蛋白质组学高效酶解新技术

【作者】 王胜；

【导师】 杨芃原；

【作者基本信息】 复旦大学 ， 分析化学， 2008， 博士

【摘要】 第一章概述了蛋白质组学目前发展的概况,简要介绍了蛋白质组学的仪器技术及其在当今生物科学研究中的地位和作用;说明了当前酶解新技术的发展现状,就新型酶解反应器和用于促进酶解的微波、超声等技术的发展进行了综述;对富集技术的发展概况进行了简要说明。提出了本论文选题的目的和意义。本论文主要发展了一系列新颖方便的高效酶解技术,还对用于磷酸化多肽富集的纳米材料进行了一些研究。主要内容摘要如下:在第二章,为用于高效蛋白酶解制备了一种四组分的纳米复合物;固定了胰蛋白酶的聚苯胺包覆的Fe₃O₄/碳纳米管复合物。在含有金属离子Fe²⁺和Fe³⁺以及碳纳米管的溶液中滴加氨水,制备了nano-Fe₃O₄/CNT纳米复合物。之后在含有胰蛋白酶的溶液中,使苯胺（PA）在nano-Fe₃O₄/CNT纳米复合物的表面组装聚合,得到固定了胰蛋白酶的PA/Fe₃O₄/CNT纳米复合物。对所得的1维超顺磁性复合物进行了多种方法的表征:TEM,SEM,XRD,以及磁性分析。以BSA、MYO为典型蛋白质的酶解-MALDI-TOF-MS实验说明由这种材料所催化的酶解可以在5 min内给出比传统溶液酶解12 h更高的肽段覆盖度,更多匹配的肽段。此材料具有很好的生物相容性和极好的分散性,这有利于在酶解过程中酶与蛋白质的重复接触。利用纳米Fe₃O₄的顺磁性,还可以在酶解后利用外磁场的帮助将此复合物与反应体系方便地分离,从而避免复合物对体系的污染并有利于后续的质谱鉴定。这种固定了胰蛋白酶的纳米复合物可以通过简单的两步沉积制备,也许它还会得到除蛋白质组学外更多的生物学应用。第三章介绍了一种纤维内芯酶反应器。内芯由玻璃纤维制作:首先在玻璃纤维表面构建壳聚糖、和海藻酸钠的静电自组装层,再将此纤维浸入胰蛋白酶溶液中以固定胰酶。将此玻璃纤维插入微量进样器的不锈钢针头内制得高效方便的酶解反应器。此针孔内的酶解器可以通过更换纤维内芯进行再生。室温下（25℃）当蛋白质（BSA和LYS）溶液流经此针头时,在针头内仅仅与内芯接触5 s。对流出的溶液进行质谱分析的结果表明,蛋白质在针头中被酶解,并且在质谱分析中得到了正确的鉴定,并且其序列覆盖度与传统溶液酶解方法的结果相近。该针内酶反应器酶解的高效性,可归因于在纤维内芯上包附的高浓度的胰酶,和针内纤维的高比表面积,从而使底物和酶的能够有高频率的相互作用。另外,由于CTS和SA（两种天然高聚物）的生物相容性,最大限度地减少了被固定胰酶的变性。除了酶解的高效性,此方法的另外一个优势是它的简便性,其内芯的制备仅需在不同溶液中反复浸泡和清洗玻璃纤维,其使用和更换也都十分方便,而且多个纤维酶反应器的阵列可以来实现高通量的蛋白质组研究。在第四章,低电压交流电（AC,通常为5V）产生的交变电场（AEF）被用来提高用于肽谱分析的胰蛋白酶酶解的效率。在蛋白质的溶液酶解过程中,在溶液中插入一对电极,并在电极上加低压交流电产生一个交变电场。使用这种新颖的AEF辅助酶解方法,结合MALDI-TOF-MS,对几种标准蛋白质和SDS-PAGE胶内的HSA蛋白质进行了酶解和肽谱分析。结果显示交变电场可以显著地加速溶液酶解并使酶解时间缩短至5 min。得到的序列覆盖度接近12 h传统溶液酶解的结果,也以电泳分离的人血清白蛋白为例证实了该方法对实际蛋白质样品的适应性。本酶解方法十分简便高效,必将在蛋白质鉴定中得到广泛的应用。在第五章,我们发展了低压交流电辅助的靶上酶解。AC辅助靶上酶解是一种十分新颖的技术,与MALDI-TOF MS联用的它是蛋白质肽谱分析的有力工具。在交流电的帮助下,酶解时间可以从传统溶液酶解的12 h缩短至5 min。靶上酶解需要的样品量很少,这也是本方法的优点。本章也对AEF对酶解的促进原理作了探讨,认为酶解效率的提高主要是因为交变电场中带电组分的运动方向和偶极距的方向都会周期性地改变（50 Hz导致每秒100次的方向转换）,从而增加了蛋白质和酶的相互作用,而且在蛋白质和多肽上的带电基团如氨基羧基等也会受到交变电场的影响而使多肽振动。但本方法操作还不够简便。如果多个样品需要同时进行靶上酶解,则需要多个铂电极的阵列。而水电解反应在电极上的发生,也将限制本酶解方法对一些含有巯基的蛋白质的分析应用。在第六章首次提出采用红外（IR）辐照来促进肽谱分析中的胰蛋白酶（trypsin）酶解。这种红外辅助的酶解方法简单,花费低廉而效率很高,十分适用于蛋白质的高通量鉴定。红外辅助酶解的操作:在透明的eppendorf离心管中,蛋白溶液在37℃在红外灯辐照下被trypsin酶解,反应进行5 min。BSA和MYO在此方法中的酶解时间从常规的12 h被显著地缩短为5 min,证明了此新颖的酶解方法的可行性和有效性。酶解后的肽段用MALDI-TOF-MS进行分析,序列覆盖度为69%（BSA）和90%（MYO）,而这都大大高于传统溶液酶解所得的覆盖度。这说明红外辐照对酶解过程起了很大的促进作用。并且研究了红外辐照对糜蛋白酶的酶解过程的加速作用,其结果表明红外辐照对糜蛋白酶的酶解也有类似的促进作用。并且通过对人类血清进行的酶解验证了红外辐助糜蛋白酶酶解方法对复杂样品的适应性。红外线辐照对酶解效率的提高的机理应该是,红外线辐照可以提高酶解反应中反应物在反应位点上的局部振动能级,降低了反应的活化能,故而能够大大加速酶解的过程。另外,在蛋白质中,红外辐射所激发的肽链振动也可能诱使更多的酶切位点暴露给胰酶,使得酶解更加容易,从而得到较高多肽覆盖度,即匹配更多肽段。第七章发展了红外辐照辅助靶上（on-plate）酶解技术。使用红外线对在MALDI靶板上的含有trypsin的蛋白质溶液进行辐照。用BSA和Cyt-c的酶解研究了这种新颖的酶解方法的效果。酶解后的肽段用MALDI TOFMS进行分析。结果表明红外辐射显著地增强了酶解的效率并且酶解的时间被显著缩短至5min以内:肽段覆盖度为55%（BSA）和75%（Cyt-c）,这与传统溶液酶解的结果相差无几。通过对人血清和牛奶中提取的酪蛋白的酶解验证了红外辅助靶上酶解这种方法对较为复杂的样品的适应性。此种酶解方法简单而且高效,特别适合LC-MALDI,是高通量蛋白质鉴定的一个新的优良的技术平台。在第八章,针对翻译后修饰蛋白中磷酸化蛋白及肽段的分离富集问题,发展了一种制备磁性IMAC纳米材料的新方法。先用水热法制备了表面包含氨基的Fe₃O₄纳米磁球,用三氯氧磷将氨基表面改性为磷酸根表面,再将磁球在金属离子溶液中浸泡。并用所得的纳米磁珠用于对β-casein酶解肽段中磷酸化肽段的富集,将磷酸化多肽洗脱后进行MALDI-TOF分析。研究发现固定不同的金属离子制作的纳米粒子对磷酸化多肽的富集能力是不同的的。其中固定Fe（Ⅲ）的纳米磁珠表现最好:富集磷酸化多肽的灵敏度低于20 fmol/μL;β-casein和BSA以1:1000的质量比混合后,仍能够富集检测到其中的磷酸化肽,显示了其良好的抗非特异性吸附的能力。更多还原

【Abstract】 In chapter 1,a bird view of proteomics advancement and achievements were given.Instruments and methods used in proteomics were summarized,along with the impacts of proteomics on modern biological science.And the proteolysis techniques ever developed was summarized with two parts:1) the new types of enzyme reactor,2) microwave and ultrasound techniques.Enrichment techniques used in proteomics was also briefly introduced.The new strategies of high efficient proteolysis in this thesis were briefed at the end of this chapter,demonstrating their importance and usefulness. This dissertation contributes a series of new strategies of high efficient proteolysis for proteomic analysis.And a short study on nanomaterial for phosphopeptides enrichment was described.The main contents are:In Chapter 2,a four-component nanocomposite,trypsin-immobilized polyaniline-coated Fe₃O₄/carbon nanotube composite,was synthesized for highly efficient protein digestion.Fe₃O₄ was deposited by the chemical coprecipitation of Fe²⁺ and Fe³⁺ in an alkaline solution containing carbon nanotubes（CNTs） to prepare nano-Fe₃O₄/CNT composite.Subsequently,polyaniline（PA） was assembled on the Fe₃O₄/CNT composite by the in situ polymerization of aniline in the presence of trypsin to obtain trypsin-immobilized PA/Fe₃O₄/CNT nanocomposite.The novel 1D superparamagnetic biomaterial has been characterized by TEM,SEM,XRD,and magnetometric analysis.The feasibility and performance of the unique magnetic biomaterial have been demonstrated by the tryptic digestion of bovine serum albumin, myoglobin,and lysozyme within 5 min.The digests were identified by MALDI-TOF MS with sequence coverages that were comparable to those obtained from the conventional in-solution tryptic digestion.The present biocomposite offers considerable promise for protein analysis due to its high magnetic responsivity and excellent dispersibility.It can be easily isolated from the digests with the aid of an external magnetic field.Because the enzyme-immobilized nanocomposite can be prepared by a simple two-step deposition approach with low cost,it may find a wide range of biological applications including proteome research.In Chapter 3,a core-changeable needle enzymatic reactor was developed for highly efficient proteolysis.Self-assembled layers of CTS and SA were first constructed on the surface of a glass fiber;this glass fiber was then immersed in trypsin solution to immobilize trypsin.A piece of enzyme-immobilized fiber core was inserted into the needle of a syringe pump to form a flow-through bioreactor.The novel in-needle bioreactor could be regenerated by changing its fiber core.The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of bovine serum albumin and lysozyme,and the digestion time was significantly reduced to less than 5 s.The digests were identified by MALDI-TOF-MS with sequence coverage comparable to those obtained by the conventional in-solution tryptic digestion.The significantly enhanced digestion efficiency of the in-needle fiber bioreactor can be attributed to the high concentration of trypsin in the modified layer on the fiber core and the higher surface area of the fiber core in the needle,increasing the interaction frequency between trypsin and proteins.In addition,the biocompatibility of CTS and SA（two naturally occurring polymers） may provide milder environmental conditions so that the denaturation of the immobilized trypsin was minimized.Besides the high digestion efficiency,an additional advantage of the present approach is its simplicity,which is promising for the automatic high-throughput protein analysis using a platform containing multiple in-needle fiber bioreactors.In Chapter 4,Sinusoidal alternating voltages（typically 5V） were employed to enhance the efficiency of proteolysis for peptide mapping.Protein solutions containing trypsin were allowed to digest with the assistance of alternating electric fields（AEFs） between a pair of platinum wire electrodes in Eppendorf tubes.The feasibility and performance of the novel proteolysis approach were investigated by the digestion of several standard proteins.It was demonstrated that AEFs significantly accelerated in-solution proteolysis and the digestion time was substantially reduced to 5 min.The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS） with sequence coverages that were comparable to those obtained by using conventional 12-h in-solution proteolysis. The suitability of AEF-assisted proteolysis to real protein samples was demonstrated by digesting and identifying human serum albumin in gel separated from human serum by sodium dodecyl sulphate/polyacrylamide gel electrophoresis（SDS-PAGE）. The present proteolysis strategy is simple and efficient and will find a wide range of applications in protein identification.In Chapter 5,alternating current-assisted on-plate proteolysis has been developed for rapid peptide mapping.Protein solutions containing trypsin were allowed to digest directly on the spots of a stainless steel MALDI plate with the assistance of low-voltage alternating current electricity.Alternating current（AC） was allowed to pass through the protein solutions via the MALDI plate and a platinum disc electrode. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of BSA and cytochrome c（Cyt-c）.It was demonstrated that AC substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min.The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF MS） with sequence coverages of 42%（BSA） and 77%（Cyt-c） that were comparable to those obtained by using conventional in-solution tryptic digestion.The present proteolysis strategy is simple and efficient,offering great promise for MALDI-TOF-MS peptide mapping.In Chapter 6,infrared（IR） radiation was employed to enhance the efficiency of tryptic proteolysis for peptide mapping.The ease,simplicity,efficiency,and low cost of the novel proteolysis approach indicate great promise for the high-throughput protein identification.Protein solutions containing trypsin in sealed transparent Eppendorf tubes were allowed to digest under IR lamp radiation at 37℃.The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of bovine serum albumin（BSA） and myoglobin（MYO） and the digestion time was significantly reduced to 5 min.The obtained digests were identified by MALDI-TOF MS with the sequence coverage of 69%（BSA） and 90% （MYO） that were much better than those obtained by conventional in-solution tryptic digestion.The present IR-assisted proteolysis strategy is simple and efficient,offering great promise for high-throughput protein identification.Infrared（IR） radiation was also employed to enhance the efficiency of chymotryptic proteolysis for peptide mapping.The suitability of IR-assisted chymotryptic proteolysis to complex proteins was demonstrated by digesting human serum.The present proteolysis strategy is simple and efficient,offering great promise for high-throughput protein identification.The significantly enhanced digestion efficiency of the present proteolysis approach can be attributed to IR radiation,which increases the energy level of vibrations of peptide bonds so as to decrease the activation energy of proteolysis reaction,which might lead to a great increase in the reaction speed.In addition,the IR-induced vibrations of the chains in proteins might also lead to more cleavage sites exposed to enzymes,resulting in easier cleavage of peptide bonds.It might be the reason why there were more matched peptides in the PMF spectra of the digests obtained by using IR-assisted digestion.In Chapter 7,infrared（IR）-assisted on-plate proteolysis has been developed for rapid peptide mapping.Protein solutions containing trypsin were allowed to digest directly on the spots of MALDI plates under IR radiation.The feasibility and performance of the novel proteolysis approach were investigated by the digestion of bovine serum albumin（BSA） and cytochrome c（Cyt-c）.It was demonstrated that IR radiation substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min.The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF MS） with sequence coverages of 55%（BSA） and 75%（Cyt-c） that were comparable to those obtained by using conventional in-solution tryptic digestion.The suitability of IR-assisted on-plate proteolysis to complex proteins was demonstrated by digesting human serum and casein extracted from commercially available milk sample.The present proteolysis strategy is simple and efficient,offering great promise for high-throughput protein identification.In Chapter 8,to faciliate the enrichment of phosphopeptide or phosphoprotein several kinds of metal ions Ga（Ⅲ）,Al（Ⅲ）,Zr（Ⅳ）,Zn（Ⅱ）,Fe（Ⅲ）,Ni（Ⅱ）,were trapped on the surface of Fe₃O₄ magnetic nanoparticles with phosphate group binding. Amine functioned Fe₃O₄ nanoparticles were synthesized first,then the amine groups on the surface was reacted with POCl₃,resulting in a phosphate group functioned surface.Then the nanoparticles were immersed in solutions of different kinds of metal ions to immobilize metal ions on its surface.And the ability of those magnetic particles in isolating phosphopeptides was studied withβ-casein tryptic digest.Results show that only Fe（Ⅲ） modified magnetic nanoparticles work best.When the magnetic nanoparticle enrichment was coupled with MALDI TOF mass analysis,a detection limit lower than 20 fmol/μL was observed.And good specificity was demonstrated whenβ-casein digest was mixed with BSA in the ration of 1:1000.更多还原