基于新材料的翻译后修饰蛋白质、多肽富集鉴定新方法的研究
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摘要
随着人类基因组草图测序的完成与蛋白质组学概念的提出，揭示着后基因组时代的到来。而蛋白质作为生物系统活动中的执行体，调节着生物体内许多重要的生理活动。蛋白质翻译后修饰称之为继核酸、蛋白质之后的第三种重要的生命信息寄存形式。在众多的翻译后修饰中，蛋白质磷酸化及糖基化在体内分布最广、影响最多，与生命活动最为密切相关。生物质谱技术作为后基因组学研究的核心技术之一，已经被广泛的应用于蛋白质翻译后修饰的研究，然而对于纷繁复杂的生物体系而言，以质谱为主要研究手段的蛋白质磷酸化与糖基化研究,仍然面临着诸多问题，如：修饰肽段化学计量水平低、存在强的背景干扰，同时质谱检测前样本处理过程复杂且损失严重。因此特异的富集技术与快速灵敏的检测手段成为蛋白质磷酸化与糖基化研究中的关键。本研究针对蛋白质磷酸化与糖基化研究中缺乏灵敏、快速、高效的富集方法与实时、动态的检测手段等问题，开发了多种基于新型制备技术的蛋白质磷酸化高效选择性富集材料与蛋白质糖基化快速分离与原位检测新方法，并在实际生物样本中获得了成功应用，显著提高了磷酸化、糖基化翻译后修饰的鉴定规模。
第一部分为解决蛋白质磷酸化丰度低，质谱鉴定困难和离线富集步骤繁琐等问题，创新性地利用表面引发原子转移自由基聚合（SI-ATRP）反应制备了新型三维波浪状聚合物修饰的石英毛细管柱，实现了磷酸化蛋白质/肽段在线规模化富集鉴定。该新型毛细管柱与常规磷酸化富集材料相比具有以下创新点和优势：（1）首次在石英毛细管内壁利用SI-ATRP反应的高可控性原位修饰三维波浪状聚合物涂层，有效提高毛细管柱内壁比表面积的同时也维持了较低的柱压，（2）三维波浪状聚合物的覆盖显著增加了柱内富集官能团的密度以及与磷酸化肽段的接触几率，因此大幅提高了磷酸化肽段的富集效率，（3）负载量与传统的开管柱相比提高了将近一个数量级。将该新型三维波浪状聚合物修饰的石英毛细管柱应用于HepG2细胞磷酸化蛋白质的规模化分析，磷酸化肽段鉴定数量与文献报道的基于TiO_2装填柱相比明显提高。这部分研究内容已经发表在Analytical Chemistry2010,82(22)9461-9468。在此基础上为有效提高样品上样量，进一步提高磷酸化蛋白质的鉴定规模，我们还发展了TiO_2固相萃取小柱-高pH值反相色谱分离结合质谱鉴定的磷酸化肽段整体富集鉴定研究。该策略成功应用到复杂生物样本小鼠肝脏蛋白质磷酸化的富集分离中，共鉴定到11291条非冗余的磷酸化肽段和2610种磷酸化蛋白，磷酸化蛋白质鉴定数量与国际上本领域领先实验室哈弗医学院Steven P. Gygi教授采用的SCX结合IMAC技术相比提高了12%。该方法的主要特点和优势在于（1）利用SPE小柱超大的装填容量填装与磷酸化肽段具有特异亲和的TiO_2颗粒，大幅提高初始样本上样量，（2）首次将高pH值反相色谱分离技术应用到富集后的磷酸化肽段混合物的分离，有效降低了样本的复杂程度，显著提高了质谱鉴定的成功率。该技术的建立为磷酸化蛋白质组学的研究提供了有力的技术支持。
第二部分针对蛋白质糖基化研究中糖蛋白质糖链丰度偏低、质谱信号差以及现有富集方法步骤多与耗时长等问题，首创性的发展了自组装芘衍生化氧化石墨烯对糖蛋白质糖链高效、可视化富集新方法。该方法与生物质谱技术结合，成功应用于HepG2细胞糖蛋白质糖链的富集鉴定，共鉴定到糖型26种，糖型鉴定数量和信噪比获得明显提高。该方法与传统的糖蛋白质糖链富集技术相比具有以下特点：（1）首次利用氧化石墨烯超大的比表面积结合超高密度的芘丁酰氯修饰，显著提高了富集材料对糖蛋白质糖链的捕获能力（2）利用酰氯基团与糖蛋白质糖链羟基的共价结合，选择性富集糖蛋白质糖链，材料的结合能力高于HILIC、凝集素等非共价富集方法；（3）糖链的多羟基可引发功能化氧化石墨烯迅速聚集，实现了对富集过程的可视化监测；（4）显著缩短了糖链富集所需时间并简化了操作步骤。这部分研究内容已经发表在Analytical Chemistry2013,85(5)2703–2709上，该技术的开发为蛋白质糖基化分离富集技术的研究提供了新的技术支持。为进一步实时、动态、原位的研究不同类型细胞表面膜糖糖型的变化及其与疾病发生发展的关系，我们还创新性的利用SI-ATRP反应制备了高度水溶性、凝集素功能化的上转换荧光纳米颗粒，应用于细胞表面膜糖糖型的原位差异识别分析研究。与现有亲水修饰技术相比，SI-ATRP反应的高可控性可在上转换荧光纳米颗粒表面原位修饰结构规整、厚度均匀的亲水聚合物层，并保持上转换荧光颗粒内核的完整性，因此可实现有效增加其生物相容性的同时而不影响上转换荧光纳米颗粒的量子效率。此外，线性聚合物侧链所负载的大量功能化位点在有效提高固定凝集素数量的同时，也将凝集素的固定模式由传统的两维平面提升为三维立体，不同朝向固定的凝集素增加了与其特定糖型的识别几率，显著提高了功能化上转换荧光纳米材料的识别特异性和亲和力。体外与体内实验均表明，该材料可以应用于不同类型肝癌细胞表面膜糖糖型的原位差异识别。该技术的发展为研究肝癌细胞表面膜糖糖型的变化与癌症发生发展的关系提供了有力的技术支持。该部分研究内容已经投稿《ACS Nano》。
Post-translational modifications (PTMs) of proteins have been viewed as the thirdimportant life information storage following nucleic acids and proteinsand are involvedin the control ofalmost all life processes, such as proliferation, growth, differentiation,cellular signaling pathways. Among the common PTMs of proteins, phosphorylationand glycosylation are the most widely distributed and affected almost all biologicalprocess. As one of the core technologies in post genomics era, bio-mass spectrometry iswidely applied in the PTM research.However, the low stoichiometric level, strongbackground interference and tedious sample processing procedure makes it particularlydifficult for the identification of protein phosphorylation and glycosylation by massspectrometry. Therefore, the primary task of phosphorylation and glycosylation researchis to selectively and efficiently isolatethe phosphopeptides/glycopeptides from complexproteolytic peptide mixture. In this study, we developed a few highly efficient methodsfor phosphopeptides and glycans enrichment and in-situ imaging technique for proteinglycosylation using novel functional materials.
In the first chapter, we developed a new type of capillary column using thesurface-initiatedatomic transfer free radical polymerization (SI-ATRP) and successfullyapplied it in on linelarge scale phosphopeptides enrichment and identification.There is afew key features of the newly developed capillary. First, three-dimensional wavelikepolymer structures are grown on the inner wall of capillary columnsfor the firsttime, results in largely increased surface area. Second, the three-dimensionalwavelikepolymerstructureon the inner wallof capillary columns carries a large numberof densely packed enrichment functional groups, and thus, increased enrichmentefficiency of phosphopeptides is achieved. Finally, compared with conventional opentubular capillarycolumn, the loading capacity of SI-ATRP column is increased by nearlyone order of magnitude.The SI-ATRP modified capillarycolumn was successful appliedin the online phosphopeptides enrichment and identification of HepG2cell lysates andresulted in obviously increased number of identifiedphosphopeptides compared withreported on line method using TiO_2packing column. This work was publishedinAnalytical Chemistry2010,82,9461-9468. To further increase the number ofphosphorylation identification, we also developed a new off-line phosphopeptidesenrichment strategy by combining TiO_2packed columns enrichment and high pHreversed-phase chromatography separations. The new strategy was successfully appliedin mouse liver phosphorylation identification.Unique phosphopeptides (11291) andphosphoprotein (2610) were identified which are12%higher than that reported in StevenP. Gygi et al. PNAS.2007.104(5):1488-93using SCX combined IMAC technology. Thenew strategy has two advantages. First, the large loading capacity ofTiO_2packedcolumnssignificantlyincrease the initial sample loading amount.Second, the high pHreversed-phase separation technique was first applied to the separation of thephosphopeptides mixture after enrichment. The high orthogonality of this2D LCseparation efficiently reduce samplecomplexity and increase the phosphorylationidentification in complex samples. The establishment of these new enrichment methodswill provide powerful technical support for phosphoproteomic research.
In the second chapter, we reported a rapid, highly efficient, and visualized approachfor glycans enrichment using1-pyrenebutyryl chloride functionalized free grapheneoxide (PCGO). Combined with bio-mass spectrometry, this new method, wassuccessfully applied in the enrichment and identification of glycans fromHepG2cellmembrane protein.Compared with conventional glycans enrichment methods, the newstrategy has exhibits the following features and advantages. First, Improved enrichmentefficiency is achieved by the large specific surface area of freePCGO and heavyfunctionalization of highly active1-pyrenebutyryl chloride. Second, reversible covalentbond between the hydroxyl groups of glycans and the acyl chloride groups on grapheneoxide (GO) increased the enrichment specificity comparing with HILIC orlectins based enrichment. Third, the multiple hydroxyl groups of glycans lead tocross-linking andself-assembly of free PCGO sheets into visible aggregation, therefore visualmonitoringof the enrichment process is achieved. Finally, largely reduced the time required forenrichment and simplification of the operation. This work was published in AnalyticalChemistry2013,852703-2709.To realize real time and in-situ investigationon therelationship between cell membrane glycans and disease progression,we prepared highlywater-soluble and lectin functionalized upconversionnanoparticles(UCNPs) usingSI-ATRP technique for cell membrane glycansimaging.SI-ATRP modification resultsin in situ growth of hydrophilic polymer on UCNPs surface and well defined core-shellstructure which renders UCNPs largely improved biocompatibility with intactluminance property. Furthermore, the numerous functional groups on the polymer brushshell provided large number of binding site and3D support for lectin immobilization.The densely packed lectins with diversified orientation\facilitate multivalent bindingbetween the immobilized lectin and target glycans and leads to improved labelingspecificity. Finally, thelectin-ATRP-UCNP is successfully applied in in vitro and in vivoimaging of glycans on hepatocellular carcinoma cells (HCC) and distinct difference inglycan profile was found betweenHCC with and normal liver cells. The establishment ofthis new imaging method will provide powerful technical support for investigation therelationship between cell surface glycans patterns and cancer development.
第一部分为解决蛋白质磷酸化丰度低，质谱鉴定困难和离线富集步骤繁琐等问题，创新性地利用表面引发原子转移自由基聚合（SI-ATRP）反应制备了新型三维波浪状聚合物修饰的石英毛细管柱，实现了磷酸化蛋白质/肽段在线规模化富集鉴定。该新型毛细管柱与常规磷酸化富集材料相比具有以下创新点和优势：（1）首次在石英毛细管内壁利用SI-ATRP反应的高可控性原位修饰三维波浪状聚合物涂层，有效提高毛细管柱内壁比表面积的同时也维持了较低的柱压，（2）三维波浪状聚合物的覆盖显著增加了柱内富集官能团的密度以及与磷酸化肽段的接触几率，因此大幅提高了磷酸化肽段的富集效率，（3）负载量与传统的开管柱相比提高了将近一个数量级。将该新型三维波浪状聚合物修饰的石英毛细管柱应用于HepG2细胞磷酸化蛋白质的规模化分析，磷酸化肽段鉴定数量与文献报道的基于TiO_2装填柱相比明显提高。这部分研究内容已经发表在Analytical Chemistry2010,82(22)9461-9468。在此基础上为有效提高样品上样量，进一步提高磷酸化蛋白质的鉴定规模，我们还发展了TiO_2固相萃取小柱-高pH值反相色谱分离结合质谱鉴定的磷酸化肽段整体富集鉴定研究。该策略成功应用到复杂生物样本小鼠肝脏蛋白质磷酸化的富集分离中，共鉴定到11291条非冗余的磷酸化肽段和2610种磷酸化蛋白，磷酸化蛋白质鉴定数量与国际上本领域领先实验室哈弗医学院Steven P. Gygi教授采用的SCX结合IMAC技术相比提高了12%。该方法的主要特点和优势在于（1）利用SPE小柱超大的装填容量填装与磷酸化肽段具有特异亲和的TiO_2颗粒，大幅提高初始样本上样量，（2）首次将高pH值反相色谱分离技术应用到富集后的磷酸化肽段混合物的分离，有效降低了样本的复杂程度，显著提高了质谱鉴定的成功率。该技术的建立为磷酸化蛋白质组学的研究提供了有力的技术支持。
第二部分针对蛋白质糖基化研究中糖蛋白质糖链丰度偏低、质谱信号差以及现有富集方法步骤多与耗时长等问题，首创性的发展了自组装芘衍生化氧化石墨烯对糖蛋白质糖链高效、可视化富集新方法。该方法与生物质谱技术结合，成功应用于HepG2细胞糖蛋白质糖链的富集鉴定，共鉴定到糖型26种，糖型鉴定数量和信噪比获得明显提高。该方法与传统的糖蛋白质糖链富集技术相比具有以下特点：（1）首次利用氧化石墨烯超大的比表面积结合超高密度的芘丁酰氯修饰，显著提高了富集材料对糖蛋白质糖链的捕获能力（2）利用酰氯基团与糖蛋白质糖链羟基的共价结合，选择性富集糖蛋白质糖链，材料的结合能力高于HILIC、凝集素等非共价富集方法；（3）糖链的多羟基可引发功能化氧化石墨烯迅速聚集，实现了对富集过程的可视化监测；（4）显著缩短了糖链富集所需时间并简化了操作步骤。这部分研究内容已经发表在Analytical Chemistry2013,85(5)2703–2709上，该技术的开发为蛋白质糖基化分离富集技术的研究提供了新的技术支持。为进一步实时、动态、原位的研究不同类型细胞表面膜糖糖型的变化及其与疾病发生发展的关系，我们还创新性的利用SI-ATRP反应制备了高度水溶性、凝集素功能化的上转换荧光纳米颗粒，应用于细胞表面膜糖糖型的原位差异识别分析研究。与现有亲水修饰技术相比，SI-ATRP反应的高可控性可在上转换荧光纳米颗粒表面原位修饰结构规整、厚度均匀的亲水聚合物层，并保持上转换荧光颗粒内核的完整性，因此可实现有效增加其生物相容性的同时而不影响上转换荧光纳米颗粒的量子效率。此外，线性聚合物侧链所负载的大量功能化位点在有效提高固定凝集素数量的同时，也将凝集素的固定模式由传统的两维平面提升为三维立体，不同朝向固定的凝集素增加了与其特定糖型的识别几率，显著提高了功能化上转换荧光纳米材料的识别特异性和亲和力。体外与体内实验均表明，该材料可以应用于不同类型肝癌细胞表面膜糖糖型的原位差异识别。该技术的发展为研究肝癌细胞表面膜糖糖型的变化与癌症发生发展的关系提供了有力的技术支持。该部分研究内容已经投稿《ACS Nano》。
Post-translational modifications (PTMs) of proteins have been viewed as the thirdimportant life information storage following nucleic acids and proteinsand are involvedin the control ofalmost all life processes, such as proliferation, growth, differentiation,cellular signaling pathways. Among the common PTMs of proteins, phosphorylationand glycosylation are the most widely distributed and affected almost all biologicalprocess. As one of the core technologies in post genomics era, bio-mass spectrometry iswidely applied in the PTM research.However, the low stoichiometric level, strongbackground interference and tedious sample processing procedure makes it particularlydifficult for the identification of protein phosphorylation and glycosylation by massspectrometry. Therefore, the primary task of phosphorylation and glycosylation researchis to selectively and efficiently isolatethe phosphopeptides/glycopeptides from complexproteolytic peptide mixture. In this study, we developed a few highly efficient methodsfor phosphopeptides and glycans enrichment and in-situ imaging technique for proteinglycosylation using novel functional materials.
In the first chapter, we developed a new type of capillary column using thesurface-initiatedatomic transfer free radical polymerization (SI-ATRP) and successfullyapplied it in on linelarge scale phosphopeptides enrichment and identification.There is afew key features of the newly developed capillary. First, three-dimensional wavelikepolymer structures are grown on the inner wall of capillary columnsfor the firsttime, results in largely increased surface area. Second, the three-dimensionalwavelikepolymerstructureon the inner wallof capillary columns carries a large numberof densely packed enrichment functional groups, and thus, increased enrichmentefficiency of phosphopeptides is achieved. Finally, compared with conventional opentubular capillarycolumn, the loading capacity of SI-ATRP column is increased by nearlyone order of magnitude.The SI-ATRP modified capillarycolumn was successful appliedin the online phosphopeptides enrichment and identification of HepG2cell lysates andresulted in obviously increased number of identifiedphosphopeptides compared withreported on line method using TiO_2packing column. This work was publishedinAnalytical Chemistry2010,82,9461-9468. To further increase the number ofphosphorylation identification, we also developed a new off-line phosphopeptidesenrichment strategy by combining TiO_2packed columns enrichment and high pHreversed-phase chromatography separations. The new strategy was successfully appliedin mouse liver phosphorylation identification.Unique phosphopeptides (11291) andphosphoprotein (2610) were identified which are12%higher than that reported in StevenP. Gygi et al. PNAS.2007.104(5):1488-93using SCX combined IMAC technology. Thenew strategy has two advantages. First, the large loading capacity ofTiO_2packedcolumnssignificantlyincrease the initial sample loading amount.Second, the high pHreversed-phase separation technique was first applied to the separation of thephosphopeptides mixture after enrichment. The high orthogonality of this2D LCseparation efficiently reduce samplecomplexity and increase the phosphorylationidentification in complex samples. The establishment of these new enrichment methodswill provide powerful technical support for phosphoproteomic research.
In the second chapter, we reported a rapid, highly efficient, and visualized approachfor glycans enrichment using1-pyrenebutyryl chloride functionalized free grapheneoxide (PCGO). Combined with bio-mass spectrometry, this new method, wassuccessfully applied in the enrichment and identification of glycans fromHepG2cellmembrane protein.Compared with conventional glycans enrichment methods, the newstrategy has exhibits the following features and advantages. First, Improved enrichmentefficiency is achieved by the large specific surface area of freePCGO and heavyfunctionalization of highly active1-pyrenebutyryl chloride. Second, reversible covalentbond between the hydroxyl groups of glycans and the acyl chloride groups on grapheneoxide (GO) increased the enrichment specificity comparing with HILIC orlectins based enrichment. Third, the multiple hydroxyl groups of glycans lead tocross-linking andself-assembly of free PCGO sheets into visible aggregation, therefore visualmonitoringof the enrichment process is achieved. Finally, largely reduced the time required forenrichment and simplification of the operation. This work was published in AnalyticalChemistry2013,852703-2709.To realize real time and in-situ investigationon therelationship between cell membrane glycans and disease progression,we prepared highlywater-soluble and lectin functionalized upconversionnanoparticles(UCNPs) usingSI-ATRP technique for cell membrane glycansimaging.SI-ATRP modification resultsin in situ growth of hydrophilic polymer on UCNPs surface and well defined core-shellstructure which renders UCNPs largely improved biocompatibility with intactluminance property. Furthermore, the numerous functional groups on the polymer brushshell provided large number of binding site and3D support for lectin immobilization.The densely packed lectins with diversified orientation\facilitate multivalent bindingbetween the immobilized lectin and target glycans and leads to improved labelingspecificity. Finally, thelectin-ATRP-UCNP is successfully applied in in vitro and in vivoimaging of glycans on hepatocellular carcinoma cells (HCC) and distinct difference inglycan profile was found betweenHCC with and normal liver cells. The establishment ofthis new imaging method will provide powerful technical support for investigation therelationship between cell surface glycans patterns and cancer development.
引文
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[1] Walsh CT, Garneau-Tsodikova S, Gatto GJ, Jr. Protein posttranslational modifications: thechemistry of proteome diversifications. Angew Chem Int Ed Engl.2005;44(45):7342-7372.
[2] Mann M, Jensen ON. Proteomic analysis of post-translational modifications. Nat Biotechnol.2003;21(3):255-261.
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[4] Linding R, Jensen LJ, Ostheimer GJ, et al. Systematic discovery of in vivo phosphorylationnetworks. Cell.2007;129(7):1415-1426.
[5] Ficarro SB, McCleland ML, Stukenberg PT, et al. Phosphoproteome analysis by mass spectrometryand its application to Saccharomyces cerevisiae. Nat Biotechnol.2002;20(3):301-305.
[6] Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature.2003;422(6928):198-207.
[7] Beausoleil SA, Villen J, Gerber SA, et al. A probability-based approach for high-throughput proteinphosphorylation analysis and site localization. Nat Biotechnol.2006;24(10):1285-1292.
[8] Pereira Navaza A, Ruiz Encinar J, Sanz-Medel A. Absolute and accurate quantification of proteinphosphorylation by using an elemental phosphorus standard and element mass spectrometry. AngewChem Int Ed Engl.2007;46(4):569-571.
[9] Villen J, Beausoleil SA, Gerber SA, et al. Large-scale phosphorylation analysis of mouse liver. ProcNatl Acad Sci U S A.2007;104(5):1488-1493.
[10] Tao WA, Wollscheid B, O'Brien R, et al. Quantitative phosphoproteome analysis using adendrimer conjugation chemistry and tandem mass spectrometry. Nat Methods.2005;2(8):591-598.
[11] Li S, Zeng D. Chemoenzymatic enrichment of phosphotyrosine-containing peptides. Angew ChemInt Ed Engl.2007;46(25):4751-4753.
[12] Schaefer BC, Paulson E, Strominger JL, et al. Constitutive activation of Epstein-Barr virus (EBV)nuclear antigen1gene transcription by IRF1and IRF2during restricted EBV latency. Mol Cell Biol.1997;17(2):873-886.
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