Dissecting the hydrogen exchange properties of insulin under amyloid fibril forming conditions: A site-specific investigation by mass spectrometry

We have examined the hydrogen exchange properties of bovine insulin under s olution conditions that cause it to aggregate and eventually form amyloid f ibrils. The results have been obtained at the residue-specific level using peptic digestion and mass spectrometry. A total of 19 peptides were assigne d to regions of the protein and their exchange properties monitored for a p eriod of 24 hours. The results of the peptic digestion show that residues A 13 to A21 and B11 to B30 are more susceptible to proteolysis than the N-ter minal regions of the protein. A total of 15 slowly exchanging amides were o bserved for insulin under these solution conditions. Location of the protec ted amides was carried out using a peptic-digestion protocol at low pH. Chr omatographic 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 assi gned to helix A2-A8. Taken together the results from digestion and hydrogen exchange show that at low pH and relatively high concentrations the C term ini of both chains are susceptible to proteolysis but that the solution str ucture contains the native state helices. More generally the results demons trate 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. (C) 2000 Aca demic Press.