| Knowledge of the 3D structures of polypeptides is significant for understanding their functions. In the present research hydrogen/deuterium exchange (HDX) in conjunction with mass spectrometry (MS) was used to discover the solution phase conformation of the C-terminal tail of occludin (C-occludin), tachykinins (eledoisin, neurokinin A, neurokinin A4-10) and the related peptide (beta-amyloid25-35). In addition to these studies, the proteins involved in lipid trafficking from endoplasmic reticulum to Golgi complex were also characterized using MS-based proteomics.; To probe the 3D structure of C-occludin, time-resolved HDX experiments were performed on intact protein and analyzed by MALDI-TOF mass spectrometry to obtain kinetic data. In order to study the site-specific deuterium incorporation, a protein fragmentation/MS approach was used. The residue numbers 34-54, 58-76 and 69-85 were found to be involved in the formation of the secondary and tertiary structures, whereas fragments 1-10, 84-94, 95-105 and 145-150 were in extended conformations. These results were corroborated by a model of C-occludin generated by FUGUE software that predicts the 3D structure of the polypeptides based on a de novo approach.; For conformational analysis of the tachykinin and related peptides, HDX was performed in aqueous and membrane-like environment and monitored by ESI-MS and MS/MS. The HDX-MS studies in all of the four peptides indicated that they were mostly present in a random coil conformation in the aqueous solvent, except for neurokinin A which was found to contain some elements of secondary structure. In membrane-mimetic solvents, the ordered arrangement of the amino acid residues in a peptide increased. ESI MS/MS revealed that the N-terminal segment of the tachykinins peptides was involved in the secondary structure formation. However, this does not hold true for beta-amyloid25-35. Among the tachykinin pepides, more significant similarities were observed in the message domain than in the address domain. Again, these similarities were not observed with the beta-amyloid25-35.; In order to characterize the proteins that are involved in lipid trafficking, an in vitro model of PCTVs budding was generated using ER, Golgi complex and cytosolic proteins collected from rat's enterocytes. The cytosolic proteins were then fractionated using various chromatographic steps, viz., heparin, size-exclusion and ion-exchange chromatography. After each fractionation, individual fractions and their combinations were used in the above-mentioned in vitro model instead of the crude mixture of cytosolic proteins to identify the active fractions that contain proteins involved in the budding of PCTVs. The proteins present in these active fractions were further fractionated using RP-HPLC and characterized by mass spectrometry. A total of eight proteins have been identified that possess PCTVs budding activity. These are liver-fatty acid binding protein, cyclosporin A binding protein, acyl-Co A-binding protein, glutathione S-tranferase, ubiquitin, profilin I, lactococcin biosynthesis protein and translocation protein. |
