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Individual samples were injected in triplicate and the average angiotensin peptide concentrations and standard error are reported. All reagents used for LC-MS/MS analysis were of Mass Spectrometry Grade and all unlabeled angiotensin peptide standards and reagents were purchased from Sigma-Aldrich (St. Louis, MO). Table 1 Instrument parameters for multiple reaction monitoring (MRM) Statistical analysis All data are expressed as the mean?��?standard error of the mean (SEM) and were analyzed by Student's t-test for two-group comparisons or by ANOVA followed by Newman�CKeuls correction for multiple comparisons. Statistical analyses were performed using GraphPad Prism 5.0 (La Jolla, CA) statistical and graphical software. Differences Ficain were considered significant at P?Small molecule library screening system, AngII can be further metabolized to other angiotensin peptides which have been shown to contribute to cardiovascular function (Campagnole-Santos et?al. 1989; Santos et?al. 2000; Wilson 2005; Ruiz-Ortega et?al. 2007; Marc et?al. 2012; Gao et?al. 2014). For example, AngII can be cleaved by angiotensin-converting enzyme 2 (ACE2) to form Ang-1-7 or by aminopeptidase A to form Ang III, which is further Idelalisib metabolized by aminopeptidase N to form Ang IV (Fig.?(Fig.1).1). Although the primary objective of this study was to examine the stability of exogenous AngII in neuronal cell culture media, it was also important to determine if exogenous AngII is metabolized to these other angiotensin peptides. As shown in the representative chromatogram (Fig.?(Fig.2),2), we were able to successfully separate and detect all four angiotensin peptides from a mixture containing the commercially available peptides ranging in concentration from 12�C16?nmol/L. AngII is an octapeptide which under the conditions of the chromatography should accumulate in the +3 charged state due to the positive charges at the terminal amino group as well as the Arg and His side chains. Upon loss of the C-terminus Phe, rendering Ang-1-7, the peptide is much less hydrophobic and thus binds less strongly to the C-18 side chains of the HPLC column particles. Since Phe is more hydrophobic than Asp, removal of Asp instead of Phe from AngII makes the Ang III peptide less hydrophobic than AngII but more than Ang-1-7. Therefore, Ang III elutes between Ang-1-7 and AngII (Fig.?(Fig.2).2). The hexapeptide Ang IV is generated from the removal of the N-terminus positive side chain Arg from Ang III, while keeping the hydrophobic residues Ile and Phe.