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

我们建立了一种质谱学的差异肽 一种直接检测组织内多肽变化的显示方法 在施加特定的生理刺激后，进行匀浆。 我们采用了基质辅助激光解吸质谱 (MALDI-TOF MS)，使用单一参考肽与 仔细扫描基质/分析物的整个晶缘 准备，以半定量的方式确定在 红豆杉未经分离的甲醇提取物中的多肽含量 盐负荷后大鼠神经中间叶(NIIL)的变化。 在盐负荷的大鼠，脑电活动强度显著降低。 观察到6种NIIL提取物的分子种类，与 控制局面。这些分子离子对应于 大量的加压素，催产素，神经生理素，和一种不明身份的 质子化质量为5930Da。对这个进行提纯 未知的分子，然后是埃德曼的降解，产生了 猪瘟病毒羧基末端糖肽的氨基酸序列 加压素前体。利用串联质谱仪，主要的碳水化合物 多肽由HeX3HexNAc5Fuc组成。要举例说明 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 提取。此外，通过结合埃德曼降解和 NLkLDI-MS分析纯化多肽的酶消化(即， 从前列腺素衍生的神经物理素以及两个截断的 从原物理素导出的神经物理素有类似物 在结构上确定了。最后，使用直接NLkLDI质量分析 神经叶和单个黑素细胞的n-~钩刺， 可以将多肽的子集分配给各种不同的 NI1L的解剖间隔。

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..