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

我们建立了一个质谱差异肽 直接检测组织中肽变化的展示方法 匀浆，在施加限定的生理刺激后。 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 制备，以半定量的方式测定 在未分级甲醇提取物中肽的水平 大鼠神经中间叶（NIIL）。 在盐负荷大鼠中， 观察到NIIL提取物的6种分子种类， 控制的情况。 这些分子离子对应于 大量的后叶加压素催产素神经垂体素和一种 该分子的质子化质量为5930 Da。 未经鉴定的分子，然后进行Edman降解，得到了 的羧基末端糖肽的氨基酸序列， 加压素前体 使用串联质谱， 确定肽由HeX 3 HexNAc 5 Fuc组成。 为了说明 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的， 未分级NEL中分子离子的串联MS分析 提取物 此外，通过Edman降解和 纯化肽的酶消化的NLkLDI-MS分析（即， 源自前列腺素的neurophysin以及两个截短的 衍生自prooxyphysin的neurophysin类似物）， 结构识别。 最后，使用直接NLkLDI质量分析 神经叶和单个黑素细胞的n-cropunks， 可以将肽的子集分配给各种不同的 NI 1 L的解剖隔室。

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..