:a predictor of order-disorder in component peak distribution in multidimensional separation. J. Chromatogr. A, 1995,703:3-15
[49] Giddings J C, Concepts and comparisons in multi-dimensional separation. J. High Resout Chromatogr Commun, 1987, 10: 319-323
[50] Giddings J C. Two-dimensional separations: concept and promise. Anal. Chem., 1984, 56 (12): 1258A-1270A
[51] Giddings J C. Concepts and comparisons in multidimensional separation. Journal of High Resolution Chromatography, 1987, 10: 319-407
[52] Sweeney A P, Shalliker R A. Development of a two-dimensional liquid chromatography system with trapping and sample enrichment capabilities. J. Chromatogr. A, 2002, 968:41-52
[53] Murphy R E, Schure M R, Foley J P. Effect of sampling rate on resolution in comprehensive two-dimensional liquid chromatography. Anal. Chem., 1998, 70(8): 1585-1592
[54]孟兆玲,刘道杰.多维液相色谱-质谱联用技术及其在复杂体系分离分析中的应用.药物分析杂志. 2007, 27(10):1674-1679
[55] Ma S, Chen L X, Luo G A, et al. Offline comprehensive two-dimensional high-performance liquid chromatography system with size exclusion column and reverse phase column for separation of complex traditional Chinese medicine Qingkailing injection. J. Chromatogr. A, 2006, 1127(1-2):207-213
[56] I m K, Park H.W., Kim Y, et al. Comprehensive two-dimensional liquid chromatography analysis of a block copolymer. Anal. Chem., 2007, 79(3): 1067-1072
[57]郭菲,王彦,王刃锋,等.二维液相色谱-质谱联用分离中药复方葛根芩连汤中的有效成分.色谱, 2008, 26(1) : 152-160
[58] Zhou L, Beuerman R, Chew A P, et al. Quantitative analysis of N-Linked glycoproteins in tear fluid of climatic droplet keratopathy by glycopeptide capture and iTRAQ. J. Proteome Res., 2009, 8: 1992-2003
[59]吴剑威,赵润怀,陈波,等.多维液相色谱及其在中药研究中的应用.中草药, 2010, 41, 6: 1019-1023
[60] Stroink T, Wiesea G, Lingemanb H, et al.Develoment of an on-line size exclusion chromatographic-reversed-phase liquid chromatographic two-dimensional system for the quantitative determination of peptides with concentration prior to re-versed-phase liquid chromatographic separation.Anal Chim Acta, 2001, 444(2):193-203
[61] Suzanne A, Odile V T, Herve′C. Heart-cutting 2D-CE with on-line preconcentration for the chiral analysis of native amino acids. Electrophoresis, 2010,31, 1029-1035
[62]徐溢,申纪伟,曹明霞,等.含双T切换接口的胶束电动色谱-毛细管区带电泳二维电泳芯片上混合氨基酸的分离富集.分析化学研究报告, 2009, 10: 1421- 1425
[63] Dickerson J A, Dovichi N J. Capillary sieving electrophoresis and micellar electrokinetic capillary chromatography produce highly correlated separation of tryptic digests. Electrophoresis, 2010, 31, 2461-2464
[64] Lemmon A V, Jorgenson J W. Transverse flow gating interface for the coupling of microcolumn LC with CZE in a comprehensive two-dimensional system. Anal. Chem., 1993, 65 (11):1576-1581
[65] Chambers, Andrew G.; Mellors, J. Scott; Henley, W. Hampton; Ramsey, J. Michael. 238th ACS National Meeting, Washington DC, United States, August 16-20, 2009
[66]黄爽,徐韶瑛,张祥民.毛细管高效液相色谱-毛细管电泳二维分离接口的优化.分析化学, 2000, 28(12): 1467-1471
[67] Wang Y J, Balgley B M, Rudnick P A, et al. Integrated capillary isoelectric focusing/nano-resersed phase liquid chromatography coupled with ESI-MS for characterization of intact Yeast proteins. J. Proteome Res., 2005, 4 (1) :33-42
[68] Chen J Z, Balgley B M , Devoe D L , et al. Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis. Anal. Chem., 2003 , 75(13) : 3145-3152
[69] Sheng L, Pawliyn J. Comprehensive two dimensional separation based on coupling micellar electrokinetic chromatography with capillary isoelectric focusing. Analyst, 2002, 127:1159-1163
[70]刘和春,杨春,杨青,等.毛细管等电聚焦/毛细管无胶筛分电泳二维蛋白质分离平台的构建.分析化学研究报告, 2004, 32(3) : 273-277
[76]刘和春,杨春,张维冰.毛细管等电聚焦/毛细管区带电泳二维蛋白质分离平台的构建及性能考察.高等学校化学学报, 2004, 25(8) :1451-1453
[72] Yang C, Liu H C, Yang Q, et al. On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface. Anal. Chem., 2003, 75: 215-218
[73] Dickerson J A, Ramsay L M, Dada O O, et al. Two-dimensional capillary electrophoresis: Capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection. Electrophoresis, 2010, 31:2650-2654
[74] Mohan D, Lee C S. On-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis: A two-dimensional separation system for proteomics. Electrophoresis, 2002, 23:3160-3167
[75] Zhang M Q, Rassi Z El. Two-dimensional microcolumn separation platform forproteomics consisting of on-line coupled capillary isoelectric focusing and capillary electrochromatography. 1. Evaluation of the capillary-based two-dimensional platform with proteins, peptides, and human serum. J. Proteome Res.. 2006, 5: 2001-2008
[76] Stahl M, Jakob A, Von Brocke A, et al. Comparison of different setups for one- and two-dimensional capillary high-performance liquid chromatography and pressurized capillary electrochromatography coupled on-line with mass spectrometry. Electrophoresis, 2002, 23: 2949-2962
[77]吴漪,王彦,谷雪,等.强阳离子交换毛细管液相色谱-反相加压毛细管电色谱二维系统的构建及其在中药黄柏提取物分离中的应用色谱. 2010, 28(3): 226-230
[78] Righetti, P G, Glanazza, E, Gelfi, C, et al. Isoelectric focusing in immobilized pH gradients. Anal. Chem., 1989, 61:1602-1612
[79] Zhu M D, Rodriguez R, Wehr T. Optimizing separation parameters in capillary iso- electric focusing. J. Chromatogr A, 1991, 559:479-488
[80] Mazzeo J R, Krull J S. Capillary isoelectric focusing of proteins in uncoated fused-silica capillaried using polymeric assitices. Anal. Chem., 1991, 63:2852-2857
[81]Shen Y F, Berger S J, Anderson G A, et al. High-efficiency capillary isoelectric focusing of peptides. Anal. Chem., 2000, 72:2154-2159
[82] Graf M, Watzig H. Capillary isoelectric focusing reproducibility and protein adsorption. Electrophoresis, 2004, 25: 2959-2964
[83]曹枫,张维冰,阎超,等.压力对加压电色谱分离选择性影响的研究.分析化学, 2004, 32 (2) : 143-148
[84] Wu X P, Wang L, Xie Z H, et al. Rapid separation and determination of carbamate insecticides using isocratic elution pressurized capillary electrochromatography. Electrophoresis, 2006, 27: 768-777
[85] Bradford M M. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72: 24-32
[86] Li M J, Zhou J Y, Gu X, et al. Quantitative capillary electrophoresis and its application in analysis of alkaloids in tea, coffee, coca cola and theophylline tablets. J. Sep. Sci., 2009, 32: 267-274
[87] Hu S, Wang Z L, Li P B, et al. Amperometric detection in capillary electrophoresis with an etched joint. Anal. Chem., 1997, 69 (2): 264-267.
[88] Chen J, Lee C S, Shen Y, et al. Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis, 2002, 23: 3143-3148