液相色谱法对不同状态蛋白的快速分离和鉴定
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摘要
基因重组蛋白的复性问题是生物工程下游技术中的一个瓶颈。对于蛋白复性过程中所形成的中间体(M state)的研究是解决促进蛋白进行正确折叠的关键。蛋白折叠色谱是近年来出现的一个新的复性方法,它不仅可以实现蛋白折叠过程中不同分子构象状态的分离,而且该方法也可用于研究蛋白的分子构象变化,并用计量置换理论(stoichiometric displacement theory,SDT)中的两个线性参数Z和logI对蛋白变性及复性过程中产生的不同分子构象状态其进行表征,所以用蛋白质折叠液相色谱法和SDT来对蛋白折叠及折叠中间体进行研究是当前生物技术中的一个新方法。二维及多维液相色谱法已成为分析化学中复杂体系组分分离和分析的强有力工具,尤其在蛋白质组学研究中对整体(intact)天然蛋白(N态)的预分离方面。由于传统的二维及多维液相色谱本身存在的一些缺陷而限制了其在蛋白质组学研究方面的广泛应用,因此对二维液相色谱方法的改进及发展目前仍为国内外蛋白组学及色谱研究的热点之一。
论文包括以下5个部分:
1.文献综述:蛋白折叠过程中产生的中间体在很大程度上阻碍了变性蛋白(U态)向具有生物活性的天然态的转变,而且由于中间体的寿命非常短使得难以对其进行分离和捕获,因此对蛋白折叠及折叠中间体的研究具有重大的理论意义和重要的实际价值,目前的研究还一直处于经验摸索的阶段。本文从蛋白折叠及折叠中间体形成的原因、研究策略、方法及进展进行了全面地综述。在二维液相色谱(two dimensional liquid chromatography,2DLC)方面,全面介绍了目前二维及多维液相色谱(MDLC)发展的方向以及存在的问题,共包括文献160篇。
2.用疏水相互作用色谱法对脲变α-糜蛋白酶(α-Chy)折叠中间体进行了研究,通过HIC在动态条件下在线制备和纯化出一个稳定的α-Chy中间体。在不同的脲变条件下测定了天然和中间体α-Chy的Z、lgI和j值,结果发现,当脲浓度不同时,天然和中间体α-Chy与固定相表面的这三个不同参数有很大区别,其中天然α-Chy的Z和lgI之间有很好的线性关系,而中间体却没有,并且中间体的Z和lgI值要比天然态的小得多,根据这三个参数的不同可以用来区分蛋白的不同分子构象状态,建立了一个在线折叠液相色谱表征α-Chy中间体的新方法。
3.以脲变α-糜蛋白酶(α-Chy)为模型蛋白,用蛋白折叠液相色谱法研究了该蛋白在6种不同固体表面上的折叠及其在折叠过程中形成的中间体,选用疏水相互作用色谱(HPHIC)固定相为吸附剂,在动态条件下着重研究了疏水色谱固定相TSK和PEG-600表面对脲变α-Chy复性效率的贡献。用基质辅助激光解吸附离子化飞行时间质谱对3.0 mol/L脲变α-Chy,在经HPHIC柱复性并同时分离的收集组分进行确认后,仅有一种稳定的脲变α-Chy折叠中间体。并通过活性测定发现该折叠中间体难以复性并具有低的生物活性,但PEG-600固定相表面较TSK固定相对α-Chy复性效果好。证实了疏水性强度及固体表面配基的结构对蛋白折叠起着关键性的作用。
4.通过实验合成了三种具有弱阳(WCX)和疏水(HIC)性质的双功能色谱固定相(bi-functional stationary phase),通过对这几种固定相的表征发现该双功能固定相无论在IEC模式下还是在HIC模式下对蛋白都有很高的分辨率,因此这种色谱柱称之为WCX～HIC柱,并简要描述了WCX～HIC柱对蛋白的分离原理。蛋白在IEC和HIC的保留强弱可以根据配基的种类,加入的量以及合成条件的不同进行控制。实验测得该类型的双功能色谱固定相在两种模式下都具有很大的动力学吸附量,而且通过对标准蛋白的质量回收率测定发现,这几种标准蛋白分别在这两种模式下的质量回收率都大于96%,表明该类型的固定相对蛋白的不可逆吸附很小。在此基础上首次提出了用一根色谱柱来实现离子交换和疏水色谱快速分离整体蛋白的新方法。在选择合适的固定相、流动相、缓冲液交换方法及二次进样的条件下,在1小时内用同一根色谱柱完成了通常用两根色谱柱和在两种流动相条件才能实现的“二维色谱”分离,并且能维持蛋白原有的三维和四维分子结构。通过和最好的商品TSK离子交换和疏水色谱柱比较发现,本实验合成的双功能固定相在单独的IEC和HIC模式下可以和TSK柱相媲美。而且从峰容量来看,由于双功能色谱柱可以用于二维色谱分离,因此在IEC～HIC或HIC～IEC的二维色谱模式下其峰容量(n1×n2)大大超过一根色谱柱,比如TSK柱。
5.首次将合成的双功能色谱柱用于特殊模式的2DLC蛋白分离,即蛋白单柱二维液相色谱法(two-dimensional LC by a single column,2DLC-1C)。该2DLC-1C既可以在离线,也可以在在线模式下对蛋白进行分离,后者称之为在线2DLC-1C。通过解决在线缓冲液的快速交换,实现了大体积的样品或收集液(0.001～100mL)进行连续或非连续的在线进样。首次将2DLC-1C用于分析和制备规模上的在线蛋白二维分离。通过离线和在线模式下实验了对标准蛋白的二维分离以及对实际样品人血清的快速预分离,取得了非常满意的效果。这种2DLC-1C的优点有:(1)整个蛋白分离都是在密封的体系中进行的,从而防止了样品的污染;(2)所有的样品组分都是定量地转移到下一步的操作;(3)所有的蛋白都保持其天然状态;(4)原样的复杂性大大简化,比如1/10～1/100,甚至更小;(5)每一种蛋白的相对丰度,尤其蛋白质组学中可以提高低丰度蛋白的浓度10～100倍,甚至更高;(6)容易实现操作自动化;(7)可以在线进行天然或整体蛋白的快速分离;(8)该方法不但可以提高信息的可靠性,而且可以获得蛋白组学研究的纯的蛋白;(9)对天然产物或动物体液的分离,可以获得高的质量回收率和活性回收率;(10)费用非常低。
Protein renaturation is a recognized bottle-neck technique in the down-stream in biotechnology.The key point here is how to make the stable intermediates(M state) of protein fold into its correctly state.Protein folding liquid chromatography(PFLC) is a recently developed new method for protein renaturation.It is not only employed for carrying out the separation of protein in different states,but is also for the investigating the changes in molecular conformation of proteins.With some linear parameters of stoichiometric displacement theory(SDT),the different molecular conformations of proteins can be characterized.Thus,using the PFLC and SDT to study on the proteins folding and the folded intermediates is a new approach in biotechnology and molecular biology.Two-dimensional liquid chromatography (2DLC)and multi-dimensional liquid chromatography(mDLC)have become very strong tools for the separation and analysis of some components in a complex system in analytical chemistry,especially for the pre-fractionation of intact proteins in proteomies.Due to some shortages of the present separation of proteins by 2DLC or mDLC,such as high cost,long separation process,unsatisfactory reproducibility,their applications are greatly limited.Therefore,the method for improving 2DLC,or mDLC has become one of researching hot-point in both proteomics and LC in the world.
The thesis contains five parts as follows:
1.Literature review:The intermediate(M state)formed during protein folding retards its corresponding denatured protein(U state)to convert to its native state (N state).Because the lifetime of the M state is so short that it is very difficult to be captured and separated,the study on the folded M state has a greatly theoretical signification and widely applied value.This research in this field still places at an experimental stage.The formation,researching strategy,method and recent development of the M state during protein folding were comprehensively reviewed in this chapter.Some unsolved difficult problems,and in the future of 2DLC mDLC for the separation of proteins in N state were also introduced and commented.The thesis contains 160 references.
2.The M state of urea-denaturedα-chymotrypsin(α-Chy)was selected as a model M state and its character was investigated by PFLC.By using hydrophobic interaction chromatography(HIC),a stable M state ofα-Chy was prepared and separated on-line under a dynamically chromatographic condition.The three linear parameters of Z(A constant corresponding to moles of water is squeezed out at the contact region between protein and stationary phase as one mole protein is adsorbed by stationary phase),lgI(a batch of constant corresponding to the affinity of one mole protein to stationary phase),and j(a batch of constant corresponding to the affinity of one mole water to stationary phase)in SDT ofα-Chy in M and N states were determined under different concentrations of urea. The obtained result demonstrates that the three parameters of theα-Chy in M and N states are quite different with each other,a very good linearity relation(Slope is j)was found to exist between the Z and lg I of theα-Chy in N state,but not for its M state.It was also found that the value of Z and lgI of theα-Chy in M state is less than its corresponding N state.Then the different molecular conformations of theα-Chy can be distinguished by the different of the three parameters,and established a new method for the characterization ofα-Chy intermediate by online PFLC
3.The retention of the obtained M state ofα-Chy during protein folding by six different kinds of HIC stationary phases was compared with each other.The surfaces of the HIC stationary phase of PEG-600 and TSK-phenyl were selected as the two typical solid surfaces to study the folding efficiency of the urea-denaturedα-Chy andα-Chy in M state.The obtained result indicates that the character of a solid surface has a significant contribution to protein folding. Compared with the TSK column,the surface of PEG-600 stationary phase is efficient for the renaturation of the urea-denaturedα-Chy and for its quality control duringα-Chy folding.Matrix-assisted laser desorption ionization time of a flight mass spectrometer(MALTI-TOF MS)was employed to measure the molecular mass of each collections after HPHIC separation by the two kinds of stationary phases and to confirm that there is only one stable folded intermediate from the mixture of the urea-denaturedα-Chy.With the comparison of specific bioactivity of the refoldedα-Chy,the surface of HIC PEG-600 stationary phase was proved to be better than that of TSK again.It further demonstrates that a suitable hydrophobic surface with a suitable hydrophobic strength and a ligand structure plays a key role in protein folding.
4.Three kinds of specific stationary phase having the character of both weak cation-exchange chromatography(WCX)and HIC,respectively,were synthesized. It was proved by experiment that these stationary phases have very good resolution for proteins separation,ether in ion-exchange chromatography(IEC) mode,or HIC mode,and this column is expressed as WCX～HIC column.The principle of protein separation with the WCX～HIC column was briefly described. The character of protein retention on the stationary of the WCX～HIC column can be controlled by the kinds of ligands,the amount of added reagent amount and the differently synthesized condition.It was found that this WCX～HIC column has a great dynamic column capacity under the two chromatographic modes, respectively.Furthermore,the mass recovery of several proteins under the two modes was found to be higher than 96%,this result indicates that the in-reversible adsorption on this stationary phase may be ignored.Based on all of the foregoing results,a new method that with only a single chromatographic column to accomplish the separation of intact proteins by the combination of WCX and HIC is firstly presented.With selecting a suitable stationary phase,mobile phase, buffer exchange condition,and sample injection by two times,the protein separation with usually 2DLC mode can now be accomplished using the single column only in one hour.By compared with the very good commercial TSK WCX and HIC columns,respectively,the resolution of the WCX～HIC column can be comparable with the two TSK columns.Due to the WCX～HIC column can be used in 2DLC with the column capacity(n1×n2),the peak capacity of the WCW～HIC column is much greater than one normal column.
5.When the synthesized single WCX～HIC column is employed for a 2DLC separation of proteins,it is called as the two-dimensional liquid chromatography by a single column and expressed by 2DLC-1C.The 2DLC-1C can be employed by either off-line,or on-line for protein separation.The latter is called as on-line 2DLC-1C.With solving the on-line buffer exchange,sample and/or collection of large volume(0.001-100mL)on-line injection by either,continuously,or discontinuously,the 2DLC-1C is firstly used for on-line 2DLC for protein separation in both analytical and preparative scales.Moreover,the 2DLC-1C was applied for standard proteins and blood serum rapid separation;the obtained results were proved to be excellent.The advantages of the 2DLC-1C are that all of operations are:(1),Whole protein separations are carried out in a closed system, prevent from any contaminations;(2),All of components in original sample are quantitatively transferred to the subsequent operation;(3),Each protein remains native state;(4),The complexity of the original sample is simplified very much, such as from 1/10～1/100,even much less;(5),The relative enrichment of each proteins,especially for very low enrichment proteins in proteomics can be increased up to 10～100 folds,even much more;(6),It is easily for operation atomization;(7),fast Native and/or intact protein can be fast separated by means of on-line matter;(8),It can not only obtain reliable information,but also obtain pure proteins in proteomics;(9),A high mass and bioactivity recovery for isolation from natural product,or animal fluids can be obtained;(10),Its expense is very low.
论文包括以下5个部分:
1.文献综述:蛋白折叠过程中产生的中间体在很大程度上阻碍了变性蛋白(U态)向具有生物活性的天然态的转变,而且由于中间体的寿命非常短使得难以对其进行分离和捕获,因此对蛋白折叠及折叠中间体的研究具有重大的理论意义和重要的实际价值,目前的研究还一直处于经验摸索的阶段。本文从蛋白折叠及折叠中间体形成的原因、研究策略、方法及进展进行了全面地综述。在二维液相色谱(two dimensional liquid chromatography,2DLC)方面,全面介绍了目前二维及多维液相色谱(MDLC)发展的方向以及存在的问题,共包括文献160篇。
2.用疏水相互作用色谱法对脲变α-糜蛋白酶(α-Chy)折叠中间体进行了研究,通过HIC在动态条件下在线制备和纯化出一个稳定的α-Chy中间体。在不同的脲变条件下测定了天然和中间体α-Chy的Z、lgI和j值,结果发现,当脲浓度不同时,天然和中间体α-Chy与固定相表面的这三个不同参数有很大区别,其中天然α-Chy的Z和lgI之间有很好的线性关系,而中间体却没有,并且中间体的Z和lgI值要比天然态的小得多,根据这三个参数的不同可以用来区分蛋白的不同分子构象状态,建立了一个在线折叠液相色谱表征α-Chy中间体的新方法。
3.以脲变α-糜蛋白酶(α-Chy)为模型蛋白,用蛋白折叠液相色谱法研究了该蛋白在6种不同固体表面上的折叠及其在折叠过程中形成的中间体,选用疏水相互作用色谱(HPHIC)固定相为吸附剂,在动态条件下着重研究了疏水色谱固定相TSK和PEG-600表面对脲变α-Chy复性效率的贡献。用基质辅助激光解吸附离子化飞行时间质谱对3.0 mol/L脲变α-Chy,在经HPHIC柱复性并同时分离的收集组分进行确认后,仅有一种稳定的脲变α-Chy折叠中间体。并通过活性测定发现该折叠中间体难以复性并具有低的生物活性,但PEG-600固定相表面较TSK固定相对α-Chy复性效果好。证实了疏水性强度及固体表面配基的结构对蛋白折叠起着关键性的作用。
4.通过实验合成了三种具有弱阳(WCX)和疏水(HIC)性质的双功能色谱固定相(bi-functional stationary phase),通过对这几种固定相的表征发现该双功能固定相无论在IEC模式下还是在HIC模式下对蛋白都有很高的分辨率,因此这种色谱柱称之为WCX～HIC柱,并简要描述了WCX～HIC柱对蛋白的分离原理。蛋白在IEC和HIC的保留强弱可以根据配基的种类,加入的量以及合成条件的不同进行控制。实验测得该类型的双功能色谱固定相在两种模式下都具有很大的动力学吸附量,而且通过对标准蛋白的质量回收率测定发现,这几种标准蛋白分别在这两种模式下的质量回收率都大于96%,表明该类型的固定相对蛋白的不可逆吸附很小。在此基础上首次提出了用一根色谱柱来实现离子交换和疏水色谱快速分离整体蛋白的新方法。在选择合适的固定相、流动相、缓冲液交换方法及二次进样的条件下,在1小时内用同一根色谱柱完成了通常用两根色谱柱和在两种流动相条件才能实现的“二维色谱”分离,并且能维持蛋白原有的三维和四维分子结构。通过和最好的商品TSK离子交换和疏水色谱柱比较发现,本实验合成的双功能固定相在单独的IEC和HIC模式下可以和TSK柱相媲美。而且从峰容量来看,由于双功能色谱柱可以用于二维色谱分离,因此在IEC～HIC或HIC～IEC的二维色谱模式下其峰容量(n1×n2)大大超过一根色谱柱,比如TSK柱。
5.首次将合成的双功能色谱柱用于特殊模式的2DLC蛋白分离,即蛋白单柱二维液相色谱法(two-dimensional LC by a single column,2DLC-1C)。该2DLC-1C既可以在离线,也可以在在线模式下对蛋白进行分离,后者称之为在线2DLC-1C。通过解决在线缓冲液的快速交换,实现了大体积的样品或收集液(0.001～100mL)进行连续或非连续的在线进样。首次将2DLC-1C用于分析和制备规模上的在线蛋白二维分离。通过离线和在线模式下实验了对标准蛋白的二维分离以及对实际样品人血清的快速预分离,取得了非常满意的效果。这种2DLC-1C的优点有:(1)整个蛋白分离都是在密封的体系中进行的,从而防止了样品的污染;(2)所有的样品组分都是定量地转移到下一步的操作;(3)所有的蛋白都保持其天然状态;(4)原样的复杂性大大简化,比如1/10～1/100,甚至更小;(5)每一种蛋白的相对丰度,尤其蛋白质组学中可以提高低丰度蛋白的浓度10～100倍,甚至更高;(6)容易实现操作自动化;(7)可以在线进行天然或整体蛋白的快速分离;(8)该方法不但可以提高信息的可靠性,而且可以获得蛋白组学研究的纯的蛋白;(9)对天然产物或动物体液的分离,可以获得高的质量回收率和活性回收率;(10)费用非常低。
Protein renaturation is a recognized bottle-neck technique in the down-stream in biotechnology.The key point here is how to make the stable intermediates(M state) of protein fold into its correctly state.Protein folding liquid chromatography(PFLC) is a recently developed new method for protein renaturation.It is not only employed for carrying out the separation of protein in different states,but is also for the investigating the changes in molecular conformation of proteins.With some linear parameters of stoichiometric displacement theory(SDT),the different molecular conformations of proteins can be characterized.Thus,using the PFLC and SDT to study on the proteins folding and the folded intermediates is a new approach in biotechnology and molecular biology.Two-dimensional liquid chromatography (2DLC)and multi-dimensional liquid chromatography(mDLC)have become very strong tools for the separation and analysis of some components in a complex system in analytical chemistry,especially for the pre-fractionation of intact proteins in proteomies.Due to some shortages of the present separation of proteins by 2DLC or mDLC,such as high cost,long separation process,unsatisfactory reproducibility,their applications are greatly limited.Therefore,the method for improving 2DLC,or mDLC has become one of researching hot-point in both proteomics and LC in the world.
The thesis contains five parts as follows:
1.Literature review:The intermediate(M state)formed during protein folding retards its corresponding denatured protein(U state)to convert to its native state (N state).Because the lifetime of the M state is so short that it is very difficult to be captured and separated,the study on the folded M state has a greatly theoretical signification and widely applied value.This research in this field still places at an experimental stage.The formation,researching strategy,method and recent development of the M state during protein folding were comprehensively reviewed in this chapter.Some unsolved difficult problems,and in the future of 2DLC mDLC for the separation of proteins in N state were also introduced and commented.The thesis contains 160 references.
2.The M state of urea-denaturedα-chymotrypsin(α-Chy)was selected as a model M state and its character was investigated by PFLC.By using hydrophobic interaction chromatography(HIC),a stable M state ofα-Chy was prepared and separated on-line under a dynamically chromatographic condition.The three linear parameters of Z(A constant corresponding to moles of water is squeezed out at the contact region between protein and stationary phase as one mole protein is adsorbed by stationary phase),lgI(a batch of constant corresponding to the affinity of one mole protein to stationary phase),and j(a batch of constant corresponding to the affinity of one mole water to stationary phase)in SDT ofα-Chy in M and N states were determined under different concentrations of urea. The obtained result demonstrates that the three parameters of theα-Chy in M and N states are quite different with each other,a very good linearity relation(Slope is j)was found to exist between the Z and lg I of theα-Chy in N state,but not for its M state.It was also found that the value of Z and lgI of theα-Chy in M state is less than its corresponding N state.Then the different molecular conformations of theα-Chy can be distinguished by the different of the three parameters,and established a new method for the characterization ofα-Chy intermediate by online PFLC
3.The retention of the obtained M state ofα-Chy during protein folding by six different kinds of HIC stationary phases was compared with each other.The surfaces of the HIC stationary phase of PEG-600 and TSK-phenyl were selected as the two typical solid surfaces to study the folding efficiency of the urea-denaturedα-Chy andα-Chy in M state.The obtained result indicates that the character of a solid surface has a significant contribution to protein folding. Compared with the TSK column,the surface of PEG-600 stationary phase is efficient for the renaturation of the urea-denaturedα-Chy and for its quality control duringα-Chy folding.Matrix-assisted laser desorption ionization time of a flight mass spectrometer(MALTI-TOF MS)was employed to measure the molecular mass of each collections after HPHIC separation by the two kinds of stationary phases and to confirm that there is only one stable folded intermediate from the mixture of the urea-denaturedα-Chy.With the comparison of specific bioactivity of the refoldedα-Chy,the surface of HIC PEG-600 stationary phase was proved to be better than that of TSK again.It further demonstrates that a suitable hydrophobic surface with a suitable hydrophobic strength and a ligand structure plays a key role in protein folding.
4.Three kinds of specific stationary phase having the character of both weak cation-exchange chromatography(WCX)and HIC,respectively,were synthesized. It was proved by experiment that these stationary phases have very good resolution for proteins separation,ether in ion-exchange chromatography(IEC) mode,or HIC mode,and this column is expressed as WCX～HIC column.The principle of protein separation with the WCX～HIC column was briefly described. The character of protein retention on the stationary of the WCX～HIC column can be controlled by the kinds of ligands,the amount of added reagent amount and the differently synthesized condition.It was found that this WCX～HIC column has a great dynamic column capacity under the two chromatographic modes, respectively.Furthermore,the mass recovery of several proteins under the two modes was found to be higher than 96%,this result indicates that the in-reversible adsorption on this stationary phase may be ignored.Based on all of the foregoing results,a new method that with only a single chromatographic column to accomplish the separation of intact proteins by the combination of WCX and HIC is firstly presented.With selecting a suitable stationary phase,mobile phase, buffer exchange condition,and sample injection by two times,the protein separation with usually 2DLC mode can now be accomplished using the single column only in one hour.By compared with the very good commercial TSK WCX and HIC columns,respectively,the resolution of the WCX～HIC column can be comparable with the two TSK columns.Due to the WCX～HIC column can be used in 2DLC with the column capacity(n1×n2),the peak capacity of the WCW～HIC column is much greater than one normal column.
5.When the synthesized single WCX～HIC column is employed for a 2DLC separation of proteins,it is called as the two-dimensional liquid chromatography by a single column and expressed by 2DLC-1C.The 2DLC-1C can be employed by either off-line,or on-line for protein separation.The latter is called as on-line 2DLC-1C.With solving the on-line buffer exchange,sample and/or collection of large volume(0.001-100mL)on-line injection by either,continuously,or discontinuously,the 2DLC-1C is firstly used for on-line 2DLC for protein separation in both analytical and preparative scales.Moreover,the 2DLC-1C was applied for standard proteins and blood serum rapid separation;the obtained results were proved to be excellent.The advantages of the 2DLC-1C are that all of operations are:(1),Whole protein separations are carried out in a closed system, prevent from any contaminations;(2),All of components in original sample are quantitatively transferred to the subsequent operation;(3),Each protein remains native state;(4),The complexity of the original sample is simplified very much, such as from 1/10～1/100,even much less;(5),The relative enrichment of each proteins,especially for very low enrichment proteins in proteomics can be increased up to 10～100 folds,even much more;(6),It is easily for operation atomization;(7),fast Native and/or intact protein can be fast separated by means of on-line matter;(8),It can not only obtain reliable information,but also obtain pure proteins in proteomics;(9),A high mass and bioactivity recovery for isolation from natural product,or animal fluids can be obtained;(10),Its expense is very low.
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