In this study, we present a new isotope-coded carbamidomethylation (iCCM)-based quantitative proteomics, as acomplementary strategy for conventional isotope labeling strategies, with providing the simplicity, ease of use, and robustness. In iCCM-based quantification, two proteome samples can be separately isotope-labeled by means of covalently reaction of allcysteinyl residues in proteins with iodoacetamide (IAA) and its isotope (IAA-13C2, D2), denoted as CM and iCCM, respectively,leading to a mass shift of all cysteinyl residues to be + 4 Da. To evaluate iCCM-based isotope labeling in proteomic quantification,6 protein standards (i.e., bovine serum albumin, serotransferrin, lysozyme, beta-lactoglobulin, beta-galactosidase, andalpha-lactalbumin) isotopically labeled with IAA and its isotope, mixed equally, and followed by proteolytic digestion. Theresulting CM-/iCCM-labeled peptide mixtures were analyzed using a nLC-ESI-FT orbitrap-MS/MS. From our experimentalresults, we found that the efficiency of iCCM-based quantification is more superior to that of mTRAQ, as a conventional nonisobariclabeling method, in which both of a number of identified peptides from 6 protein standards and the less quantitative variationsin the relative abundance ratios of heavy-/light-labeled corresponding peptide pairs. Finally, we applied the developediCCM-based quantitative method to lung cancer serum proteome in order to evaluate the potential in biomarker discovery study
Microglia are the confined immune cells of the central nervous system (CNS). In response to injury or infection,microglia readily become activated and release proinflammatory mediators that are believed to contribute to microglia-mediatedneurodegeneration. In the present study, inflammation was induced in the immortalized murine microglial cell line BV-2 bylipopolysaccharide (LPS) treatment. We firstly performed phosphoproteomics analysis and phosphoinositide lipidomics analysiswith LPS activated microglia in order to compare phosphorylation patterns in active and inactive microglia and to detect the patternof changes in phosphoinositide regulation upon activation of microglia. Mass spectrometry analysis of the phosphoproteomeof the LPS treatment group compared to that of the untreated control group revealed a notable increase in the diversity ofcellular phosphorylation upon LPS treatment. Additionally, a lipidomics analysis detected significant increases in the amounts ofphosphoinositide species in the LPS treatment. This investigation could provide an insight for understanding molecular mechanismsunderlying microglia-mediated neurodegenerative diseases.
Three different dutasteride extraction methods were compared and a method based on liquid-liquid extraction (LLE)using methyl tert-butyl ether and methylene chloride was proved to be more effective than others for the extraction of dutasterideand finasteride, the internal standard (IS), from rat plasma. Additionally, a method composed of the LLE extraction, liquid chromatography,and multiple reaction monitoring (MRM) to target dutasteride and IS was validated by assessing specificity, linearity(r2 = 0.9993, 5 - 400 ng/mL), sensitivity (the limit of detection: 4.03 ng/mL; the limit of quantitation: 12.10 ng/mL), accuracy(intra-day: 89.4 - 105.9%; inter-day: 84.9 - 100.9%), precision (intra-day: 0.8 - 6.9%; inter-day: 2.9 - 15.9%), and recovery (84.7- 107.8%). Since the validated method was successfully applied to a pharmacokinetic study of dutasteride, it can be useful for thepharmacokinetic evaluation of newly developed dutasteride formulations.
Licoricidin isolated from Glycyrrhiza uralensis is known to have anticancer, anti-nephritic, anti-Helicobacter pylori,and antibacterial effects. In this study, a cocktail probe assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to investigate the modulating effect of licoricidin on cytochrome P450 (CYP) enzymes in human livermicrosomes. When licoricidin was incubated at 0-25 μM with CYP probes for 60 min at 37oC, it showed potent inhibitoryeffects on CYP2B6-catalyzed bupropion hydroxylation and CYP2C9-catalyzed diclofenac 4’-hydroxylation with half maximalinhibitory concentration (IC50) values of 3.4 and 4.0 μM, respectively. The inhibition mode of licoricidin was revealed as competitive,dose-dependent, and non-time-dependent, and following the pattern of Lineweaver-Burk plots. The inhibitory effect oflicoricidin has been confirmed in human recombinant cDNA-expressed CYP2B6 and 2C9 with IC50 values of 4.5 and 0.73 μM,respectively. In conclusion, this study has shown the potent inhibitory effect of licoricidin on CYP2B6 and CYP2C9 activitycould be important for predicting potential herb-drug interactions with substrates that mainly undergo CYP2B- and CYP2C9-mediated metabolism.
Lincomycin is one of the major species among the Pharmaceuticals and Personal Care Products (PPCPs) detectedfrom the four major rivers in Korea. The structure characterization was performed of six degradation products of lincomycinformed under the irradiation of electron beam, and the degradation efficiency as a function of the various irradiation dose andsample concentration was investigated. Electron beam (10 MeV, 0.5 mA and 5 kW) experiments for the structural characterizationof degradation products that are fortified with lincomycin, were performed at the dose of 10 kGy. The separation of degradationproducts and lincomycin was carried out using a C18 column (2.1×100 mm, 3.5 μm), using gradient elution with 20 mMammonium acetate and acetonitrile. The structures of six degradation products of lincomycin were proposed by interpretation ofmass spectra and chromatograms by LC-MS/MS. The mass fragmentation pathways of mass spectra in tandem mass spectrometrywere also proposed. Experiments were performed of the degradation efficiency as a function of the irradiation dose intensityand the initial concentration of lincomycin in an aqueous environment. In addition, increased degradation efficiency wasobserved with a higher dose of electron beam and lower concentration.