CPTR - Mass Spectrometry

CPTR-质谱法

• 批准号: 8938482
• 负责人: Lisa Jenkins
• 金额: $ 28.37万
• 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/8938482
• 关键词: Acetylation; Active Sites; Autophagocytosis; Basic Science; Biochemistry; Biological; Blood capillaries; C-terminal; CCR; Cell Aging; Cell Death; Cell Line; Cells; Cellular biology; Collaborations; Communication; Communities; Complex; Data; Dominant-Negative Mutation; Down-Regulation; Drug usage; Experimental Designs; Fibroblasts; Glycine; Human; Immunoassay; Ions; Journals; Label; Laboratories; Length; Liquid substance; Lysine; Manuscripts; Mass Spectrum Analysis; Messenger RNA; Methods; Methylation; Modification; Molecular Chaperones; Multi-Drug Resistance; N-terminal; Nature; Oncogenes; Orphan Drugs; Outcome; P-Glycoprotein; Peptides; Phosphorylation; Post-Translational Modification Site; Post-Translational Protein Processing; Preparation; Process; Production; Protein Isoforms; Proteins; Proteome; Proteomics; Publications; Publishing; Reactive Oxygen Species; Regulation; Research; Research Personnel; Resources; Sampling; Selenocysteine; Signal Transduction; Signaling Protein; Source; Sulfhydryl Compounds; Techniques; Technology; Testing; Time; Transactivation; Translational Research; Ubiquitination; United States Food and Drug Administration; Vesicle; Work; base; capillary; carcinogenesis; design; detector; follow-up; glutathione peroxidase; glycosylation; improved; inhibitor/antagonist; insight; instrument; killings; knock-down; mass analyzer; mass spectrometer; multicatalytic endopeptidase complex; protein function; research study; response; senescence; ubiquitin ligase; ubiquitin-protein ligase

In the last year, the mass spectrometry facility added a new mass spectrometer for use - a nanospray Orbitrap Fusion (Thermo) tribrid mass spectrometer. This instrument combines a quadropole mass filter with ion trap and Orbitrap mass analyzers, which allows the two detectors to operate in parallel, improving both sensitivity and selectivity. In addition to CID and HCD methods for peptide fragmentation, the Orbitrap Fusion is configured with an ETD source that facilitates analysis of larger peptides and post-translational modifications, particularly phosphorylation and glycosylation. Overall, the expertise of the mass spectrometry facility is being widely used to further the research of multiple groups within the CCR. In FY2014, the mass spectrometry facility within CPTR collaborated in 35 different projects, with more than 1100 samples processed and analyzed. These projects are being performed in collaboration with 23 different CCR investigators, including 4 tenure-track investigators. These projects include several to identify sites of post-translational modification, including phosphorylation, ubiquitination, acetylation, and methylation, to better understand signal transduction and protein regulation. The facility is also being used to identify protein interactors, including identification of those that change following post-translational modification. These studies will further research into protein function and regulation. Finally, mass spectrometry is being used extensively for large-scale quantitative proteomics projects. In these, labeled or label-free methods are used to comprehensively identify the proteome of a vesicle, fluid, or protein interaction network. 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. In the past year, two studies incorporating results obtained in the mass spectrometry facility have been published. In addition, several other projects are nearing completion and will be prepared as manuscripts for publication. The first study, published in the Journal of Biological Chemistry, is a collaboration with Dr. Michael Gottesmann in the Laboratory of Cell Biology. Here, mass spectrometry was used as one technique to investigate the mechanism by which tiopronin elicits collateral sensitivity in multidrug-resistant (MDR) cells. Collateral sensitivity is the selective targeting of MDR cells, while the sensitive parental cells are unaffected. Previously, the group had observed that some MDR cell lines showed collateral sensitivity towards tiopronin, a thiol-substituted N-propanoyl form of glycine and an Food and Drug Administration-registered orphan drug used for over 30 years to treat a diverse range of pathophysiological conditions. Although tiopronin showed MDR-selective activity against some P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1)-expressing cells, it did not kill all P-gp-expressing cell lines tested, and inhibition of P-gp did not abrogate selective killing. Rather, using mass spectrometry, we showed that tiopronin inhibits glutathione peroxidase (Gpx) by reacting with the selenocysteine residue in the active site, which is followed by intramolecular transfer to a proximal lysine residue, resulting in its covalent modification. This inhibition of Gpx resulted in increased production of reactive oxygen species (ROS), leading to cell death. The second study is the result of a collaboration with Dr. Curtis Harris, Laboratory of Human Carcinogenesis, to study the biological regulation of the delta133 isoform of p53. This isoform lacks the N-terminal transactivation domain and acts as a dominant negative inhibitor of full-length p53. Previously, we had observed that endogenous delta133p53 protein was down-regulated during replicative senescence, but not upon oncogene-induced senescence, in human fibroblasts; this down-regulation was not due to changes in mRNA level. Since delta133p53 was not degraded by the proteasome, we investigated whether it could be degraded by autophagic mechanisms. 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. Contrary to initial hypotheses, the interaction with the Hsp70 complex and STUB1 actually protects delta133p53 from degradation, rather than promoting degradation. When STUB1 was knocked down, the delta133p53 level was decreased and p53-dependent senescence increased. Further studies to identify the ubiquitin ligase and mechanism for delta133p53 autophagic degradation are ongoing. This work has been accepted for publication in Nature Communications.

去年，质谱设施增加了一个新的质谱仪供使用 - 纳米喷雾轨道融合（Thermo）Tribrid质谱仪。该仪器将四倍体质量过滤器与离子陷阱和Orbitrap质量分析仪相结合，这使两个检测器可以并行运行，从而提高灵敏度和选择性。除了CID和HCD方法用于肽破碎化外，Orbitrap融合还配置了ETD源，可促进对较大肽和翻译后修饰的分析，尤其是磷酸化和糖基化。总体而言，质谱设施的专业知识已被广泛用于进一步研究CCR中多个组的研究。在2014财年，CPTR中的质谱设施在35个不同的项目中合作，对1100多个样本进行了处理和分析。这些项目正在与23位不同的CCR调查人员合作执行，其中包括4位终身轨道调查人员。这些项目包括几个识别翻译后修饰部位的部位，包括磷酸化，泛素化，乙酰化和甲基化，以更好地了解信号转导和蛋白质调节。该设施还用于识别蛋白质相互作用，包括鉴定翻译后修饰后改变的蛋白质相互作用。这些研究将进一步研究蛋白质功能和调节。最后，质谱法已广泛用于大规模定量蛋白质组学项目。在其中，使用标记或无标记的方法用于全面识别囊泡，流体或蛋白质相互作用网络的蛋白质组。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。在过去的一年中，已经发布了两项结合质谱设施中获得的结果的研究。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《生物化学杂志》上，是与迈克尔·戈特斯曼博士在细胞生物学实验室中的合作。在这里，质谱法被用作一种研究一种技术来研究tiorpronin在多药耐药（MDR）细胞中引起侧支敏感性的机制。侧支灵敏度是MDR细胞的选择性靶向，而敏感的亲本细胞不受影响。以前，该小组观察到一些MDR细胞系对ti-蛋白素表现出侧支敏感性，这是一种硫代基建立的N-丙酰甘氨酸形式的甘氨酸形式以及食品和药物管理注册的孤儿药物，用于治疗多样化的病理生理条件范围。尽管tiorponin对某些P-糖蛋白（P-GP）和多药抗性蛋白1（MRP1）表达细胞的MDR选择性活性，但它并未杀死所有测试的表达P-gp的细胞系，并且对P-gp的抑制并未消除精选的杀伤。相反，使用质谱法，我们表明硫素通过与活性位点中的硒代半胱氨酸残基反应来抑制谷胱甘肽过氧化物酶（GPX），然后将分子内转移至近端赖氨酸残基，从而导致其共价修饰。 GPX的这种抑制导致活性氧（ROS）的产生增加，导致细胞死亡。第二项研究是与人类癌变实验室柯蒂斯·哈里斯（Curtis Harris）合作的结果，以研究p53的Delta133同工型的生物学调节。这种同工型缺乏N末端的反式激活结构域，并充当全长p53的主要负抑制剂。以前，我们已经观察到内源性Delta133p53蛋白在复制衰老过程中被下调，但在人成纤维细胞中不进行癌基因引起的衰老。这种下调不是由于mRNA水平的变化。由于Delta133p53没有被蛋白酶体降解，因此我们研究了它是否可以被自噬机制降解。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。与初始假设相反，与HSP70复合物和Stub1的相互作用实际上保护了Delta133p53免受降解的影响，而不是促进降解。当Stub1被击倒时，Delta133p53水平降低，p53依赖性衰老增加。进一步的研究以鉴定泛素连接酶和Delta133p53自噬降解的机制。这项工作已被接受用于自然传播中的出版。