title: Dissecting the hydrogen exchange properties of insulin under amyloid fibril forming conditions: a site-specific investigation by mass spectrometry.
abstract: We have examined the hydrogen exchange properties of bovine insulin under solution conditions that cause it to aggregate and eventually form amyloid fibrils. The results have been obtained at the residue-specific level using peptic digestion and mass spectrometry. A total of 19 peptides were assigned to regions of the protein and their exchange properties monitored for a period of 24 hours. The results of the peptic digestion show that residues A13 to A21 and B11 to B30 are more susceptible to proteolysis than the N-terminal regions of the protein. A total of 15 slowly exchanging amides were observed for insulin under these solution conditions. Location of the protected amides was carried out using a peptic-digestion protocol at low pH. Chromatographic separation was not required. This enabled a direct comparison of the peptides within the same mass spectrum. From kinetic analysis of the rates slow exchange has been located to 4(+/-1) backbone amides in the A13-A19 helix and 6(+/-1) in the B chain helix. The remaining 5(+/-1) are assigned to helix A2-A8. Taken together the results from digestion and hydrogen exchange show that at low pH and relatively high concentrations the C termini of both chains are susceptible to proteolysis but that the solution structure contains the native state helices. More generally the results demonstrate that mass spectrometry can be applied to study site-specific hydrogen exchange properties of proteins even under conditions where they are known to be partially folded and aggregate extensively in solution.
authors.affiliation: Oxford Centre for Molecular Sciences, New Chemistry Laboratory, South Parks Road, Oxford, OX1 3QT, UK.
authors.forename: P
authors.initials: P
authors.lastname: Tito
authors.forename: E J
authors.initials: EJ
authors.lastname: Nettleton
authors.forename: C V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003903:Deuterium / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D058225:Plaque, Amyloid / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D023302:Protein Footprinting; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D007328:Insulin; D010446:Peptide Fragments; D012996:Solutions; D006859:Hydrogen; D003903:Deuterium; D010434:Pepsin A