Application of Differential Isotopic Mass Splitting (DiMaS),a New Mass Spectrometry Methodology for the Analysis of Truncated Peptides,to Amyloid Precursor Protein Cleavage by Human Bleomycin Hydrolase and Neprilysin

A simple method for the analysis of peptides based on mass spectrometry has been applied to the study of interactions between human bleomycin hydrolase (hBH) or neprilysin (NEP; EC 3.4.24.11) and peptides Ac-HHQKLVFFAG-NH₂, Ac-SEVNLDAEFG-NH² and Ac-GGVVIATVIG-NH², three substrates cleaved by α, β and γ-secretase, respectively. These proteases are involved in the catabolism of amyloid protein precursor (APP), a protein implicated in Alzheimer's disease. The results indicate that hBH does not cleave the three substrates tested. Conversely, NEP cleaves Ac-HHQKLVFFAG-NH² at multiple sites and Ac-SEVNLDAEFG-NH² at only one position, whereas no cleavage was observed with Ac-GGVVIATVIG-NH². The fragments generated by cleavage with NEP were clearly identified without any further analysis by tandem mass spectrometry. The implication of the role of NEP in the Alzheimer detoxification process is discussed. This technique provides useful information on peptide cleavage points and could be applied easily to a mixture of peptides for the determination of amino acid sequences preferred by any protease.