Neuropsychiatric Disorders--protein Structure/activity Studies

蛋白质/多肽的明确鉴定对蛋白质组的定义至关重要。利用ProteinChip阵列技术（SELDI），我们开发了实验方案并探索了在ProteinChip阵列上进行蛋白质鉴定所需的测试条件。我们能够通过特定的蛋白酶（如胰蛋白酶、Glu C和Asp N）直接消化芯片表面上的蛋白质/肽，并通过简单的激光解吸/电离质谱仪直接分析获得被研究样品的肽质量指纹图谱。此外，通过ProteinChip接口，采用碰撞四极杆飞行时间（Qq-TOF） MS/MS，可以对所得的色氨酸进行串联质谱分析，从而精确鉴定样品中的亲本蛋白。此外，我们能够直接在ProteinChip表面上用羧基肽酶Y消化后，结合SELDI-TOF质谱分析，在天然和变性条件下鉴定肽的c端序列。利用芯片消化方法，我们检查了疾病和可能诊断疾病过程的蛋白质的生理形式之间的结构差异。通过MS/MS分析，两种形式的蛋白质的初级结构可能是相同的；然而，翻译后的修饰，如改变的n链糖基化模式，可能被证明是有用的。通过耦合酶切（包括肽n -糖苷酶F和胰蛋白酶）和ProteinChip技术，这些改变的蛋白质形式是可识别的。此外，我们已经开发了研究蛋白质与它们的配体或受体的病理和生理形式的相互作用的方法。病理形式的生物学特性可能因其结合特性而改变。利用SELDI，我们还检测了患者血浆中的肽成分。为了充分表征这些分子，我们开发了一种微制备程序（采用液相色谱和SELDI），提供了这些肽的分离。对这些多肽进行微测序和质谱/质谱分析，已经确定了几个分子，这些分子是较大蛋白质的片段，似乎与疾病和对照组不同。结合实验室的资源，我们已经清楚地展示了我们分析各种疾病状态样本的能力，如阿尔茨海默病和多发性硬化症。我们目前正在评估从脑脊液中检测到的蛋白/肽数量最大化的方法。这些方法将用于检查患者的脑脊液以及其他生物液体和组织，以确定可能用于早期诊断的生物标志物。通过结合SELDI分析和芯片上免疫分析，我们希望清楚地识别出不同于患者和对照组的物种，以及那些翻译后改变的物种。

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.