AP/MALDI‐MS complete characterization of the proteolytic fragments produced by the interaction of insulin degrading enzyme with bovine insulin

Abstract

The prominent role that insulin degrading enzyme (IDE) has in the clearance of insulin as well as of other molecules such as amyloid-beta has recently drawn much interest in the scientific community toward this protease. In order to give an insight into the manner of interaction of IDE with its substrates, several papers have focused on the structure of the IDE/insulin complex. In this scenario, although the cleavage sites involved in the interaction of insulin with IDE are known, a convenient experimental method that is able to identify in a complete and unambiguous way, all the peptide fragments generated by such interaction has yet to be found. MS-based experiments have often represented to be invaluable tools for the assessment of the cleavage sites, but the reported MS-spectra always show a partial coverage of all the peptide fragments generated by the enzyme interaction, lacking a complete characterization. In this work, we report a new experimental procedure by which an unambiguous as well as complete assignment of all the peptide fragments generated by the interaction of insulin with IDE is described. Atmospheric pressure/matrix-assisted laser desorption ionization (AP/MALDI) mass spectra are reported and the data recorded, together with the introduction of a reduction/alkylation step, allows us to fully characterize the cleavage sites of the bovine insulin interacting with IDE. Different experimental conditions are screened and some insights into the IDE/insulin system regarding preference of the cleavage and its dependence on particular experimental conditions used are also given. Investigation on the tendency that different insulin fragments have toward aggregation is also carried out. Good reproducibility, global and unambiguous assignment, low time-consuming experimental procedure, and requirements of enzyme in small amounts are some of the advantages of the proposed AP/MALDI based approach.

Metal ions affect insulin-degrading enzyme activity

“…4. The principal fragments (see Table 1) are B20-25 (m/z: 754.3) and B26-30 (m/z: 725.4), indicating that two of the principal insulin cleavage sites (namely B (25)(26) and B (24)(25)) are specific cleavage sites for the interaction between IDE and insulin [13]. Other less specific peptide fragments also appear in the spectrum for a very long incubation time (Fig.…”

“…IDE, His-Tag, rat, recombinant, from Spodoptera frugiperda was purchased from Calbiochem and its activity was verified by carrying out enzymatic digestion of insulin solutions according to the experimental procedure previously reported [13]. In order to improve the reproducibility of the enzymatic assays carried out in the presence of metal ions, we have chosen to use the recombinant enzyme of a same commercial lot to minimize the differences on the absolute proteolytic activity of IDE found in the different commercial lots purchased (see Results and discussion section).…”

“…In order to improve the reproducibility of the enzymatic assays carried out in the presence of metal ions, we have chosen to use the recombinant enzyme of a same commercial lot to minimize the differences on the absolute proteolytic activity of IDE found in the different commercial lots purchased (see Results and discussion section). It is also important to highlight that, although the recombinant enzyme used has been already tested as a good model for human IDE [13], the latter could, in principle, behave differently in the presence of metal ions. However, we have assumed that the His-Tag of the recombinant enzyme should not alter the IDE capability to bind metal ions because the concentration of the latter in the enzymatic assays are always at least about three orders of magnitude higher than the IDE concentration.…”

“…It is also interesting to outline that if we compare these parameters with those obtained for the amyloid peptides Aβ [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] and Aβ [16][17][18][19][20][21][22][23][24][25][26][27][28] several similarities and some differences emerge [54]. In particular, the overall proteolytic activity toward the main cleavage sites (i.e., Phe 24 [16][17][18][19][20][21][22][23][24][25][26][27][28] ) is somewhat higher in the case of the B20-30 peptide due to a slightly more efficient cleavage rate-limiting step (see Table 2), whereas the substrate affinity of B20-30 appears intermediate between that of Aβ [1][2][3]…”

“…Since the discovery of insulin and Aβ cleavage by IDE [11], much effort has been put in trying to understand some key questions regarding cleavage sites [12,13], kinetics of substrate interaction [14,15] and the mechanism of IDE modulation [16,17].…”