BETABELLINS 15D & 16D, DE NOVO DESIGNED BETA SANDWICH PROTEINS

贝塔贝林 15D

• 批准号: 6478950
• 负责人: DANIEL A KIRSCHNER
• 金额: $ 5.36万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6478950
• 关键词: Mammalia; biological products; biomaterials; biomedical resource; carbohydrates; human tissue; minerals; nervous system; proteins; spectrometry; technology /technique

We have established a mass spectrometric differential peptide display method to detect changes of peptides directly in tissue homogenates, after application of a defined physiological stimulus. We employed matrix-assisted laser desorption mass spectrometry (MALDI-TOF MS), using a single reference peptide in combination with careful scanning of the whole crystal rim of the matrix/analyte preparation, to determine in a semi-quantitative way the changes in the levels of peptides in an unfractionated methanol extract of the rat neurointermediate lobe (NIIL) after salt-loading of the animals. In the salt-loaded rat, a considerable decrease in the intensities of 6 molecular species of the NIIL extract was observed as compared to the control situation. These molecular ions corresponded to the masses of vasopressin, oxytocin, the neurophysins, and an unidentified molecule with a protonated mass of 5930 Da. Purification of this unidentified molecule, followed by Edman degradation, yielded the amino acid sequence of the carboxyl terminal glycopeptide of the vasopressin precursor. Using tandem MS, the major carbohydrate on the peptide was determined to consist of HeX3HexNAc5Fuc. To illustrate the potential of an MS-based differential display strategy, vasopressin and oxytocin were structurally characterized by direct tandem MS analysis of the molecular ions in the unfractionated NEL extract. Moreover, by means of a combination of Edman degradation and NLkLDI-MS analysis of the enzymatic digest of purified peptides (i.e., the neurophysin derived from propressophysin as well as two truncated analogs of the neurophysin derived from prooxyphysin) were structurally identified. Finally, using direct NLkLDI mass analysis of n-~cropunches of the neural lobe and of single melanotrope cells, it was possible to assign subsets of peptides to various distinct anatomical compartments of the NI1L..

我们建立了质谱差异肽 直接检测组织中肽变化的显示方法 在施加规定的生理刺激后进行匀浆。 我们采用基质辅助激光解吸质谱法 (MALDI-TOF MS)，使用单一参考肽与 仔细扫描基质/分析物的整个晶体边缘 准备，以半定量的方式确定变化 未分级的甲醇提取物中肽的含量 动物加盐后的大鼠神经中间叶（NIIL）。 在高盐大鼠中，盐的强度显着降低。 与 NIIL 提取物相比，观察到 6 种分子种类 控制情况。 这些分子离子对应于 大量加压素、催产素、神经素和一种身份不明的物质 质子化质量为 5930 Da 的分子。纯化此 不明分子，随后进行埃德曼降解，产生 羧基末端糖肽的氨基酸序列 加压素前体。 使用串联 MS，对主要碳水化合物进行分析 肽经确定由HeX3HexNAc5Fuc组成。 举例说明 基于 MS 的差异显示策略的潜力， 加压素和催产素的结构特征是直接 普通 NEL 中分子离子的串联 MS 分析 提炼。 此外，通过 Edman 退化和 对纯化肽的酶消化物进行 NLkLDI-MS 分析（即 神经素衍生自前素素以及两个截短的 衍生自原氧生理素的神经生理素的类似物）是 结构上已确定。 最后，使用直接 NLkLDI 质量分析 神经叶和单个黑素细胞的 n-~cropunches， 可以将肽的子集分配给各种不同的 NI1L 的解剖结构..