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

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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..