TOP-DOWN PROTEOMICS USING A NOVEL ESI QQQ-FTMS

使用新型 ESI QQQ-FTMS 进行自上而下的蛋白质组学

• 批准号: 7601996
• 负责人: PETER B. O'CONNOR
• 金额: $ 0.43万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-03 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601996
• 关键词: Accounting; Amino Acid Sequence; Anaphase; Biochemical; Biological; Cancer Biology; Cell Cycle; Cells; Chemistry; Cleaved cell; Collaborations; Complex; Complex Mixtures; Computer Retrieval of Information on Scientific Projects Database; Condition; Dissection; Electrospray Ionization; Ensure; Funding; Gel; Genomics; Grant; Histone H2B; Human; Human Cell Line; Institution; Laboratories; Light-Chain Immunoglobulins; Liquid Chromatography; Mass Spectrum Analysis; Measures; Methods; Mitosis; Modification; Molecular; Patients; Peptide Sequence Determination; Peptides; Physiologic pulse; Post-Translational Protein Processing; Prealbumin; Process; Protein Splicing; Proteins; Proteolysis; Proteomics; Pulse taking; RNA Splicing; Research; Research Personnel; Resolution; Resources; SHFM1 gene; Sampling; Side; Source; Time; United States National Institutes of Health; Variant; anaphase-promoting complex; base; instrument; interest; mass spectrometer; medical schools; novel; research study; ubiquitin ligase; ubiquitin-protein ligase

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 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；传统方法遵循自下而上的策略，该策略非常耗时，因为在大多数情况下，需要进行凝胶分离，然后进行蛋白水解，然后需要进行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水解产物的质量和/或使用片段化方法对这些小蛋白水解产物进行测序以分配蛋白质身份。主要问题是使用单次蛋白水解自下而上实验实现 100% 序列覆盖率是极不可能的，即通常不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肽。此外，任何与预测质量不匹配的肽都必须进一步研究，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该过程。实验室构建的 ESI qQq-FTMS 仪器对于自上而下的蛋白质组学具有多种优势。该仪器提供多种类型的片段化，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有助于实现完整的序列覆盖，因为蛋白质内的某些基序和序列比另一种片段化方法更适合一种片段化方法，因此拥有所有可用选项可增加获得 100% 序列覆盖率的可能性。此外，如果正在研究翻译后修饰，“较软”的断裂方法可能是优选的，这样修饰就不会从肽的侧链上裂解下来。事实上，这种新颖的仪器具有具有单位分辨率能力的解析四极杆，允许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，在将感兴趣的物种转移到 ICR 细胞之前，碰撞池中可以积累感兴趣的物种。这使得可以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来优化该仪器上的条件和脉冲序列，以便我们拥有对生物样品中的蛋白质进行测序的方法。一些市售蛋白质已获得几乎 100% 的序列覆盖率。为了对来自人类细胞系和患者的样品进行自上而下的实验，已经设计了分离经过修饰的完整蛋白质的方法。与哈佛大学 Marc W. Kirschner 教授合作。医学院，我们之前设计了一种生化和分子生物学方法来分离复合物，例如后期促进复合物，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），已知该复合物在细胞周期中被高度翻译后修饰。我们已经对该复合物的相互作用伙伴（即 UbCH10（一种 E2 泛素连接酶））进行了自上而下的实验。 APC 等生物复合物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。自上而下的分析还用于表征氧化 p21ras、组蛋白 H2B 和淀粉样蛋白（例如免疫球蛋白轻链和运甲状腺素蛋白）。