CPTR - Mass Spectrometry Unit

CPTR - 质谱仪

• 批准号: 9556842
• 负责人: Lisa Jenkins
• 金额: $ 39.18万
• 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/9556842
• 关键词: ATP phosphohydrolase; Acetylation; Actins; Adaptor Signaling Protein; Affinity; Ankyrin Repeat; Archives; BTG2 Gene; Bacteriophage lambda; Basic Science; Binding; Biochemistry; C-terminal; CCR; Catalytic RNA; Cell Extracts; Cell Separation; Cells; Chimeric Proteins; Chromosomes, Human, Pair 2; Clathrin Adaptors; Co-Immunoprecipitations; Collaborations; Colorectal Cancer; Communities; Complex; Cytoskeleton; DNA Binding; DNA Damage; DNA biosynthesis; Data Analyses; Dimerization; Experimental Designs; Family; Fluorescence; GTP-Binding Proteins; Gene Targeting; Genetic; Glutathione; HIV; Human; Hybrids; Immobilization; In Vitro; Investigation; Journals; Kinesin; Label; Laboratories; Manuscripts; Mass Spectrum Analysis; Methodology; Methods; Methylation; MicroRNAs; Mitochondria; Molecular; Molecular Biology; Molecular Chaperones; Molecular and Cellular Biology; Muscarinic Acetylcholine Receptor; N-terminal; Nucleic Acids; Organelles; Outcome; PH Domain; Paper; Phosphorylation; Phosphorylation Site; Plant Resins; Plasma; Population; Post-Translational Protein Processing; Preparation; Process; Proteins; Proteome; Proteomics; Publishing; RNA; RNA Phages; RNA Splicing; RNA-Binding Proteins; Recombinants; Regulation; Research; Research Personnel; Resources; Sampling; Sepharose; Signal Transduction; Site; Site-Directed Mutagenesis; Sorting - Cell Movement; T-Lymphocyte; TP53 gene; Techniques; Technology; Time; Translational Research; Ubiquitination; Untranslated RNA; Vaccines; Vesicle; Vibrio cholerae; Viral; Winged Helix; Work; X Chromosome; Yeasts; base; cancer cell; cell motility; crosslink; design; dimer; exosome; experimental study; follow-up; genetic analysis; glucosamine 6-phosphate; in vivo; insight; interest; knock-down; macrophage; matrin 3; member; monocyte; nanoscale; novel; particle; patient population; protein complex; protein function; response; small molecule inhibitor; trafficking; tumor growth

Overall, the expertise of the Mass Spectrometry Unit is being widely used to further the research of multiple groups within the CCR. In FY2017, the unit collaborated in 41 different projects, with more than 1750 samples processed and analyzed. These projects are being performed in collaboration with 32 different investigators. Among these are projects to characterize the post-translational modifications of target proteins, including sites of phosphorylation, ubiquitination, acetylation, and methylation, to better understand signal transduction, protein regulation, and the effects of small molecule inhibitors. 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 additionally being used extensively for large-scale quantitative proteomics projects. In these, labeled or label-free methods are used to comprehensively identify whole proteomes, such as the protein composition of a subcellular organelle or vesicle (e.g., mitochondria and exosomes) or a whole cell. Additionally, we are collaborating on a global quantitative phosphoproteome study, 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. In the past year, five collaborative studies have been published, and several other projects have been completed and the corresponding manuscripts are under review. The first study, a collaboration with Dr. Paul Randazzo, Laboratory of Cellular and Molecular Biology, identified a novel interaction between Kinesin-like protein 2A (Kif2A) and Arf GAP with GTP-binding protein-like, ankyrin repeats and PH domains 1 (AGAP1) and elucidated the molecular function of this interaction. Mass spectrometry was used to screen for interactors of the GLD and PH domains of AGAP1, an Arf GAP that binds directly to the clathrin adaptor protein AP-3 to form a vesicular coat or to muscarinic receptor to modulate its trafficking. Among the identified interactors were a Ras family GTP-binding protein, actin and actin-associated proteins, and a kinesin. Yeast-2-hybrid analyses of binding partners of AGAP proteins also identified kinesin proteins as interactors, and several kinesins were shown to bind to AGAP1 by co-immunoprecipitation. Kif2A increased GAP activity of AGAP1, whereas the GLDPH domains of AGAP1 increased ATPase activity of Kif2A. Knockdown of either protein slowed cell migration and accelerated cell spreading, indicating that the Kif2A-AGAP1 complex contributes to control of cytoskeleton remodeling involved in cell movement. In this study, the initial mass spectrometry analysis to identify binding parts of AGAP1 was essential to the investigation of this family as modulators of AGAP1 activity. The study was published in the Journal of Biological Chemistry and selected as paper of the week. In the second study, mass spectrometry was used to demonstrate the utility of a new method for sorting viral particles. The laboratory of Dr. Marjorie Robert-Guroff, Vaccine Branch, used nanoscale fluorescence-activated cytometric cell sorting to sort infectious HIV from monocytes/macrophages, T cells, as well as from archived plasma. Sorted populations from human plasma were subsequently analyzed both genetically and using mass spectrometry. Although the yield of HIV was low, the mass spectrometry analysis confirmed the presence of HIV proteins and the results were consistent with the genetic analysis. Further, the analysis demonstrates the potential to sort patient populations of HIV and perform proteomics analyses with them. This study was published in JCI Insight. The third study used crosslinking-mass spectrometry analyses to demonstrate that the N- and C-terminal regions of the Vibrio cholerae DNA replication initiator protein RctB could interact in trans. RctB specifically initiates replication of V. cholerae Chromosome 2 (Chr2); RctB binds to both 12-mer sites to promote replication as well as to 39-mer sites to inhibit Chr2 replication. Additionally, binding of the chaperone DnaJK promotes both effects. Using mass spectrometry in combination with crosslinking analysis, we demonstrated that the N-terminal K-I site of RctB, which binds to DnaK, directly contacts the C-terminal winged-helix domain, which binds DNA. Other identified crosslinks were formed in regions shown to be important for dimerization and DNA binding. These results suggest that the RctB K-I site could be directly contacting the region responsible for 39-mer binding to cause autoinhibition. These findings, performed in collaboration with Dr. Dhruba Chattoraj, Laboratory of Biochemistry and Molecular Biology, were published in mBio. We have further used mass spectrometry to characterize the protein interactors of PINCR, a p53-regulated long noncoding RNA (lncRNA). PINCR is an 2.2 kb spliced intergenic lncRNA transcribed from the X-chromosome that was observed to be induced approximately 100-fold following DNA damage in a p53-dependent manner. Further, PINCR was found to exert a prosurvival function in human colorectal cancer cells (CRC) in vitro and to promote tumor growth in vivo. Mass spectrometry analysis was used to identify the protein interactors of PINCR following pulldown of a biotinylated form of the RNA. Among those interactors, we identified the RNA-binding protein Matrin 3, which in turn associates with p53. Through its interaction with Matrin 3, and thereby working also through p53, PINCR induces the expression of a subset of p53 target genes, including BTG2, GPX1 and RRM2B. This study demonstrates the presence of a critical prosurvival function of a p53/PINCR/Matrin 3 comeplex in response to DNA damage in CRC cells. This research, performed in collaboration with Dr. Ashish Lal, Genetics Branch, was published in eLIFE. Finally, we worked with Dr. Pascale Legault, Universite de Montreal, to develop a novel methodology to pulldown and identify the protein interactors of RNAs. In the method, specific RNA molecules are in vitro transcribed with an ARiBo tag, which contains an activatable glmS ribozyme and the BoxB RNA from bacteriophage lambda. The lambda boxB RNA allows immobilization of the RNA on glutathione sepharose via its high affinity to a recombinant lambda N-GST fusion protein, whereas the glmS ribozyme can be activated by glucosamine-6-phosphate to free the RNA of interest from the resin. Thus, the RNA of interest can be immobilized on resin for incubation with cell extract and pulldown, then the RNA-protein complexes eluted by activation of the ribozyme. Protein interactors are then identified by mass spectrometry analysis. To demonstrate the utility of the method, we identified the protein interactome of two members of the let-7 family of miRNAs, let-7a1 and let-7g. Mass spectrometry analysis demonstrated the specific enrichment of several known interactors of both miRNAs, including lin28A and lin28B with low background binding to the ARiBo tag alone. Thus, this method represents a new means to identify the protein interactors of specific RNA molecules. This research was published in RNA.

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2017财年，该部门在41个不同的项目中进行了合作，并进行了1750多个样品处理和分析。这些项目正在与32位不同的研究人员合作执行。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，以更好地理解信号转导，蛋白质调节以及小分子抑制剂的作用。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鉴定全蛋白质组，例如亚细胞器细胞器或囊泡的蛋白质组成（例如，线粒体和外泌体）或整个细胞。此外，我们正在进行一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。在过去的一年中，已经发表了五项合作研究，其他几个项目也完成了，相应的手稿正在审查中。第一项研究是与细胞和分子生物学实验室的Paul Randazzo博士的合作，确定了驱动蛋白样蛋白2a（KIF2A）与ARF GAP与GTP结合蛋白类蛋白，Ankyrin重复序列和pH域1（AGAP1）之间的新型相互作用，并赋予了这种相互作用的分子功能。质谱法用于筛选AGAP1的GLD和pH结构域的相互作用，这是一种直接与网格蛋白适配器蛋白AP-3结合的ARF间隙，形成囊泡外套或毒蕈碱受体调节其运输。在已确定的相互作用中，有RAS家族GTP结合蛋白，肌动蛋白和肌动蛋白相关蛋白以及驱动蛋白。 AGAP蛋白的结合伴侣的酵母-2杂交分析也将动力蛋白蛋白鉴定为相互作用，并且通过共免疫沉淀显示了几种可与AGAP1结合的动力素。 KIF2A增加了AGAP1的间隙活性，而AGAP1的GLDPH结构域增加了KIF2A的ATPase活性。敲低的蛋白质减慢了细胞迁移和加速细胞扩散，表明KIF2A-AGAP1复合物有助于控制细胞运动中涉及的细胞骨架重塑。在这项研究中，鉴定AGAP1结合部分的初始质谱分析对于将该家族作为AGAP1活性调节剂的研究至关重要。该研究发表在《生物化学杂志》上，并被选为本周的论文。在第二项研究中，质谱法用于证明一种新方法对病毒颗粒进行分类的实用性。疫苗分支Marjorie Robert-Guroff博士的实验室使用了纳米级荧光激活的细胞仪细胞分类，从单核细胞/巨噬细胞，T细胞以及存档的血浆中分类感染性HIV。随后在遗传和质谱法上分析了人血浆中的分类种群。尽管HIV的产量很低，但质谱分析证实了HIV蛋白的存在，结果与遗传分析一致。此外，该分析表明了分类HIV患者种群并与其进行蛋白质组学分析的潜力。这项研究发表在JCI Insight中。第三项研究使用交联的质谱分析来证明颤音霍乱DNA复制引发剂蛋白RCTB的N-和C末端区域可能在反式中相互作用。 RCTB专门启动了霍乱弧菌染色体2（CHR2）的复制； RCTB与两个12-MER位点结合，以促进复制以及39-MER位点抑制CHR2复制。另外，伴侣DNAJK的结合促进了这两种作用。使用质谱与交联分析结合使用质谱，我们证明了与DNAK结合的RCTB的N端K-I位点直接接触C端翼螺旋螺旋结构域，该结构结合了DNA。在证明对二聚化和DNA结合很重要的区域中形成了其他鉴定的交联。这些结果表明，RCTB K-I位点可以直接接触负责39-MER结合的区域以引起自身抑制。这些发现与生物化学和分子生物学实验室Dhruba Chattoraj博士合作，发表在MBIO中。我们进一步使用了质谱法来表征PINCR的蛋白质相互作用者，PINCR是P53调节的长无编码RNA（LNCRNA）。 PINCR是从X染色体转录的2.2 Kb剪接的基因间LNCRNA，观察到以p53依赖性方式在DNA损伤后被观察到约100倍。此外，发现PINCR在体外在人类结直肠癌细胞（CRC）中发挥了生存功能，并在体内促进肿瘤生长。质谱分析用于鉴定PILLED的RNA的蛋白质相互作用者的蛋白质相互作用者。在这些相互作用的人中，我们鉴定了RNA结合蛋白基质蛋白3，这又与p53相关。通过它与Matrin 3的相互作用，从而通过P53进行工作，PINCR诱导了包括BTG2，GPX1和RRM2B在内的p53靶基因子集的表达。这项研究证明了p53/pincr/matrin 3 comeplex的关键生存功能，响应CRC细胞中的DNA损伤。这项与遗传学分支Ashish Lal博士合作进行的研究发表在Elife中。最后，我们与蒙特利尔大学的Pascale Legault博士合作，开发了一种新颖的方法，以使RNA的蛋白质相互作用者下调和识别。在该方法中，特定的RNA分子在体外用Aribo TAG转录，该标签包含可激活的GLMS核酶和来自噬菌体lambda的BoxB RNA。 Lambda Boxb RNA允许将RNA通过其高亲和力通过其对重组Lambda N-GST融合蛋白的高亲和力固定，而GLMS核酶可以通过葡萄糖胺-6-磷酸盐激活GLMS核酶，从而从感兴趣的RAS中释放RNA。因此，可以将感兴趣的RNA固定在树脂上，以与细胞提取物和下拉孵育，然后通过核酶激活洗脱的RNA蛋白络合物。然后，通过质谱分析来识别蛋白质相互作用。为了证明该方法的效用，我们确定了miRNA，let-7a1和let-7g的两个家族的两个成员的蛋白质相互作用组。质谱分析证明了miRNA的几个已知相互作用者的特异性富集，包括Lin28a和Lin28b，其背景低于Aribo TAG。因此，该方法代表了一种新手段，以鉴定特定RNA分子的蛋白质相互作用。这项研究发表在RNA中。