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

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.