The exponentially modified Gaussian (EMG) peak shape is widely used for peak approximation in chromatography. We constructed the EMG peak deconvolution routine for chromatography using a combination of two EMG formulas and linear optimization methods. A convenient way to determine the position of the EMG peak top was found. The routine accounts for the maximum linear range of the detector and can work with out-of-range peaks, where the detector saturation cuts off the top of the peak.The optimization routine is applied to the reconstruction of out-of-range peaks using correctly measured points, so that an analyst can get an idea of the height, area and concentration of such peaks as well as the prediction error in similar cases. Peak reconstruction helps in reducing the number of chromatographic runs during method development and routine work. The possibility of reconstructing out-of-range peaks using the pre-defined peak shape obtained while calibrating is also discussed.
Chronic imbalance between production and degradation of the human amyloid-beta peptide (Abeta) is assumed to play an important role in pathogenesis of Alzheimer's disease (AD). Post-translational modifications of Abeta could influence its interactions with specifically cleaving proteases and, therefore, perturb the Abeta homeostasis. The angiotensin-converting enzyme (ACE) was previously shown to degrade non-modified Abeta in vitro and in cells. In the presented work, we investigated the effect of isomerization of Asp-7, a common non-enzymatic age-related modification found in AD-associated Abeta species, on hydrolysis of Abeta by ACE. Two synthetic peptides corresponding to the Abeta region 1-16 with either Asp or isoAsp residues in position 7 were examined as monomeric soluble substrates for the N- as well as for the C-domain of ACE. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with the (18)O-labeled internal standard approach has allowed us to show that (i) the N-domain of ACE (N-ACE), but not the C-domain, selectively cleaves the Arg-5-His-6 bond in both peptides, and that (ii) N-ACE hydrolyzes the isoAsp-7 analogue more efficiently than the non-modified one. Our results demonstrate a new endopeptidase activity of N-ACE as well as high preference of the domain to recognize and hydrolyze the isomerized Abeta species that were earlier suggested to promote AD pathogenesis. The results suggest the need for further analysis of biological effects of isomerized Abeta and its interaction with ACE in AD pathogenesis.
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