Mass spectrometric investigation of biomolecules and dru

生物分子和药物的质谱研究

• 批准号: 7153595
• 负责人: Sonja Hess
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 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/7153595
• 关键词: DNA binding protein; DNA footprinting; adipocytes; antiAIDS agent; biotechnology; cerebrospinal fluid; conformation; drug discovery /isolation; health science research support; human immunodeficiency virus; integrase; intermolecular interaction; mass spectrometry; phosphorylation; podocyte; protein structure function; proteomics; secretory protein; small molecule

In our Facility, we use mass spectrometry to analyze and characterize biomolecules such as proteins and DNA. We also maintain a small molecule unit that analyzes chemical drug candidates synthesized by various researchers. We provide scientific and technical support in these areas. Furthermore, we successfully promote collaborations to increase our understanding of fundamental biological processes, mostly related but not limited to diabetes, digestive and kidney diseases. We concluded and published our investigations of the secretory proteome of adipocytes: A total of 84 proteins was identified as secreted adipokines. This large number of secretory proteins comprise multiple functional categories. Comparative proteomics of 18O proteolytic labeling allows the detection of different levels of many secreted proteins as exemplified by the difference between basal and insulin treatment of adipose cells. Our proteomic approach is able to identify and quantify the comprehensive secretory proteome of adipose cells. Thus, our data support the endocrine role of adipose cells in pathophysiological states through the secretion of signaling molecules. This successful approach is currently being adapted to investigate the secretory proteome of podocytes from kidneys. As for the adipocytes, we expect to gain further insights in the fundamentals of podocyte secretion. We also concluded a study on the cerebrospinal fluid proteome associated with chronic fatigue and related syndromes, which yielded the first predictive biomarkers for these syndromes. Using a logisitic model, we found that detection of >1 of a select set of 5 CFS-related proteins predicted CFS status with 80% concordance. We also expanded on our previously established protein-ligand interaction studies using HIV integrase as an important target for antiviral therapies. We found that pyridoxal-5-phosphate binds to a unique lysine (K244), located in the C-terminal domain, which signifantly impairs the formation of IN-DNA complex. Using a similar footprinting method, we also studied the hyperphosphorylation-induced conformational changes of full length native and hyperphosphorylated hRPA (human replication protein A). We found that three residues in the DNA binding domain B (K343, R335 and R382) were significantly shielded by hyperphosphorylation when compared to native hRPA. This led us to conclude that significant conformational changes involving the ssDNA binding cleft are induced after hyperphosphorylation. We provided analyses to more than 50 principal investigators of our Institute. Some of them, like the identification of a novel protein that forms complexes with MATER (Maternal Antigen That Embryos Require) are considered significant contributions. Many of these analyses are considered to be service and do not result in any publication.

在我们的设施中，我们使用质谱分析和表征生物分子，如蛋白质和DNA。我们还拥有一个小分子单元，用于分析各种研究人员合成的化学候选药物。我们在这些领域提供科学和技术支持。此外，我们成功地促进合作，以增加我们对基本生物过程的理解，主要涉及但不限于糖尿病，消化和肾脏疾病。 我们总结并发表了我们对脂肪细胞分泌蛋白质组的研究：共鉴定出84种蛋白质为分泌型脂肪因子。这种大量的分泌蛋白包括多个功能类别。18 O蛋白水解标记的比较蛋白质组学允许检测许多分泌蛋白的不同水平，如脂肪细胞的基础和胰岛素治疗之间的差异所例示的。我们的蛋白质组学方法能够识别和量化脂肪细胞的全面分泌蛋白质组。因此，我们的数据支持内分泌作用的脂肪细胞在病理生理状态下，通过分泌的信号分子。 这一成功的方法目前正被用于研究肾脏足细胞的分泌蛋白质组。至于脂肪细胞，我们期望获得进一步的了解足细胞分泌的基本原理。 我们还总结了一项关于慢性疲劳和相关综合征相关脑脊液蛋白质组的研究，该研究首次为这些综合征提供了预测生物标志物。使用logisitic模型，我们发现检测到5种CFS相关蛋白中的>1种可以预测CFS状态，具有80%的一致性。 我们还扩展了我们以前建立的蛋白质-配体相互作用的研究，使用HIV整合酶作为抗病毒治疗的重要靶点。我们发现，吡哆醛-5-磷酸结合到一个独特的赖氨酸（K244），位于C-末端结构域，这显着损害IN-DNA复合物的形成。 使用类似的足迹法，我们还研究了全长天然和过度磷酸化的hRPA（人复制蛋白A）的过度磷酸化诱导的构象变化。我们发现，与天然hRPA相比，DNA结合结构域B中的三个残基（K343、R335和R382）被过度磷酸化显著屏蔽。这使我们得出结论，过度磷酸化后会诱导涉及ssDNA结合裂缝的显着构象变化。 我们向我们研究所的50多名主要研究人员提供了分析。其中一些，如鉴定一种与MATER（胚胎所需的母体抗原）形成复合物的新蛋白质，被认为是重大贡献。其中许多分析被认为是服务性的，不会导致任何出版物。