TOP-DOWN PROTEOMICS USING A NOVEL ESI QQQ-FTMS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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工作。大多数蛋白质不能单靠质量来鉴定，因为基于基因组序列预测的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，然后是蛋白质分解，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测量的蛋白质分解产物的质量和/或使用碎裂方法来对这些小的蛋白质分解产物进行排序，以指定蛋白质的特性。主要的问题是，使用单次蛋白分解自下而上实验，100%的序列覆盖率是极不可能的，即通常没有考虑蛋白质序列的某些部分，因为没有观察到跨越这些区域的多肽。此外，任何与预测质量不匹配的多肽都必须进一步研究，以确定蛋白质修饰或序列变异。与自下而上的方法不同，另一种自上而下的质谱学(MS)可以通过省去几个湿化学步骤并确保完整的序列覆盖来显著简化这一过程。实验室建立的ESI QQQ-FTMS仪器对自上而下的蛋白质组学具有几个优点。该仪器上有几种类型的片段，包括掠夺器片段、Q2 CAD、MSAD、IRMPD、ECD和SORI-CAD-许多不同的片段方法的可用性促进了完整的序列覆盖，因为蛋白质中的某些基序和序列比另一种片段方法更适合于一种片段方法，因此拥有所有可用的选项增加了获得100%序列覆盖的可能性。此外，如果正在研究翻译后修饰，“较软”的碎片法可能更可取，这样修饰就不会从多肽的侧链上断裂。这种新型仪器有一个具有单位分辨能力的分辨四极杆，这一事实允许人们使用质谱仪的前端从相当复杂的混合物中分离出感兴趣的蛋白质，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种转移到ICR池之前积累感兴趣的物种。这使得对低丰度蛋白质进行自上而下的研究成为可能。我们正在使用商业上可用的蛋白质来优化这台仪器上的条件和脉冲序列，以便我们有适当的方法对生物样本中的蛋白质进行测序。一些商业上可用的蛋白质已经获得了几乎100%的序列覆盖率。为了对人类细胞系和患者的样本进行自上而下的实验，已经设计出了经过修改的分离完整蛋白质的方法。与哈佛大学马克·W·基什内尔教授合作。在此之前，我们已经设计了一种生化和分子生物学的方法来分离复合体，如后期促进复合体，并将检测一个人类复合体，后期促进复合体(APC-一种E3泛素连接酶)，它在细胞周期中被高度翻译后修饰。我们已经在这个复合体的相互作用伙伴UbCH10(一种E2泛素连接酶)上进行了自上而下的实验。对APC等生物复合体的解剖与癌症生物学有关，因为它们控制着细胞周期中的关键转变，即APC启动后期和退出有丝分裂。自上而下的分析也被用于表征氧化的p21ras、组蛋白H2B和淀粉样蛋白，如免疫球蛋白轻链和转甲状腺素。

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.