CPTR - Mass Spectrometry Unit

CPTR - 质谱仪

• 批准号: 9154335
• 负责人: Lisa Jenkins
• 金额: $ 35.47万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9154335
• 关键词: Acetylation; Acids; Automation; Autophagocytosis; Back; Basic Science; Binding; Biological; Biological Assay; Biological Markers; Biological Process; Biological Sciences; Bulla; C-terminal; CCR; Cell Cycle Checkpoint; Cells; Cellular biology; Chromosomes; Clinical; Collaborations; Communication; Communities; Complex; Conditioned Culture Media; DNA biosynthesis; DNA replication fork; Data; Deuterium; Development; Experimental Designs; Genome Stability; Genomic Instability; Histone Deacetylase; Human; Hydrogen; Incubated; Injection of therapeutic agent; International; Journals; Label; Laboratories; Ligands; Liquid substance; Lysine; Macao; Manuscripts; Mass Spectrum Analysis; Methods; Methylation; Mitochondria; Molecular Chaperones; Mutation; National Institute of Diabetes and Digestive and Kidney Diseases; Nature; Nucleic Acids; Organelles; Outcome; Pepsin A; Peptides; Phenotype; Phosphorylation; Phosphorylation Site; Play; Post-Translational Protein Processing; Preparation; Process; Protein Conformation; Protein Dynamics; Protein Isoforms; Proteins; Proteomics; Protons; Publications; Publishing; Reaction; Regulation; Replication Origin; Repression; Research; Research Personnel; Resources; Robotics; Role; S Phase; Sampling; Signal Transduction; Signaling Protein; Site; Solutions; Solvents; Suction; Surface; Taiwan; Techniques; Technology; Temperature; Time; Translational Research; Ubiquitination; Universities; Vesicle; base; carcinogenesis; design; follow-up; insight; interest; mass spectrometer; protein function; research study; response; tumor; ubiquitin-protein ligase; uptake

Overall, the expertise of the Mass Spectrometry Unit is being widely used to further the research of multiple groups within the CCR. In FY2015, the unit collaborated in 40 different projects, with more than 1700 samples processed and analyzed. These projects are being performed in collaboration with 27 different CCR investigators. Among these are projects to characterize the post-translational modifications of target proteins, including sites of phosphorylation, ubiquitination, acetylation, and methylation, better understand signal transduction and protein regulation. The resource is also being used to identify protein interactors of both proteins and nucleic acids, including identification of those that change following post-translational modification. These studies help provide critical insight into protein function and regulation. Mass spectrometry is being used extensively for large-scale quantitative proteomics projects. In these, labeled or label-free methods are used to comprehensively identify whole proteomics, such as the protein composition of conditioned media, biological fluids (in particular, suction blister fluid), a subcellular organelle or vesicle (i.e., mitochondria and exosomes), or a whole cell. Additionally, we are collaborating on two global quantitative phosphoproteome studies, in which the global level of phosphorylation is compared. These discovery-oriented studies, which are sample- and time-intensive, provide broad information for defining new hypotheses and provide new insight into global protein activities and cellular responses. Finally, we are collaborating on two projects that make use of targeted quantitation to compare the protein level of particular proteins in complex samples; these studies are used for quantitation of a small number of specific target proteins in a large number of clinical samples to help validate potential biomarkers. The Mass Spectrometry Unit is also adding hydrogen-deuterium exchange mass spectrometry (HDX-MS) to the techniques available to CCR collaborators. In an HDX-MS experiment, the protein of interest is incubated with a solution of deuterium for varying times, allowing the surface-accessible protons to exchange with deuterium in the solvent. Exchange is quenched by treatment with acid, then the protein is digested with pepsin and the peptides analyzed by mass spectrometry. By analyzing the change in deuterium uptake over time, protein dynamics and conformational changes can be probed. Furthermore, by comparing the exchange profile of a protein in different conditions or in the presence of a binding partner or ligand, more detailed information about changes in the protein conformation can be obtained. For these experiments, we have a robotic liquid handling apparatus that allows automation of the sample preparation steps, from deuterium exchange reaction to injection onto the mass spectrometer, and provides temperature control which is critical to minimize back-exchange of the deuterium to hydrogen. We are also purchasing a Q Exactive Plus mass spectrometer to be primarily dedicated to these experiments. In the past year, two collaborative studies have been published. In addition, several other projects are nearing completion and will be prepared as manuscripts for publication. The first study, published in Nature Communications, is the result of an on-going collaboration with Dr. Curtis Harris, Laboratory of Human Carcinogenesis, to study the biological regulation of the delta133 isoform of p53. Using cell-based methods, we demonstrated that indeed delta133p53 was degraded by selective autophagy, and mass spectrometry experiments identified two ubiquitinated lysine residues in the C-terminal regulatory domain that were responsible for this effect. Further, mass spectrometric identification of interacting proteins indicated that delta133p53 forms a complex with the Hsp70 chaperone complex and the STUB1 E3 ubiquitin ligase. Mutation of the identified ubiquitination sites did reduce the level of delta133p53, but it did not abolish it. Thus, we are continuing to characterize the post-translational modification of delta133 p53 to look for other sites of ubiquitination. In addition, we are looking at additional protein interactors of this isoform of p53 to better understand its biological functions. In a second study, we collaborated with Drs. Ettore Appella, Laboratory of Cell Biology, and Chu-Xia Deng, formerly of NIDDK and currently at the University of Macau, SAR of People's Republic of China. Mass spectrometry was used to investigate the interactors of SIRT1, a histone deacetylase involved in numerous biological functions. Previously, Dr. Deng had demonstrated that SIRT1 deficiency resulted in genome instability, leading to the development of tumors, but the precise mechanisms for this phenotype was unknown. Among the protein interactors of SIRT1 identified were several proteins involved in the DNA replication, such as TopBP1, which plays an essential role in DNA replication fork protection and replication origin firing. Biological assays demonstrated that loss of SIRT1 resulted in increased replication origin firing, asymmetric fork progression, defective intra-S-phase checkpoint, and chromosome damage. In addition, characterization of the sites of acetylation of TopBP1 identified seven modified lysine residues, some of which were deacetylated by SIRT1. Thus, we were able to show that SIRT1 deficiency results in increased acetylation of TopBP1, which in turn causes repression of replication origin firing, indicating that SIRT1 acts upstream of TopBP1 to maintaining genome stability by modulating DNA replication fork initiation and the intra-S-phase cell cycle checkpoint. This research was published in the International Journal of Biological Sciences.

总体而言，质谱单元的专业知识被广泛用于进一步研究CCR中多个组的研究。在2015财年，该部门合作进行了40个不同的项目，并经过了1700多个样品进行处理和分析。这些项目正在与27位不同的CCR调查人员合作执行。其中包括旨在表征靶蛋白的翻译后修饰的项目，包括磷酸化，泛素化，乙酰化和甲基化的位点，更好地了解信号转导和蛋白质调节。该资源还用于鉴定蛋白质和核酸的蛋白质相互作用，包括鉴定在翻译后修饰后改变的蛋白质相互作用。这些研究有助于对蛋白质功能和调节的关键见解。质谱法已广泛用于大规模定量蛋白质组学项目。在这些中，使用标记或无标记的方法用于全面识别全蛋白质组学，例如条件培养基的蛋白质组成，生物流体（尤其是抽吸泡沫液），细胞细胞器或囊泡（即线粒体和外膜）或整个细胞。此外，我们正在进行两项全球定量磷蛋白组研究，其中比较了全球磷酸化水平。这些面向发现的研究和时间密集型的研究提供了广泛的信息，以定义新的假设，并为全球蛋白质活性和细胞反应提供新的见解。最后，我们正在协作两个项目，这些项目利用靶向定量比较复杂样品中特定蛋白质的蛋白质水平。这些研究用于定量大量临床样品中的少量特定靶蛋白，以帮助验证潜在的生物标志物。质谱单元还正在为CCR合作者可用的技术增加氢 - 偏见交换质谱（HDX-MS）。在HDX-MS实验中，感兴趣的蛋白质与氘的溶液在不同的时间内孵育，从而使表面可访问的质子可以在溶剂中与氘交换。用酸处理淬灭，然后用胃蛋白酶消化蛋白质，并通过质谱法分析肽。通过分析氘摄取随时间的变化，可以探测蛋白质动力学和构象变化。此外，通过比较在不同条件下或在结合伴侣或配体的情况下比较蛋白质的交换曲线，可以获得有关蛋白质构象变化的更详细信息。对于这些实验，我们有一个机器人液体处理设备，该设备可以自动化样品制备步骤，从氘交换反应到注射到质谱仪上，并提供温度控制，这对于最大程度地减少氘的反向交换至氢而言至关重要。我们还购买了一个Q Extive和质谱仪，主要用于这些实验。在过去的一年中，已经发表了两项合作研究。此外，其他几个项目即将完成，并将作为手稿准备出版。第一项研究发表在《自然通信》上，是与人类致癌实验室库尔蒂斯·哈里斯（Curtis Harris）进行的持续合作，以研究p53的Delta133同工型的生物学调节。使用基于细胞的方法，我们证明了Delta133p53的确被选择性自噬降解，并且质谱实验鉴定了C端调节域中的两个泛素赖氨酸残基，这些赖氨酸残基负责，这些赖氨酸残基是负责此作用的。此外，相互作用蛋白的质谱鉴定表明Delta133p53与HSP70伴侣复合物和Stub1 E3泛素连接酶形成复合物。确定的泛素化位点的突变确实降低了Delta133p53的水平，但并未废除。因此，我们继续表征Delta133 p53的翻译后修饰，以寻找其他泛素化位点。此外，我们正在研究该p53同工型的其他蛋白质相互作用者，以更好地了解其生物学功能。在第二项研究中，我们与Drs合作。 Ettore Appella，细胞生物学实验室和Chu-Xia Deng，前身为NIDDK，目前在澳门，澳门，中华人民共和国SAR。质谱法用于研究SIRT1的相互作用，这是一种与许多生物学功能有关的组蛋白脱乙酰基酶。以前，邓博士证明SIRT1缺乏导致基因组不稳定性，导致肿瘤的发展，但是该表型的确切机制尚不清楚。在鉴定出的SIRT1的蛋白质相互作用中，有几种参与DNA复制的蛋白质，例如TopBP1，它们在DNA复制叉保护和复制起源中起着至关重要的作用。生物学测定表明，SIRT1的损失导致复制起源发射增加，不对称叉进程，缺陷的S相检查点和染色体损伤。此外，TOPBP1乙酰化位点的表征确定了7种改良的赖氨酸残基，其中一些是通过SIRT1脱乙酰基的。因此，我们能够证明SIRT1缺乏会导致TOPBP1的乙酰化增加，这反过来又导致了复制起源的抑制，这表明SIRT1在TOPBP1上游起作用，以通过调节DNA复制fork启动和内置的细胞周期检查点来维持基因组稳定性。这项研究发表在《国际生物科学杂志》上。