In the present study, collisionally-activated dissociation (CAD) experiments were performed under low energy collisionconditions in six peptides containing a disulfide bond. Fragments produced as a result of the cleavage of a disulfide bondwere obtained after CAD in four peptides (bactenecin, TGF-α, cortistantin, and linearly linked peptide, Scheme 1) with basicamino acid residues. In contrast, the CAD analysis of two peptides with no basic residue (oxytocin and tocinoic acid) rarely producedfragments indicative of cleavage of a disulfide bond. These results are consistent with the mobile proton model suggestedby the McLuckey and O’Hair groups (ref. 22 and 23); nonmobile protons sequestered at basic amino acid residues appear to promotethe cleavage of disulfide bonds.
Trimethylboroxine (TMB) is a six-membered ring compound containing Lewis acidic boron and Lewis basic oxygenatoms that can bind halide anion and alkali metal cation, respectively. We employed Fourier transform ion cyclotron resonancespectroscopy to study the gas-phase binding of LiBrLi+ and F−(KF)2 to TMB. TMB forms association complexes with bothLiBrLi+ and F–(KF)2 at room temperature, providing direct evidence for the ditopic binding. Interestingly, the TMB·F−(KF)2anion complex is formed 33 times faster than the TMB·Li+BrLi cation complex. To gain insight into the ditopic binding of an ionpair, we examined the structures and energetics of TMB·Li+, TMB·F–, TMB·LiF (the contact ion pair), and Li+·TMB·F– (the separatedion pair) using Hartree–Fock and density functional theory. Theory suggests that F– binds more strongly to TMB than Li+and the contact ion-pair binding (TMB·LiF) is more stable than the separated ion-pair binding (Li+·TMB·F–).
The active components in a plant extract can be represented as mass profiles. We introduce here a new, multi-compounddiscovery method known as Scaling of Correlations between Activity and Mass Profiles (SCAMP). In this method, a correlationcoefficient is used to quantify similarities between the extract activity and mass profiles. The method was evaluated byfirst measuring the anti-oxidation activity of eleven fractions of an Astragali Radix extract using DPPH assays. Next, 15 T Fouriertransformion cyclotron resonance (FT-ICR) MS was employed to generate mass profiles of the eleven fractions. A comparison ofcorrelation coefficients indicated two compounds at m/z 285.076 and 286.076 that were strong antioxidants. Principal componentanalyses of these profiles yielded the same result. FT-ICR MS, which offers a mass resolving power of 500,000, was used todiscern isotopic fine structures and indicated that the molecular formula corresponding to the peak at m/z 285.076 was C16H13O5. SCAMP in combination with high-resolution MS can be applied to any type of mixture to study pharmacological activity and is apowerful tool for active compound discovery in plant extract studies.
As background significantly affects measurement accuracy and a detection limit in determination of the trace amountsof uranium, it is necessary to minimize the impurities in the filaments used for thermal ionization mass spectrometry (TIMS). Wehave varied the degassing condition such as the heating currents and duration times to reduce the backgrounds from the filamentsprepared with zone-refined rhenium tape. The most efficient degassing condition of the heating current and the duration time wasdetermined as 3.5 A and 60 min, respectively. The TIMS measurement combined with the isotope dilution mass spectrometry(IDMS) technique showed that the uranium backgrounds were determined to be in a few fg level from blank rhenium filaments. The background minimized filaments were utilized to measure the uranium isotope ratios of a U030 (NIST) standard sample. The excellent agreement of the measurement with the certified isotope ratios showed that the degassing procedure optimized inthis study efficiently reduced the impurity signals of uranium from blank rhenium filaments to a negligible level.
The development of clinical biomarkers involves discovery, verification, and validation. Recently, multiple reactionmonitoring (MRM) coupled with stable isotope dilution mass spectrometry (IDMS) has shown considerable promise for the directquantification of proteins in clinical samples. In particular, multiple biomarkers have been tracked in a single experiment usingMRM-based MS approaches combined with liquid chromatography. We report here a highly reproducible, quantitative, anddynamic MRM system for validating multi-biomarker proteins using Nanoflow HPLC-Microfluidics Chip/Triple-QuadrupoleMS. In this system, transitions were acquired only during the retention window of each eluting peptide. Transitions with the highestMRM-MS intensities for the five target peptides from colon cancer biomarker candidates were automatically selected usingOptimizer software. Relative to the corresponding non-dynamic system, the dynamic MRM provided significantly improvedcoefficients of variation in experiments with large numbers of transitions. Linear responses were obtained with concentrationsranging from fmol to pmol for five target peptides.
The high-throughput identification and accurate quantification of proteins are essential strategies for exploring cellularfunctions and processes in quantitative proteomics. Stable isotope tagging is a key technique in quantitative proteomicresearch, accompanied by automated tandem mass spectrometry. For the differential proteome analysis of mouse neuronal celllines, we used a multiplexed isobaric tagging method, in which a four-plex set of amine-reactive isobaric tags are available forpeptide derivatization. Using the four-plex set of isobaric tag for relative and absolute quantitation (iTRAQ) reagents, we analyzedthe differential proteome in several stroke time pathways (0, 4, and 8 h) after the mouse neuronal cells have been stressed usinga glutamate oxidant. In order to obtain a list of the differentially expressed proteins, we selected those proteins which had apparentlychanged significantly during the stress test. With 95% of the peptides showing only a small variation in quantity before andafter the test, we obtained a list of eight up-regulated and four down-regulated proteins for the stroke time pathways. To validatethe iTRAQ approach, we studied the use of oxidant stresses for mouse neuronal cell samples that have shown differential proteome inseveral stroke time pathways (0, 4, and 8 h). Results suggest that histone H1 might be the key protein in the oxidative injurycaused by glutamate-induced cytotoxicity in HT22 cells.
Cross reacting antibodies can cause an overestimation of the results of immunoassays. Therefore, alternative methodsare needed for the accurate quantification of steroids. Gas chromatography combined with isotope-dilution mass spectrometry(GC-IDMS) is developed to quantify urinary active androgens, testosterone, epitestosterone and dihydrotestosterone, which areclinically relevant androgens to both hair-loss and prostate diseases. The method devised involves enzymatic hydrolysis with β-glucuronidase, solid-phase extraction, liquid-liquid extraction using methyl tert-butyl ether and subsequent conversion to pentafluorophenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive and selective analysis in selected-ionmonitoring mode. Flophemesyl-TMS derivatization not only eliminates matrix interference but also has a good peak resolutionwithin a 6 min-run. A selective and sensitive GC technique with flophemesyl-TMS derivatives also allows accurate quantitativeanalysis of three active androgens when combined with IDMS. The limit of quantification of the three analytes was <50 pg/mL,and extraction recoveries ranged from 91.9 to 102.1%. The precision and accuracy were 1.2~6.5% and 89.0~106.7%, respectively. This GC-IDMS method can be useful for evaluating the drug efficacy and monitoring the biological processes responsiblefor male-pattern baldness and prostate diseases.
Wood charcoal was investigated to determine its potential as an alternative matrix for matrix-assisted laser desorption/ionization of various samples. Wood charcoal was an effective matrix for analyzing glucose, sucrose, arginine, and polyethyleneglycols (PEGs), with detection levels of 100 pmol for glucose, 1 nmol for sucrose, 100 pmol for arginine, 100 pmol for PEG 400,1 pmol for PEG 1540, and 10 pmol for PEG 3350. No analyte signal was observed for peptides or proteins.