蛋白质的毛细管阵列液相色谱分离及相互作用新方法研究
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摘要
随着人类基因组计划的完成,生命科学研究进入了后基因组时代,即蛋白质组学的研究。蛋白质组学中有两条技术路线：一条是基于肽段的"bottom-up",另一条是基于蛋白质水平的"top-down"。基于蛋白质水平的分离鉴定能够获得蛋白质完整的信息,使蛋白质的定量、蛋白与蛋白相互作用以及翻译后修饰研究更加高效可靠。因此,对蛋白质水平的分离鉴定成为近几年的研究主流方向。然而,蛋白质比肽段复杂得多。如何采用高效、高通量、高分辨的分离手段,实现对一个复杂蛋白质组在蛋白质水平上的有效分离给色谱工作者提出了一个新的挑战。构建高通量的阵列式多维液相色谱平台成为极具潜力的大规模蛋白质组学样品分析的关键。
在蛋白质组学研究中,阐明蛋白质间的相互作用是最有挑战性而又重要的任务之一。研究蛋白质相互作用的方式和程度,将有助于蛋白质功能的分析、疾病致病机理的阐明和治疗以及新型药物的开发等众多难题的解决,因此,确定蛋白质间相互作用关系、绘制相互作用图谱已成为蛋白质组学研究的热点。现有的技术研究周期长、成本高、代价昂贵,限制了对蛋白质相互作用高通量、高效的研究。
本论文针对蛋白质组学研究领域的热点难点问题,研制了新型的蛋白质捕集柱,搭建了蛋白质水平分离的两维阵列式毛细管液相色谱平台,建立了基于蛋白质固定与蛋白质荧光标记的研究蛋白质相互作用的新方法,对正常及高脂大鼠的鼠肝提取蛋白进行了基于质谱的无标记定量分析。全文共分五章,主要内容如下：
第一章,文献综述。本章总结了整体柱的发展概况及整体柱在微分离分析领域的应用,概述了多维液相色谱技术在蛋白质组学领域的进展,介绍了蛋白质相互作用的研究方法。阐述了本论文的选题意义。
第二章,商品捕集柱以及我们已研制的捕集柱主要用于肽水平样品的捕集,无法满足蛋白质水平的样品馏分高效捕集与洗脱。论文采用溶胶凝胶法制备了新型的蛋白质捕集柱并成功地对实际鼠肝蛋白质进行了高效捕集与洗脱。首先通过优化溶胶凝胶的配方及改善工艺条件,制得了由薄层溶胶凝胶涂覆的蛋白质捕集柱。柱与柱之间,批次与批次之间重现性好,同时捕集柱保持了较高的机械强度。然后将该捕集柱应用于在线捕集-分离检测的毛细管液相色谱系统,以四种标准蛋白质的混合物为研究对象,考察了捕集柱对标准蛋白质的回收率、最大上样量、柱子的重现性等参数。一根5mm长的捕集柱对标准蛋白质的平均回收率为99.3%,对四种蛋白质混合物的最大上样量为30μg。最后该在线捕集-分离检测的毛细管液相系统成功用于实际鼠肝提取蛋白的在线除盐、捕集,获得了满意的结果。
第三章,搭建了蛋白质水平分离的阵列式毛细管二维液相色谱分离技术平台,将上述研制的蛋白质捕集柱应用于蛋白的在线除盐、富集,对鼠肝组织的蛋白质组进行了高效分离。在此基础上,采用本实验室发展的靶上酶解技术,对分离后的蛋白质进行酶解,将蛋白质高通量的分离与MALDI-TOF-TOF-MS检测有效的结合起来,从而实现了蛋白质从分离、酶解到鉴定这样一个完整的"top-down"路线分析。该平台以强阴离子交换色谱(SAX)为第一维色谱分离模式,以十八根并行式的反相液相色谱(RPLC)为第二维分离模式,可快速完成进样、除盐富集、分离、酶解及鉴定,实现真正的蛋白质组学研究所需的高通量分析。SAX柱采用阶梯式盐梯度洗脱,蛋白质样品经过0.5mm内径SAX柱分离之后,洗脱下来的馏分通过两个十通阀的切换被依次保留在18根自制蛋白捕集柱的柱头,对样品进行在线除盐、捕集。馏分经捕集柱捕集除盐后,通过一个十八通道分流器,被并行反冲到阵列式的毛细管反相柱上进行第二维分离。样品分析通量提高了18倍。利用自动点样仪将经过RPLC的洗脱液直接收集到MALDI靶板上,点样频率为1分钟/,经过靶上单点酶解之后用MALDI-TOF-TOF-MS鉴定。整个系统对鼠肝蛋白进行了分离鉴定,共鉴定出1030种蛋白质。
第四章,探索了研究蛋白质相互作用的新方法。将目标蛋白质固定在氨基材料上,将与之发生相互作用的蛋白质进行固相荧光衍生,然后将两者孵育,洗脱后,用荧光显微镜进行检测。应用谷胱甘肽转移酶(GST)及其抗体对该方法的可行性进行了验证,该方法有望应用于研究蛋白质相互作用,实现对大量蛋白质高通量的筛选。首先合成了氨基纳米磁性微球,通过化学反应在氨基磁球上连接上戊二醛,然后将蛋白质连接到戊二醛上,实现了在氨基纳米磁球上固定蛋白质。通过比较蛋白质溶液与材料反应前后对280nm紫外光的吸收强度,可以估算出磁球表面固定蛋白的量。然后采用本实验室发展的固相荧光衍生法,在毛细管中制备了用于蛋白荧光标记的固相载体填充床,以荧光素异硫氰酸酯(FITC)作为荧光衍生试剂,实现了蛋白的固相荧光衍生。并白行填充了毛细管体积排阻色谱柱,对过量荧光试剂进行了有效去除。将固定在氨基材料上的GST与标记上荧光的GST抗体在Tris-HCl缓冲液(pH7.5,100mM NaCl,4℃)中过夜孵育、洗脱后,在荧光显微镜下观察,材料上可见荧光。应用该方法对固定在氨基材料上的人血清蛋白(HSA)与标记上FITC的血浆馏分之间的相互作用进行了初步探索。
第五章,采用APEX方法,建立了无标记的纳流毛细管液相色谱-电喷雾离子阱串联质谱联用分析鉴定正常及高脂鼠肝组织中的差异蛋白。将20周龄正常及高脂鼠肝组织提取蛋白酶解后的肽段使用强阳离子交换/相纳升级液相色谱(SCX/nano HPLC)进行电喷雾串联质谱分析(ESI-MS-MS)。分析步骤如下：肽段干粉使用60μL上样液(5mM甲酸铵,5%ACN水溶液,pH3.0,第一个浓度的盐梯度)溶解后每次上样19μL。第一维上样流速为20μL/min,洗脱5分钟,未被SCX保留的肽段直接被捕集柱捕集,同时经过捕集柱的盐溶液直接排入废液。经过阀切换,使捕集柱和分析柱相连,同时启动Nano泵进行反相分离,启动质谱采集在线检测肽段。进样器吸取20μL第二种浓度的盐溶液进行第二个盐梯度,重复进行以上步骤。实验共进行10个盐梯度(包括第一次上样的梯度)。盐梯度之后仍会有部分疏水性较强的肽段保留在SCX柱上,为了回收这部分肽段,最后我们添加了一个ACN浓度较高的盐梯度。对串级质谱鉴定的数据库分析后,高脂鼠肝组织中的蛋白与正常鼠肝组织中的相比,含量两倍上调的有21种,两倍下调的有10种。
综上所述,本论文针对蛋白质组学研究领域的热点难点问题,研制了新型的蛋白质捕集柱并成功应用于实际鼠肝蛋白的捕集,搭建了蛋白质水平分离的两维阵列式毛细管液相色谱平台,建立了基于蛋白质固定与蛋白质荧光标记的研究蛋白质相互作用的新方法。为利用高通量的阵列色谱平台分离复杂的蛋白质组以及研究蛋白质相互作用、蛋白质定量等打下了良好的基础。
The completion of the human genome project has facilitated the entry of biomedical research into the post-genome era-proteomics research. There are two technical routes in proteomics:peptide-centric bottom-up and intact-protein level top-down approaches."Top-down" proteomics can provide more information for intact proteins which can be used to study protein quantitation, protein-protein interactions (PPIs) and posttranslational modifications (PTMs). The strategies based on separation and identification of intact proteins has become the mainstream in proteomic research. However, proteins are more complex than peptides. The efficient and high throughput separation of complex protein mixtures at intact-protein level is a great challenge to the chromatographic field. Construction of high-throughput multi-dimensional array liquid chromatography platform has great potential for large-scale proteomics analysis.
The elucidation of PPIs is one of the most challenging and important tasks in proteomics research. Study of PPIs can provide important information for understanding protein functions, elucidating disease pathogenesis and developing new drugs. So PPIs analysis has become the focus of proteomics. The current technologies have hindered high throughput screening of PPIs because they are usually time-consuming and expensive. Therefore, it is urgent to develop new strategies for the large-scale study of PPIs.
We focus on the key and challenging problems in proteome field, a novel kind of intact-protein trapping columns was developed for trapping and desalting of proteins. We established a multidimensional capillary array liquid chromatography platform coupled by MALDI-TOF-TOF identification of intact proteins. And a new method for study of PPIs was proposed. Differential proteomics of normal and fat rat liver proteins was achieved based on label-free proteomics. The research work in this thesis is divided into five chapters.
In Chapter1, advance and application of monolithic columns were included, development and application of multidimensional liquid chromatography in the field of proteome were introduced, technologies for analyzing PPIs were summarized. The research background was demonstrated.
In Chapter2, most commercially available precolumns and our previously developed precolumns are mainly employed for trapping peptides, it is urgent to develop intact-protein trapping columns for high efficient trapping and elution of proteins. A new type of monolithic trapping columns with high mechanical strength was prepared by thin-layer sol-gel coating method and applied to trapping intact proteins for on-line capillary liquid chromatography. Hundreds times of trapping/untrapping for intact proteins were carried out. The trapping columns showed long-term stability up to300bar. Recovery, loading capacity and reproducibility of trapping columns were evaluated using four proteins. The recovery of four protein mixtures for the C8monolithic trapping columns was99.3%on average. The loading capacity of5mm×320μm i.d. C8trapping columns for the protein mixtures was30μg. The C8trapping columns were used to trap normal mouse liver intact proteins in a capillary liquid chromatography system. Results demonstrated high efficiency of the monolithic trapping columns for trapping intact proteins for proteomic analysis in on-line capillary liquid chromatography system.
In Chapter3, we established a capillary two-dimensional array liquid chromatography system for automated, high-throughput analysis of intact proteins, in which one strong-anion-exchange (SAX) chromatographic column was used as the first separation dimension and18parallel capillary reverse-phase liquid chromatographic (RPLC) columns were integrated as the second separation dimension. Proteins eluting from the SAX were trapped and desalted by18parallel intact-protein trapping columns. Then protein fractions were back-flushed simultaneously from18intact-protein trapping columns to capillary RPLC columns. The eighteenplexed capillary array chromatography system is capable of concurrently separating eighteen different samples, resulting in an18-fold increase in analytical throughput. Protein fractions eluting from RPLC columns were spotted onto the MALDI sample plate in1min intervals through an array of capillary tips. On-plate tryptic digestion technique was employed to digest the effluents. Normal mouse liver tissue proteins were analyzed by the fully multiplexed high-throughput two-dimensional liquid chromatography platform coupled by MALDI-TOF-TOF-MS identification. In total,1030proteins were identified, which proved the system's promising potential for intact-protein separation in proteomics.
In Chapter4, we proposed a new method for PPIs research. Bait protein was immobilized on amino particles, prey protein was labeled by a fluorescent reagent, and then they were incubated, eluted, detected by fluorescence microscopy. GST and its antibody were used to verify the feasibility of the method. First, amino-nano-magnetic microspheres were synthesized, glutaraldehyde was connected with amino of magnetic particles by chemical reaction, and then protein was connected to glutaraldehyde. After protein immobilization procedure was conducted, the UV absorption value of the supernatant solution was measured at280nm to calculate the amount of protein immobilized on the magnetic microspheres. A solid-support reaction was described to realize fluorescent derivatization of proteins. A simple, low-cost homemade capillary C18cartridge was fabricated as the solid-support reactor, protein was captured by this reactor and then labeled by fluorescein isothiocyanate (FITC, isomer I) on solid-support. Unwanted fluorescent intruder (excrescent FITC and products of secondary reactions) were removed from target easily. By incubating immobilized protein and FITC-labeled protein in Tris-HCl buffer (pH7.5,100mM NaCl,4℃), we investigated PPIs. This new method was applied to study PPIs between human serum albumin (HSA) and human plasma fractions.
In Chapter5, we reported on differential proteomics of normal and fat rat liver proteins. This was achieved by2D-LC-MS/MS-based label-free protein quantification. The peptides were desalted, dried and then resuspended with60μL volume of loading buffer (5mM Ammonium formate containing5%acetonitrile, pH3.0), separated and analyzed by2D strong cation-exchange (SCX)/reversed-phase (RP) nano-scale liquid chromatography/mass spectrometry (2D-nanoLC/MS). The experiments were performed on a Nano Aquity UPLC system connected to an LTQ Orbitrap XL mass spectrometer equipped with an online nano-electrospray ion source. A20μL plug was injected each time to form the salt step gradients. Ten step gradients were carried out. The plugs were loaded onto the SCX column with a loading buffer at a20μL/min flow rate for5min. A19μL peptide sample was loaded onto the SCX column before the gradient plugs were injected. The eluted peptides were captured by a Captrap Peptide column, while salts were diverted to waste. To recover hydrophobic peptides still retained on the SCX column after a conventional salt step gradient, a RP step gradient from15%to50%acetonitrile was applied to the SCX column. After all MS/MS spectrums were identified, we found quantitative differences of31proteins common to two samples, of which,21up-regulated and10down-regulated.
In summary, this thesis focuses on the key and challenging problems of proteomics. We developed a novel type of intact-protein trapping columns for desalting and trapping of intact-protein. We set up a multidimensional capillary array liquid chromatography platform coupled by MALDI-TOF-TOF identification of intact proteins. And a new method for studying PPIs was proposed.
在蛋白质组学研究中,阐明蛋白质间的相互作用是最有挑战性而又重要的任务之一。研究蛋白质相互作用的方式和程度,将有助于蛋白质功能的分析、疾病致病机理的阐明和治疗以及新型药物的开发等众多难题的解决,因此,确定蛋白质间相互作用关系、绘制相互作用图谱已成为蛋白质组学研究的热点。现有的技术研究周期长、成本高、代价昂贵,限制了对蛋白质相互作用高通量、高效的研究。
本论文针对蛋白质组学研究领域的热点难点问题,研制了新型的蛋白质捕集柱,搭建了蛋白质水平分离的两维阵列式毛细管液相色谱平台,建立了基于蛋白质固定与蛋白质荧光标记的研究蛋白质相互作用的新方法,对正常及高脂大鼠的鼠肝提取蛋白进行了基于质谱的无标记定量分析。全文共分五章,主要内容如下：
第一章,文献综述。本章总结了整体柱的发展概况及整体柱在微分离分析领域的应用,概述了多维液相色谱技术在蛋白质组学领域的进展,介绍了蛋白质相互作用的研究方法。阐述了本论文的选题意义。
第二章,商品捕集柱以及我们已研制的捕集柱主要用于肽水平样品的捕集,无法满足蛋白质水平的样品馏分高效捕集与洗脱。论文采用溶胶凝胶法制备了新型的蛋白质捕集柱并成功地对实际鼠肝蛋白质进行了高效捕集与洗脱。首先通过优化溶胶凝胶的配方及改善工艺条件,制得了由薄层溶胶凝胶涂覆的蛋白质捕集柱。柱与柱之间,批次与批次之间重现性好,同时捕集柱保持了较高的机械强度。然后将该捕集柱应用于在线捕集-分离检测的毛细管液相色谱系统,以四种标准蛋白质的混合物为研究对象,考察了捕集柱对标准蛋白质的回收率、最大上样量、柱子的重现性等参数。一根5mm长的捕集柱对标准蛋白质的平均回收率为99.3%,对四种蛋白质混合物的最大上样量为30μg。最后该在线捕集-分离检测的毛细管液相系统成功用于实际鼠肝提取蛋白的在线除盐、捕集,获得了满意的结果。
第三章,搭建了蛋白质水平分离的阵列式毛细管二维液相色谱分离技术平台,将上述研制的蛋白质捕集柱应用于蛋白的在线除盐、富集,对鼠肝组织的蛋白质组进行了高效分离。在此基础上,采用本实验室发展的靶上酶解技术,对分离后的蛋白质进行酶解,将蛋白质高通量的分离与MALDI-TOF-TOF-MS检测有效的结合起来,从而实现了蛋白质从分离、酶解到鉴定这样一个完整的"top-down"路线分析。该平台以强阴离子交换色谱(SAX)为第一维色谱分离模式,以十八根并行式的反相液相色谱(RPLC)为第二维分离模式,可快速完成进样、除盐富集、分离、酶解及鉴定,实现真正的蛋白质组学研究所需的高通量分析。SAX柱采用阶梯式盐梯度洗脱,蛋白质样品经过0.5mm内径SAX柱分离之后,洗脱下来的馏分通过两个十通阀的切换被依次保留在18根自制蛋白捕集柱的柱头,对样品进行在线除盐、捕集。馏分经捕集柱捕集除盐后,通过一个十八通道分流器,被并行反冲到阵列式的毛细管反相柱上进行第二维分离。样品分析通量提高了18倍。利用自动点样仪将经过RPLC的洗脱液直接收集到MALDI靶板上,点样频率为1分钟/,经过靶上单点酶解之后用MALDI-TOF-TOF-MS鉴定。整个系统对鼠肝蛋白进行了分离鉴定,共鉴定出1030种蛋白质。
第四章,探索了研究蛋白质相互作用的新方法。将目标蛋白质固定在氨基材料上,将与之发生相互作用的蛋白质进行固相荧光衍生,然后将两者孵育,洗脱后,用荧光显微镜进行检测。应用谷胱甘肽转移酶(GST)及其抗体对该方法的可行性进行了验证,该方法有望应用于研究蛋白质相互作用,实现对大量蛋白质高通量的筛选。首先合成了氨基纳米磁性微球,通过化学反应在氨基磁球上连接上戊二醛,然后将蛋白质连接到戊二醛上,实现了在氨基纳米磁球上固定蛋白质。通过比较蛋白质溶液与材料反应前后对280nm紫外光的吸收强度,可以估算出磁球表面固定蛋白的量。然后采用本实验室发展的固相荧光衍生法,在毛细管中制备了用于蛋白荧光标记的固相载体填充床,以荧光素异硫氰酸酯(FITC)作为荧光衍生试剂,实现了蛋白的固相荧光衍生。并白行填充了毛细管体积排阻色谱柱,对过量荧光试剂进行了有效去除。将固定在氨基材料上的GST与标记上荧光的GST抗体在Tris-HCl缓冲液(pH7.5,100mM NaCl,4℃)中过夜孵育、洗脱后,在荧光显微镜下观察,材料上可见荧光。应用该方法对固定在氨基材料上的人血清蛋白(HSA)与标记上FITC的血浆馏分之间的相互作用进行了初步探索。
第五章,采用APEX方法,建立了无标记的纳流毛细管液相色谱-电喷雾离子阱串联质谱联用分析鉴定正常及高脂鼠肝组织中的差异蛋白。将20周龄正常及高脂鼠肝组织提取蛋白酶解后的肽段使用强阳离子交换/相纳升级液相色谱(SCX/nano HPLC)进行电喷雾串联质谱分析(ESI-MS-MS)。分析步骤如下：肽段干粉使用60μL上样液(5mM甲酸铵,5%ACN水溶液,pH3.0,第一个浓度的盐梯度)溶解后每次上样19μL。第一维上样流速为20μL/min,洗脱5分钟,未被SCX保留的肽段直接被捕集柱捕集,同时经过捕集柱的盐溶液直接排入废液。经过阀切换,使捕集柱和分析柱相连,同时启动Nano泵进行反相分离,启动质谱采集在线检测肽段。进样器吸取20μL第二种浓度的盐溶液进行第二个盐梯度,重复进行以上步骤。实验共进行10个盐梯度(包括第一次上样的梯度)。盐梯度之后仍会有部分疏水性较强的肽段保留在SCX柱上,为了回收这部分肽段,最后我们添加了一个ACN浓度较高的盐梯度。对串级质谱鉴定的数据库分析后,高脂鼠肝组织中的蛋白与正常鼠肝组织中的相比,含量两倍上调的有21种,两倍下调的有10种。
综上所述,本论文针对蛋白质组学研究领域的热点难点问题,研制了新型的蛋白质捕集柱并成功应用于实际鼠肝蛋白的捕集,搭建了蛋白质水平分离的两维阵列式毛细管液相色谱平台,建立了基于蛋白质固定与蛋白质荧光标记的研究蛋白质相互作用的新方法。为利用高通量的阵列色谱平台分离复杂的蛋白质组以及研究蛋白质相互作用、蛋白质定量等打下了良好的基础。
The completion of the human genome project has facilitated the entry of biomedical research into the post-genome era-proteomics research. There are two technical routes in proteomics:peptide-centric bottom-up and intact-protein level top-down approaches."Top-down" proteomics can provide more information for intact proteins which can be used to study protein quantitation, protein-protein interactions (PPIs) and posttranslational modifications (PTMs). The strategies based on separation and identification of intact proteins has become the mainstream in proteomic research. However, proteins are more complex than peptides. The efficient and high throughput separation of complex protein mixtures at intact-protein level is a great challenge to the chromatographic field. Construction of high-throughput multi-dimensional array liquid chromatography platform has great potential for large-scale proteomics analysis.
The elucidation of PPIs is one of the most challenging and important tasks in proteomics research. Study of PPIs can provide important information for understanding protein functions, elucidating disease pathogenesis and developing new drugs. So PPIs analysis has become the focus of proteomics. The current technologies have hindered high throughput screening of PPIs because they are usually time-consuming and expensive. Therefore, it is urgent to develop new strategies for the large-scale study of PPIs.
We focus on the key and challenging problems in proteome field, a novel kind of intact-protein trapping columns was developed for trapping and desalting of proteins. We established a multidimensional capillary array liquid chromatography platform coupled by MALDI-TOF-TOF identification of intact proteins. And a new method for study of PPIs was proposed. Differential proteomics of normal and fat rat liver proteins was achieved based on label-free proteomics. The research work in this thesis is divided into five chapters.
In Chapter1, advance and application of monolithic columns were included, development and application of multidimensional liquid chromatography in the field of proteome were introduced, technologies for analyzing PPIs were summarized. The research background was demonstrated.
In Chapter2, most commercially available precolumns and our previously developed precolumns are mainly employed for trapping peptides, it is urgent to develop intact-protein trapping columns for high efficient trapping and elution of proteins. A new type of monolithic trapping columns with high mechanical strength was prepared by thin-layer sol-gel coating method and applied to trapping intact proteins for on-line capillary liquid chromatography. Hundreds times of trapping/untrapping for intact proteins were carried out. The trapping columns showed long-term stability up to300bar. Recovery, loading capacity and reproducibility of trapping columns were evaluated using four proteins. The recovery of four protein mixtures for the C8monolithic trapping columns was99.3%on average. The loading capacity of5mm×320μm i.d. C8trapping columns for the protein mixtures was30μg. The C8trapping columns were used to trap normal mouse liver intact proteins in a capillary liquid chromatography system. Results demonstrated high efficiency of the monolithic trapping columns for trapping intact proteins for proteomic analysis in on-line capillary liquid chromatography system.
In Chapter3, we established a capillary two-dimensional array liquid chromatography system for automated, high-throughput analysis of intact proteins, in which one strong-anion-exchange (SAX) chromatographic column was used as the first separation dimension and18parallel capillary reverse-phase liquid chromatographic (RPLC) columns were integrated as the second separation dimension. Proteins eluting from the SAX were trapped and desalted by18parallel intact-protein trapping columns. Then protein fractions were back-flushed simultaneously from18intact-protein trapping columns to capillary RPLC columns. The eighteenplexed capillary array chromatography system is capable of concurrently separating eighteen different samples, resulting in an18-fold increase in analytical throughput. Protein fractions eluting from RPLC columns were spotted onto the MALDI sample plate in1min intervals through an array of capillary tips. On-plate tryptic digestion technique was employed to digest the effluents. Normal mouse liver tissue proteins were analyzed by the fully multiplexed high-throughput two-dimensional liquid chromatography platform coupled by MALDI-TOF-TOF-MS identification. In total,1030proteins were identified, which proved the system's promising potential for intact-protein separation in proteomics.
In Chapter4, we proposed a new method for PPIs research. Bait protein was immobilized on amino particles, prey protein was labeled by a fluorescent reagent, and then they were incubated, eluted, detected by fluorescence microscopy. GST and its antibody were used to verify the feasibility of the method. First, amino-nano-magnetic microspheres were synthesized, glutaraldehyde was connected with amino of magnetic particles by chemical reaction, and then protein was connected to glutaraldehyde. After protein immobilization procedure was conducted, the UV absorption value of the supernatant solution was measured at280nm to calculate the amount of protein immobilized on the magnetic microspheres. A solid-support reaction was described to realize fluorescent derivatization of proteins. A simple, low-cost homemade capillary C18cartridge was fabricated as the solid-support reactor, protein was captured by this reactor and then labeled by fluorescein isothiocyanate (FITC, isomer I) on solid-support. Unwanted fluorescent intruder (excrescent FITC and products of secondary reactions) were removed from target easily. By incubating immobilized protein and FITC-labeled protein in Tris-HCl buffer (pH7.5,100mM NaCl,4℃), we investigated PPIs. This new method was applied to study PPIs between human serum albumin (HSA) and human plasma fractions.
In Chapter5, we reported on differential proteomics of normal and fat rat liver proteins. This was achieved by2D-LC-MS/MS-based label-free protein quantification. The peptides were desalted, dried and then resuspended with60μL volume of loading buffer (5mM Ammonium formate containing5%acetonitrile, pH3.0), separated and analyzed by2D strong cation-exchange (SCX)/reversed-phase (RP) nano-scale liquid chromatography/mass spectrometry (2D-nanoLC/MS). The experiments were performed on a Nano Aquity UPLC system connected to an LTQ Orbitrap XL mass spectrometer equipped with an online nano-electrospray ion source. A20μL plug was injected each time to form the salt step gradients. Ten step gradients were carried out. The plugs were loaded onto the SCX column with a loading buffer at a20μL/min flow rate for5min. A19μL peptide sample was loaded onto the SCX column before the gradient plugs were injected. The eluted peptides were captured by a Captrap Peptide column, while salts were diverted to waste. To recover hydrophobic peptides still retained on the SCX column after a conventional salt step gradient, a RP step gradient from15%to50%acetonitrile was applied to the SCX column. After all MS/MS spectrums were identified, we found quantitative differences of31proteins common to two samples, of which,21up-regulated and10down-regulated.
In summary, this thesis focuses on the key and challenging problems of proteomics. We developed a novel type of intact-protein trapping columns for desalting and trapping of intact-protein. We set up a multidimensional capillary array liquid chromatography platform coupled by MALDI-TOF-TOF identification of intact proteins. And a new method for studying PPIs was proposed.
引文
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