Neuropsychiatric Disorders--protein Structure/activity Studies

神经精神疾病--蛋白质结构/活性研究

• 批准号: 8556903
• 负责人: SANFORD P MARKEY
• 金额: $ 14.45万
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 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/8556903
• 关键词: Active Sites; Affinity; Alzheimer' s Disease; American; Binding; Biochemistry; Biologic Characteristic; Biological; Biological Markers; Biological Models; Brain Diseases; Brazil; C-terminal; Complementary DNA; Complex; Coupled; Coupling; Diagnostic; Digestion; Disease; Early Diagnosis; Endoglycosidase F; Ensure; Family; Fingerprint; Gases; Gel; Goals; High Pressure Liquid Chromatography; Human; Hydrolase; Immunoassay; Inherited; Investigation; Ion Exchange; Journals; Laboratories; Lasers; Legal patent; Length; Ligands; Link; Liquid Chromatography; Liquid substance; MALDI-TOF Mass Spectrometry; Membrane; Metalloproteases; Methodology; Methods; Molecular; Multiple Sclerosis; Mus; Nervous system structure; Parents; Pathogenesis; Pathologic; Patients; Pattern; Peptide Hydrolases; Peptides; Phase; Physiological; Plasma; Post-Translational Protein Processing; Procedures; Process; Property; Proteins; Proteome; Protocols documentation; Publishing; Rattus; Recombinants; Resources; SNAP receptor; Sampling; Scorpions; Sequence Analysis; Solid; Structure; Surface; Techniques; Technology; Testing; Time; Tissues; Toxin; Trypsin; Universities; Venoms; Work; animal tissue; base; carboxypeptidase C; expression cloning; glycosylation; human tissue; insight; ionization; lysosomal proteins; mass spectrometer; member; nervous system development; neuropsychiatry; protein aminoacid sequence; protein function; protein structure; receptor; surface enhanced laser desorption ionization; tandem mass spectrometry

The unambiguous identification of proteins/peptides is crucial for the definition of the proteome. Using ProteinChip Array technology (SELDI) we have developed experimental protocols and probed test conditions required for protein identification on ProteinChip Arrays. We are able to directly digest proteins/peptides on-chip surfaces by specific proteases such as trypsin, Glu C and Asp N and obtain the peptide mass fingerprint of the sample under investigation by direct analysis on a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry can be performed on the resulting tryptic peptides by using collision quadrupole time of flight (Qq-TOF) MS/MS via a ProteinChip interface, thus allowing the precise identification of the parent protein within the sample. In addition we are able to identify the C-terminal sequence of peptides after digestion with carboxypeptidase Y directly on ProteinChip surfaces coupled with SELDI-TOF mass spectral analysis both under native and denaturing conditions. Utilizing the on chip digestion methodology, we have examined the structural differences between the disease and physiological forms of proteins that may be diagnostic for the disease processes. The primary structure of both forms of the protein may be identical by MS/MS analysis; however, post-translational modification such as an altered N-linked glycosylation pattern could prove to be informative. By coupling enzymatic digestion(both peptide N-glycosidase F and trypsin) and ProteinChip technology these altered protein forms are identifiable. Furthermore, we have developed methods for studying the interaction of both pathologic and physiologic forms of proteins with their ligands or receptors. The biological characteristics of the pathological form may be altered with regard to their binding properties. Utilizing SELDI, we have also examined the peptide component of patient plasma. In order to fully characterize these molecules we developed a micro-preparative procedure (employing liquid chromatography and SELDI) that provided separation of these peptides. Microsequencing and MS/MS analyses of the peptides have identified several molecules that are fragments of larger proteins that would appear to be different from disease versus control. Combining the resources of the laboratory, we have clearly demonstrated our ability to analyze samples from a variety of disease states, such as Alzheimers disease and Multiple Sclerosis. We are presently evaluating methods for maximizing the number of proteins/peptides detected from CSF. These methods will be used in examining CSF from patients as well as other biological fluids and tissues to identify possible biomarkers for use in earlier diagnosis. By combining both SELDI analysis and on-chip immunoassay, we hope to clearly identify the species that are different from patient versus control as well as those that are post-translationally altered. We have succeeded in identifying and characterizing a family of metalloproteases that have unique effects on the SNARE complex in various model systems (initially rat and mouse). The proteases are capable of disrupting a number of the members of the complex in different ways depending on the particular protease. Studies have also been directed at recombinant forms of the members of the complex including Snap 25, Snap 23 and Vamp 2. Our studies may provide insight as to how portions of the complex ensure the stability and function of the proteins forming the complex. The initial work has been published in the Journal of Biological Chemistry and has been the basis for a provisional US patent application with our collaborators at East Carolina University. In addition we have a cDNA clone which encodes a portion of one of the metalloproteases and are attempting to recover a full length version of it.Recently we have also isolated a form of the metalloproteinase from the venom ofan Arizonan scorpion C. sculpturatus which we are characterizing. Due to ban on export of scorpions fom Brazil the american species will allow cloning and expression to proceed in a more reasonable manner.

蛋白质/肽的明确鉴定对于蛋白质组的定义至关重要。使用Proteinchip阵列技术（SELDI），我们开发了实验方案和蛋白质鉴定蛋白质阵列所需的测试条件。我们能够通过特定的蛋白酶（例如胰蛋白酶，GLU C和ASP N）直接消化片段表面，并通过直接分析简单的激光解吸/电离量质谱仪，从而获得所研究样品的肽质量指纹。此外，可以通过使用ProteinChip界面使用碰撞四倍飞行时间（QQ-TOF）MS/MS在产生的胰蛋白酶肽上进行串联质谱法，从而允许在样品中精确鉴定亲本蛋白。此外，我们能够直接在蛋白质芯片表面上与羧肽酶y进行消化后的C末端序列，并在天然和变性条件下与Seldi-TOF质谱分析相连。利用芯片消化方法，我们研究了疾病和蛋白质生理形式之间可能诊断为疾病过程的结构差异。通过MS/MS分析，两种形式的蛋白质的主要结构可能是相同的。但是，翻译后修饰（例如变化的N连接糖基化模式）可能是有益的。通过偶联酶促消化（肽N-糖苷酶F和胰蛋白酶）和蛋白质奇普技术，这些改变的蛋白质形式是可识别的。此外，我们开发了研究蛋白质的病理学和生理形式与配体或受体的相互作用的方法。病理形式的生物学特征可能会因其结合特性而改变。利用塞尔迪，我们还检查了患者血浆的肽成分。为了充分表征这些分子，我们开发了一种微预编程的程序（采用液相色谱和塞尔迪），从而提供了这些肽的分离。肽的微链测序和MS/MS分析已经确定了几个分子，它们是较大蛋白质的片段，似乎与疾病与对照不同。结合了实验室的资源，我们清楚地证明了我们分析来自各种疾病状态的样本的能力，例如阿尔茨海默氏病和多发性硬化症。我们目前正在评估从CSF检测到的蛋白质/肽数量最大的方法。这些方法将用于检查患者以及其他生物液和组织的CSF，以鉴定可能在早期诊断中使用的生物标志物。 通过将SELDI分析和片上免疫测定相结合，我们希望清楚地识别与患者与对照不同的物种以及后翻译后改变的物种。 我们已经成功地识别和表征了一系列金属蛋白酶，这些家族对各种模型系统（最初是大鼠和小鼠）中的圈套复合物具有独特作用。蛋白酶能够根据特定蛋白酶的不同方式破坏复合物的许多成员。研究还针对复合体成员的重组形式，包括SNAP 25，SNAP 23和VAMP 2。我们的研究可能会提供有关复合物的部分如何确保形成复合物的蛋白质的稳定性和功能的见解。最初的工作已发表在《生物化学杂志》上，是与我们在东卡罗来纳大学合作者进行临时美国专利申请的基础。此外，我们还有一个cDNA克隆，它编码了其中一个金属蛋白酶的一部分，并正在尝试恢复其全长版本。我们还从我们正在表征的象征着我们正在表征的雕塑。由于禁止出口蝎子，美国物种将允许克隆和表达以更合理的方式进行。