TOP-DOWN PROTEOMICS USING A NOVEL ESI QQQ-FTMS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 蛋白质鉴定和蛋白质的全面结构表征是使用当前蛋白质组学方法的主要努力。大多数蛋白质不能仅通过质量来鉴定，因为基于基因组序列的预测质量并不总是正确的; RNA剪接、蛋白质剪接和翻译后修饰（PTM）可以改变预期的蛋白质质量。因此，其他策略是必要的;传统的方法遵循自下而上的策略，这是非常耗时的，因为在大多数情况下，凝胶分离，然后是蛋白水解，然后需要液相色谱来表征蛋白质。这种自下而上的方法利用蛋白水解产物的测量质量和/或使用片段化方法对这些小的蛋白水解产物进行测序以分配蛋白质身份。主要的问题是，100%的序列覆盖率是极不可能使用一个单一的通过蛋白质水解自下而上的实验，即，通常蛋白质序列的某些部分没有考虑，因为没有观察到跨越这些区域的肽。此外，必须进一步研究与预测质量不匹配的任何肽，以鉴定蛋白质修饰或序列变异。替代自下而上的方法，自上而下的质谱（MS）可以通过消除几个湿化学步骤并确保完整的序列覆盖来显着简化这一过程。在实验室中构建的ESI qQQ-FTMS仪器对于自上而下的蛋白质组学具有几个优势。该仪器上提供了几种类型的片段化，包括skimmer片段化、Q2 CAD、MSAD、IRMPD、ECD和SORI-CAD -许多不同片段化方法的可用性促进了完整的序列覆盖，因为蛋白质内的某些基序和序列更适合于一种片段化方法而不是另一种，因此具有所有可用选项增加了获得100%序列覆盖的可能性。此外，如果正在研究翻译后修饰，则“较软”片段化方法可能是优选的，使得修饰不从肽的侧链裂解。这种新型仪器具有具有单位分辨率能力的分辨四极杆的事实允许使用质谱仪的前端从相当复杂的混合物中分离感兴趣的蛋白质，即从其他背景蛋白质中“纯化”蛋白质。此外，在碰撞池中，允许在将感兴趣的物质转移到ICR池中之前积累感兴趣的物质。这允许对低丰度蛋白质进行自上而下的研究。我们正在使用市售蛋白质优化该仪器的条件和脉冲序列，以便我们有方法对生物样品中的蛋白质进行测序。对于一些商业上可获得的蛋白质，已经获得了几乎100%的序列覆盖率。为了对来自人细胞系和患者的样品进行自上而下的实验，已经设计了分离具有修饰的完整蛋白质的方法。与Marc W. Kirschner，哈佛大学医学院，我们以前设计了一种生物化学和分子生物学方法来分离复合物，如后期促进复合物，并将检查人类复合物，后期促进复合物（APC-一种E3泛素连接酶），已知其在细胞周期中高度后修饰。我们已经进行了自上而下的实验，对这种复合物的相互作用的合作伙伴，即UbCH 10（E2泛素连接酶）。生物复合物如APC的解剖与癌症生物学相关，因为已知它们控制细胞周期中的关键转变，即APC启动后期并退出有丝分裂。自上而下的分析也被用于表征氧化的p21 ras，组蛋白H2 B，和淀粉样蛋白，如免疫球蛋白轻链和甲状腺素运载蛋白。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein identification and comprehensive structural characterization of proteins is a major effort using current proteomic methods. Most proteins cannot be identified by mass alone because predicted masses that are based on genomic sequences are not always correct; RNA splicing, protein splicing and Post-Translational Modifications (PTMs) can change the expected protein mass. Therefore other strategies are necessary; the conventional approach follows the bottom-up strategy which is extremely time-consuming as, in most cases gel-separation, followed by proteolysis and then liquid chromatography is required to characterize the protein. This bottom-up approach utilizes the measured masses of proteolysis products and/or uses fragmentation methods to sequence these small proteolysis products to assign protein identities. The major problem is that 100% sequence coverage is extremely unlikely using a single pass proteolysis bottom-up experiment, i.e. often certain parts of the protein sequence are not accounted for because no peptides spanning these regions are observed. Also any peptides that do not match the predicted masses have to be investigated further in order to identify protein modification or sequence variations. Instead of following the bottom-up approach, the alternative, top-down mass spectrometry (MS) can ease this process significantly by eliminating several wet chemistry steps and ensuring complete sequence coverage. The ESI qQq-FTMS instrument built in the laboratory has several advantages for top-down proteomics. Several types of fragmentation are available on this instrument including skimmer fragmentation, Q2 CAD, MSAD, IRMPD, ECD and SORI-CAD - the availability of many different methods of fragmentation facilitates complete sequence coverage as certain motifs and sequences within proteins are more amenable to one method of fragmentation versus another, thus having all the available options increases the possibility of obtaining 100% sequence coverage. In addition, if post-translational modifications are being studied, "softer" fragmentation methods might be preferable such that the modification is not cleaved from the side chain of the peptide. The fact that this novel instrument has a resolving quadrupole with unit resolution capabilities allows one to use the front-end of the mass spectrometer to isolate the protein of interest from fairly complex mixtures i.e. to "purify" the protein from other background proteins. In addition, in the collision cell allows for the accumulation of the species of interest before transferring it into the ICR cell. This allows top-down studies to be performed on low abundance proteins. We are in the process of optimizing conditions and pulse sequences on this instrument using commercially available proteins such that we have methods in place for the sequencing of proteins from biological samples. Almost 100% sequence coverage has been obtained for some commercially available proteins. In order to perform top- down experiments on samples from human cell lines and patients, methods to isolate intact proteins with modifications have been devised. In collaboration with Prof. Marc W. Kirschner, Harvard Univ. School of Medicine, we have previously devised a biochemical and molecular biological method to isolate complexes such as the Anaphase Promoting Complex and will examine a human complex, the Anaphase Promoting Complex (APC-an E3 ubiquitin ligase) which is known to be highly post-translationally modified during the cell cycle. We have already performed top-down experiments on an interaction partner of this complex i.e. UbCH10 (an E2 ubiquitin ligase). The dissection of biological complexes such as the APC have relevance to cancer biology as they are known to control crucial transitions in the cell cycle i.e. the APC initiates anaphase and exit from mitosis. Top-down analysis is also being used for characterization of oxidized p21ras, Histone H2B, and amyloidogenic proteins such as immunoglobulin light chains and transthyretin.