Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquidchromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). Weused Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidationof the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid atthe 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot ofPrx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6),and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidizedPrx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from thebasic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes afterisoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological orartificial, when an oxidized cysteine residue(s) is identified.
Eighteen of the PDE-5 inhibitors and their analogues were analyzed using GC-EI-MS. Fourteen of them could beidentified by simple GC-MS method without derivatization, but hydroxyhongdenafil, hydroxyvardenafil, xanthoanthrafil andmirodenafil could not be identified without derivatization for the high polarity due to the presence of hydroxyl groups. N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), widely used trimethylsilyl (TMS) derivatizing reagents, were used to improve the sensitivity of the hydroxylated analogues. And the analytes could be identified by GC-MS after the derivatization.
We report observation of laser desorption/ionization (LDI) of peptides from flat surfaces of tungsten silicide (WSi2). In contrast to MALDI (matrix-assisted laser desorption/ionization) and SALDI (surface-assisted laser desorption/ionization)mass spectrometry, this study did not utilize any matrices and surface nanostructures. In this work, LDI on WSi2 surfaces is demonstratedto cover a mass range up to 1,600 Da (somatostatin; monoisotopic mass = 1637.9 Da). In addition, it exhibited a high sensitivity, which could detect peptides, which could detect peptides of low femtomole levels (20 fmol for angiotensin II). The observed LDI process was discussed to be largely thermal, more specifically, due to laser-induced surface heating that is mostlikely promoted by the low thermal diffusivity (κ) of WSi2 substrate.
5α-Dihydrotestosterone (DHT) is the primary active metabolite of testosterone, catalyzed by 5α-reductase (5αR) in the skin, prostate, and liver. In this study, the 5αR activity in rat liver S9 fraction in the presence of a NADPH-generating system was evaluated and compared by gas chromatography-mass spectrometry (GC-MS)-based in vitro assays. Testosterone and a 5αR inhibitor, finasteride, were added to the S9 fractions and incubated at 37oC for 1 h. Both testosterone and DHT were quantitatively measured and compared with two different GC-MS-based steroid profiling techniques. DHT was not detected by conventional GC-MS analysis in the absence of finasteride when the concentration of testosterone in the S9 fraction was less than 0.2 μM, whereas the isotope-dilution GC-MS (GC-IDMS) system was able to evaluate the 5αR activity. Because the S9 fraction contains more reactive enzymes and is easier to collect from tissues compared with a microsomal solution, the combination of the S9 fraction and GC-IDMS technique may be a promising assay for evaluating the 5αR activity in large-scale clinical studies.
Paint Spray is developed as a direct sampling ionisation method for mass spectrometric analysis of additives in polymer-based surface coatings. The technique simply involves applying an external high voltage (5 kV) to the wetted sample placedin front of the mass spectrometer inlet and represents a much simpler ionisation technique compared to those currently available. The capabilities of Paint Spray are demonstrated herein with the detection of four commercially available hindered amine lightstabilisers; TINUVIN® 770, TINUVIN® 292, TINUVIN® 123 and TINUVIN® 152 directly from thermoset polyester-based coilcoatings. Paint Spray requires no sample preparation or pre-treatment and combined with its simplicity – requiring no specialisedequipment – makes it ideal for use by non-specialists. The application of Paint Spray for industrial use has significant potentialas sample collection from a coil coating production line and Paint Spray ionisation could enable fast quality controlscreening at high sensitivity.