一分钟整体蛋白液相色谱法的建立
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摘要
蛋白质组学、蛋白药物和来自动植物等天然产物中的蛋白的分离和纯化的研究都需要液相色谱法快速分离技术。Peter Carr小组最近提出快速分离的目标：一维液相色谱分离要在1～10 min内完成,二维液相色谱分离要在10～100 min内完成。尽管这个建议是针对多肽提出的,但是也可以作为整体蛋白快速分离的目标。在本文中,利用蛋白质在液-固界面的不连续迁移和色谱饼这种柱技术,首次建立了一分钟整体蛋白液相色谱法。一般的快速分离仅仅包含了蛋白分离过程,而一分钟整体蛋白液相色谱在1 min内完成分离过程和色谱饼再生过程。分别用标准蛋白检测了反相色谱法、离子交换色谱法和疏水相互作用色谱法三种模式的一分钟整体蛋白液相色谱法,蛋白质分离度没有明显地损失。除了分离速度快分离能力强,该技术还有负载量高的优点。
论文包括了以下6个部分：
1.绪论
说明了色谱法快速分离的意义。介绍了一维快速分离蛋白质的方法,包括非多孔固定相、整体固定相、灌注色谱法、高温快速液相色谱法和色谱饼法；还介绍了二维色谱快速分离蛋白质的方法,包括了常规的使用多根不同机理色谱柱的二维液相色谱法(2D-LC)和使用一根有两种正交分离能力色谱柱的单柱二维液相色谱法(2D-LC-1C)。
2.多孔反相填料对整体蛋白的高速、高分离度和高负载量分离
使用侧孔装填技术把3μm的C18反相多孔填料装填在超薄色谱饼(1 mm×10 mmI.D.,78μL)中,用于7种标准蛋白混合物的分离。首次比较了小柱床体积的色谱饼和大柱床体积的色谱柱(50mm×4.6mm I.D.,830μL)分离小分子和整体蛋白的效果。由于小分子在色谱柱(饼)床上的迁移是连续的,导致色谱饼分离小分子的效果远远差于色谱柱。而蛋白质在液-固界面存在“粘附能”,使得蛋白质在色谱柱(饼)床上的迁移是不连续,造成色谱饼分离蛋白质的效果与色谱柱相当。由于色谱饼内径大柱长短的特殊结构,其动力学性质远好于色谱柱,可以在高达10 mL／min的流速下使用。在优化条件下用色谱饼在30 s内分离了7种蛋白。在5 min内连续5次快速分离蛋白质。表明当固定相和蛋白质之间的作用力是非选择性作用力时,一分钟整体蛋白液相色谱法是可以实现的。在1 min内还基线分离了总量是1 mg的7种蛋白的混合物,实现了高速、高分离度和高负载量的“三高”效果。该方法有希望用于临床例行分析。
3.多孔弱阳离子交换填料对整体蛋白的“三高”分离
比较了两种弱阳离子交换商品填料TSKgel CM-5PW和PolyCAT A分离蛋白的效果,填料PolyCAT A(粒径,3μm；孔径,150 nm)更适合实现一分钟整体蛋白液相色谱法。PolyCAT A色谱饼(1 mm×10 mm I.D.)和色谱柱(50 mm×4.6 mm I.D.)的分离能力相当。然而,色谱饼的动力学性质更好。在优化的条件下,在18 s内分离了4种标准蛋白。分离速度比整体固定相和无孔填料都要快,分离效果也更好。在10 min内连续进行了10次的蛋白分离,证明了当固定相和蛋白质之间的作用力是静电相互作用力时,也可以实现一分钟整体蛋白液相色谱法。另外用PolyCAT A色谱饼在1 min内快速分离了总量1 mg的4种蛋白混合物。
4.无孔疏水填料快速分离整体蛋白
用装填2.5 gm的无孔疏水填料的色谱饼(10 mm×10 mm I.D.)和色谱柱(35 mm×4.6 mm I.D.)来分离蛋白质。在优化的条件下,用色谱柱在3.5 min内可以分离7种蛋白的混合物,而使用色谱饼仅仅需要2.1 min就可以完成类似的分离。色谱饼的动力学性质更好,可以在更大的流速下分离蛋白质,更适合快速分离。尽管得到的结果大于1min,但是比较接近一分钟整体蛋白液相色谱法的目标。5.无孔反相色谱填料对整体蛋白的“三高”分离
用装有1.0μm无孔反相填料色谱饼(7.5 mm×10 mm I.D.)首次在常规色谱条件下1min分离1μg和40μg的7种标准蛋白,并且在1 min内几乎完全分离0.5 mg该7种蛋白的混合物,达到高速、高分离度和高负载的“三高”效果。开辟了用常规液相色谱仪快速分离高负载蛋白的新途径。6.多孔二维色谱(SAX-HIC)填料及其在疏水模式下的一分钟整体蛋白色谱法
与相应模式的一维色谱填料相比,二维色谱填料有改善分离选择性的优点,因此合成了一种多孔强阴离子交换-疏水相互作用二维色谱(SAX-HIC)填料来改善填料在HIC模式下的分离能力。用该填料装填的二维色谱饼(1 mm×10 mm I.D.)在HIC模式下分离蛋白。在5 mL／min流速下1 min内不仅快速分离了6种蛋白质,还平衡了色谱柱。其分离速度和效果是一般的疏水填料难以达到的。该结果证明了由疏水相互作用力控制的疏水色谱完全可能实现一分钟整体蛋白色谱法。
The study on the separation of intact proteins in proteomics, protein drugs and the purification of proteins in native plants and animals need very fast separation by high-performance liquid chromatography (HPLC). Peter Carr's group suggested that one-dimensional liquid chromatography (1D-LC) should be accomplished in 1～10 minutes, and two-dimensional LC (2D-LC) should be accomplished in 10～100 minutes. Although it aimed at peptides, the suggestion could be also used as an important reference criterion of protein separation. Based on discontinuous migration of proteins on liquid-solid interface and new column technology of chromatographic cake, one minute-liquid chromatography (OMLC) of intact proteins which has been a dream of separation scientists for a long time is firstly established in the thesis. Compared with conventional fast separation only involving the process of protein separation itself, the OMLC includes the total time of separation and column re-generation. The OMLC was tested with reversed-phase liquid chromatography (RPLC), ion-exchange chromatography (IEC), hydrophobic-interaction chromatography (HIC) using standard proteins under the condition that in comparison with their corresponding to conventional LC, the. resolution of intact proteins in this thesis does not significantly lose. Besides high speed and high resolving power, the technology has the advantages of high loading capacity.
The thesis includes the following six sections:
1 Introduction
The significance of fast separation by LC was briefly introduced. One-dimensional methods of rapid separation of intact proteins including non-porous stationary phase, monolithic stationary phase, perfusion chromatography, high temperature fast chromatography and chromatographic cake were simply introduced and reviewed. Both conventional two-dimensional liquid chromatography (2D-LC) and two-dimensional liquid chromatography using only a single column (2D-LC-1C) which exhibits excellent resolution in two modes were also introduced and reviewed.
2 Separation of intact proteins with porous reversed-phase packings with high speed, high resolution and high sample loading
Ultrashort chromatographic cake (1 mm×10 mm I.D.,78μL) packed with 3μm porous C18 reversed-phase packings from lateral hole of the cake was employed for the separation of seven standard proteins. The resolution for intact proteins and small solutes between such a small packed bed volume of chromatographic cake and a large pack bed volume of column (50 mm×4.6 mm I.D.,830μL) were firstly compared. Continuous migration of the small molecules in the packed bed of column and cake resulted in that the resolution of small solutes was extremely poorer with the cake than the column, but due to the existence of "adhesive energy" of intact proteins on the liquid-solid interface the discontinuous migration of intact proteins resulted in almost the comparable resolution between the cake and the column. However, the cake having better hydrodynamic property than column showed moderate back pressure at high flow rate up to 10 mL/min. Under an optimized condition, the mixture of seven proteins was completely separated in 30 s. Five consecutive separations within 5 minutes were carried out with the cake also, indicating one minute-liquid chromatography (OMLC) of intact proteins can be accomplished when the molecular interactions between protein and stationary phase is non-selective force. The OMLC has been a dream of separation scientists for a long time now becomes realism. The mixture of seven proteins was well separated within 1 minute under the sample loading of 1 mg and thus the separation of intact proteins in this circumstance also achieves the "three high" purpose of high speed, high resolution and high sample loading. The method is expected to be used in clinically routine analysis.
3. Separation of intact proteins using porous weak cation-exchange packings with "three high" purpose
The comparison of separations of intact proteins with two commercial weak cation-exchange packings, TSKgel CM-5PW and PolyCAT A, was carried out. It was found that PolyCAT A (particle size,3μm; pore size,150 nm) was more suitable for achieving the OMLC. The resolution of the PolyCAT A cake (1 mm×10 mm I.D.) was comparable with the PolyCAT A column (50 mm×4.6 mm I.D.). However, the cake showed better hydrodynamic property than the column. The complete separation of 4 standard proteins was achieved in 18 s under an optimized condition with the cake. The speed and resolution is better than non-porous stationary phase and monolithic stationary phase reported in literatures. Ten consecutive separations within 10 minutes were carried out using the cake, really accomplishing the OMLC, when the molecular interaction between protein and stationary phase is electrostatic force. Also, fast separation was achieved in 1 minute under the loading of 1 mg mixture of 4 proteins.
4. Fast separation of protein with non-porous HIC packings
Both the chromatographic cake (10 mm×10 mm I.D.) and column (35 mm×4.6 mm I.D.) packed with 2.5μm non-porous HIC packings were employed for the separation of standard proteins. Under optimized conditions, the mixture of seven proteins could be separated within 3.5 min with the column, and a comparable resolution was obtained in 2.1 min with the cake. The cake, which can be used at higher flow rate, is more suitable for fast separation due to its better hydrodynamic property. Although the result is more than one minute, it is closed (2-3.5 min) to the OMLC.
5. Separation of intact proteins with "three high" purpose using non-porous RPLC packings
A chromatographic cake (7.5 mm×10 mm I.D.) having larger diameter than its thickness is firstly employed to completely separate 1.0μg and 40μg of the mixture of seven standard proteins within 1 minute under conventional chromatographic conditions. Also,0.5 mg of the mixture was almost completely separated within 1 minute. The mixture of proteins was separated with high speed, high loading and high resolution. A new approach for rapid separation of proteins was put forward using conventional HPLC under high loading.
6. Porous 2D (SAX-HIC) packings employed for the OMLC in HIC mode
Taking the advantage of two dimensional packings (2D-packings) having a better selectivity than its corresponding one dimensional packings, a kind of porous two-dimensional packings of strong anion-exchange (SAX) and HIC was synthesized.
The packed 2D (SAX-HIC) cake (1 mm×10 mm I.D.) was used to separate standard proteins in HIC mode. The mixture of six standard proteins could be almost completely separated and the cake was re-generated within one minute under the flow rate of 5 mL/min. Such a fast and good resolution could not be obtained with normally commercial HIC columns. This fact indicates that OMLC is validly carried out for the retention mechanism of protein dominated by hydrophobic-interaction force.
论文包括了以下6个部分：
1.绪论
说明了色谱法快速分离的意义。介绍了一维快速分离蛋白质的方法,包括非多孔固定相、整体固定相、灌注色谱法、高温快速液相色谱法和色谱饼法；还介绍了二维色谱快速分离蛋白质的方法,包括了常规的使用多根不同机理色谱柱的二维液相色谱法(2D-LC)和使用一根有两种正交分离能力色谱柱的单柱二维液相色谱法(2D-LC-1C)。
2.多孔反相填料对整体蛋白的高速、高分离度和高负载量分离
使用侧孔装填技术把3μm的C18反相多孔填料装填在超薄色谱饼(1 mm×10 mmI.D.,78μL)中,用于7种标准蛋白混合物的分离。首次比较了小柱床体积的色谱饼和大柱床体积的色谱柱(50mm×4.6mm I.D.,830μL)分离小分子和整体蛋白的效果。由于小分子在色谱柱(饼)床上的迁移是连续的,导致色谱饼分离小分子的效果远远差于色谱柱。而蛋白质在液-固界面存在“粘附能”,使得蛋白质在色谱柱(饼)床上的迁移是不连续,造成色谱饼分离蛋白质的效果与色谱柱相当。由于色谱饼内径大柱长短的特殊结构,其动力学性质远好于色谱柱,可以在高达10 mL／min的流速下使用。在优化条件下用色谱饼在30 s内分离了7种蛋白。在5 min内连续5次快速分离蛋白质。表明当固定相和蛋白质之间的作用力是非选择性作用力时,一分钟整体蛋白液相色谱法是可以实现的。在1 min内还基线分离了总量是1 mg的7种蛋白的混合物,实现了高速、高分离度和高负载量的“三高”效果。该方法有希望用于临床例行分析。
3.多孔弱阳离子交换填料对整体蛋白的“三高”分离
比较了两种弱阳离子交换商品填料TSKgel CM-5PW和PolyCAT A分离蛋白的效果,填料PolyCAT A(粒径,3μm；孔径,150 nm)更适合实现一分钟整体蛋白液相色谱法。PolyCAT A色谱饼(1 mm×10 mm I.D.)和色谱柱(50 mm×4.6 mm I.D.)的分离能力相当。然而,色谱饼的动力学性质更好。在优化的条件下,在18 s内分离了4种标准蛋白。分离速度比整体固定相和无孔填料都要快,分离效果也更好。在10 min内连续进行了10次的蛋白分离,证明了当固定相和蛋白质之间的作用力是静电相互作用力时,也可以实现一分钟整体蛋白液相色谱法。另外用PolyCAT A色谱饼在1 min内快速分离了总量1 mg的4种蛋白混合物。
4.无孔疏水填料快速分离整体蛋白
用装填2.5 gm的无孔疏水填料的色谱饼(10 mm×10 mm I.D.)和色谱柱(35 mm×4.6 mm I.D.)来分离蛋白质。在优化的条件下,用色谱柱在3.5 min内可以分离7种蛋白的混合物,而使用色谱饼仅仅需要2.1 min就可以完成类似的分离。色谱饼的动力学性质更好,可以在更大的流速下分离蛋白质,更适合快速分离。尽管得到的结果大于1min,但是比较接近一分钟整体蛋白液相色谱法的目标。5.无孔反相色谱填料对整体蛋白的“三高”分离
用装有1.0μm无孔反相填料色谱饼(7.5 mm×10 mm I.D.)首次在常规色谱条件下1min分离1μg和40μg的7种标准蛋白,并且在1 min内几乎完全分离0.5 mg该7种蛋白的混合物,达到高速、高分离度和高负载的“三高”效果。开辟了用常规液相色谱仪快速分离高负载蛋白的新途径。6.多孔二维色谱(SAX-HIC)填料及其在疏水模式下的一分钟整体蛋白色谱法
与相应模式的一维色谱填料相比,二维色谱填料有改善分离选择性的优点,因此合成了一种多孔强阴离子交换-疏水相互作用二维色谱(SAX-HIC)填料来改善填料在HIC模式下的分离能力。用该填料装填的二维色谱饼(1 mm×10 mm I.D.)在HIC模式下分离蛋白。在5 mL／min流速下1 min内不仅快速分离了6种蛋白质,还平衡了色谱柱。其分离速度和效果是一般的疏水填料难以达到的。该结果证明了由疏水相互作用力控制的疏水色谱完全可能实现一分钟整体蛋白色谱法。
The study on the separation of intact proteins in proteomics, protein drugs and the purification of proteins in native plants and animals need very fast separation by high-performance liquid chromatography (HPLC). Peter Carr's group suggested that one-dimensional liquid chromatography (1D-LC) should be accomplished in 1～10 minutes, and two-dimensional LC (2D-LC) should be accomplished in 10～100 minutes. Although it aimed at peptides, the suggestion could be also used as an important reference criterion of protein separation. Based on discontinuous migration of proteins on liquid-solid interface and new column technology of chromatographic cake, one minute-liquid chromatography (OMLC) of intact proteins which has been a dream of separation scientists for a long time is firstly established in the thesis. Compared with conventional fast separation only involving the process of protein separation itself, the OMLC includes the total time of separation and column re-generation. The OMLC was tested with reversed-phase liquid chromatography (RPLC), ion-exchange chromatography (IEC), hydrophobic-interaction chromatography (HIC) using standard proteins under the condition that in comparison with their corresponding to conventional LC, the. resolution of intact proteins in this thesis does not significantly lose. Besides high speed and high resolving power, the technology has the advantages of high loading capacity.
The thesis includes the following six sections:
1 Introduction
The significance of fast separation by LC was briefly introduced. One-dimensional methods of rapid separation of intact proteins including non-porous stationary phase, monolithic stationary phase, perfusion chromatography, high temperature fast chromatography and chromatographic cake were simply introduced and reviewed. Both conventional two-dimensional liquid chromatography (2D-LC) and two-dimensional liquid chromatography using only a single column (2D-LC-1C) which exhibits excellent resolution in two modes were also introduced and reviewed.
2 Separation of intact proteins with porous reversed-phase packings with high speed, high resolution and high sample loading
Ultrashort chromatographic cake (1 mm×10 mm I.D.,78μL) packed with 3μm porous C18 reversed-phase packings from lateral hole of the cake was employed for the separation of seven standard proteins. The resolution for intact proteins and small solutes between such a small packed bed volume of chromatographic cake and a large pack bed volume of column (50 mm×4.6 mm I.D.,830μL) were firstly compared. Continuous migration of the small molecules in the packed bed of column and cake resulted in that the resolution of small solutes was extremely poorer with the cake than the column, but due to the existence of "adhesive energy" of intact proteins on the liquid-solid interface the discontinuous migration of intact proteins resulted in almost the comparable resolution between the cake and the column. However, the cake having better hydrodynamic property than column showed moderate back pressure at high flow rate up to 10 mL/min. Under an optimized condition, the mixture of seven proteins was completely separated in 30 s. Five consecutive separations within 5 minutes were carried out with the cake also, indicating one minute-liquid chromatography (OMLC) of intact proteins can be accomplished when the molecular interactions between protein and stationary phase is non-selective force. The OMLC has been a dream of separation scientists for a long time now becomes realism. The mixture of seven proteins was well separated within 1 minute under the sample loading of 1 mg and thus the separation of intact proteins in this circumstance also achieves the "three high" purpose of high speed, high resolution and high sample loading. The method is expected to be used in clinically routine analysis.
3. Separation of intact proteins using porous weak cation-exchange packings with "three high" purpose
The comparison of separations of intact proteins with two commercial weak cation-exchange packings, TSKgel CM-5PW and PolyCAT A, was carried out. It was found that PolyCAT A (particle size,3μm; pore size,150 nm) was more suitable for achieving the OMLC. The resolution of the PolyCAT A cake (1 mm×10 mm I.D.) was comparable with the PolyCAT A column (50 mm×4.6 mm I.D.). However, the cake showed better hydrodynamic property than the column. The complete separation of 4 standard proteins was achieved in 18 s under an optimized condition with the cake. The speed and resolution is better than non-porous stationary phase and monolithic stationary phase reported in literatures. Ten consecutive separations within 10 minutes were carried out using the cake, really accomplishing the OMLC, when the molecular interaction between protein and stationary phase is electrostatic force. Also, fast separation was achieved in 1 minute under the loading of 1 mg mixture of 4 proteins.
4. Fast separation of protein with non-porous HIC packings
Both the chromatographic cake (10 mm×10 mm I.D.) and column (35 mm×4.6 mm I.D.) packed with 2.5μm non-porous HIC packings were employed for the separation of standard proteins. Under optimized conditions, the mixture of seven proteins could be separated within 3.5 min with the column, and a comparable resolution was obtained in 2.1 min with the cake. The cake, which can be used at higher flow rate, is more suitable for fast separation due to its better hydrodynamic property. Although the result is more than one minute, it is closed (2-3.5 min) to the OMLC.
5. Separation of intact proteins with "three high" purpose using non-porous RPLC packings
A chromatographic cake (7.5 mm×10 mm I.D.) having larger diameter than its thickness is firstly employed to completely separate 1.0μg and 40μg of the mixture of seven standard proteins within 1 minute under conventional chromatographic conditions. Also,0.5 mg of the mixture was almost completely separated within 1 minute. The mixture of proteins was separated with high speed, high loading and high resolution. A new approach for rapid separation of proteins was put forward using conventional HPLC under high loading.
6. Porous 2D (SAX-HIC) packings employed for the OMLC in HIC mode
Taking the advantage of two dimensional packings (2D-packings) having a better selectivity than its corresponding one dimensional packings, a kind of porous two-dimensional packings of strong anion-exchange (SAX) and HIC was synthesized.
The packed 2D (SAX-HIC) cake (1 mm×10 mm I.D.) was used to separate standard proteins in HIC mode. The mixture of six standard proteins could be almost completely separated and the cake was re-generated within one minute under the flow rate of 5 mL/min. Such a fast and good resolution could not be obtained with normally commercial HIC columns. This fact indicates that OMLC is validly carried out for the retention mechanism of protein dominated by hydrophobic-interaction force.
引文
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