A metabolomics study was conducted to identify urinary biomarkers for breast cancer, using gas chromatographymassspectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), analyzed by principal components analysis(PCA) as well as a partial least squares-discriminant analysis (PLS-DA) for a metabolic pattern analysis. To find potentialbiomarkers, urine samples were collected from before- and after-mastectomy of breast cancer patients and healthy controls. Androgens, corticoids, estrogens, nucleosides, and polyols were quantitatively measured and urinary metabolic profiles wereconstructed through PCA and PLS-DA. The possible biomarkers were discriminated from quantified targeted metabolites with ametabolic pattern analysis and subsequent screening. We identified two biomarkers for breast cancer in urine, β-cortol and 5-methyl-2-deoxycytidine, which were categorized at significant levels in a student t-test (p-value < 0.05). The concentrations ofthese metabolites in breast cancer patients significantly increased relative to those of controls and patients after mastectomy. Biomarkers identified in this study were highly related to metabolites causing oxidative DNA damage in the endogenous metabolism. These biomarkers are not only useful for diagnostics and patient stratification but can be mapped on a biochemical chartto identify the corresponding enzyme for target identification via metabolomics.
The Cu-binding site in the [Cu·dCMP·dCMP-H]1− complex was investigated. The tandem mass (MS/MS) spectra ofthe [Cu·dCMP·dCMP-H]1− parent ion showed [dCMP·Cu·H2PO4 + CONH]1− fragment ions. Therefore, we propose that the Cucation is simultaneously coordinated to the phosphate site and cytosine moiety in the stable geometry of the [Cu·dCMP·dCMPH]1− complex. Three geometries for the complex were considered in an attempt to optimize the structure of the[Cu·dCMP·dCMP-H]1− complex. The ab initio calculations were performed at the B3LYP/6-311G** level.
In the present study, we report that the charge-directed (assisted) peptide dissociation products, such as b- and y-type peptidebackbone fragments, were the major products in MS/MS and MS3 applications of some o-TEMPO-Bz-C(O)-peptide ions, whileradical-driven dissociation products, such as a/x and c/z-type fragments, were previously shown to be the major products in the freeradical initiated peptide sequencing mass spectrometry (FRIPS MS). Those o-TEMPO-Bz-C(O)-peptides share a common feature intheir sequences, that is, the peptides do not include an arginine residue that has the highest proton affinity among free amino acids. The appearance of b- and y-type fragments as major products in FRIPS MS can be understood in terms of the so-called “mobile-protonmodel”. When the proton is highly mobilized by the absence of arginine, the chare-directed peptide dissociation pathways appearto be more competitive than the radical-driven dissociation pathways, in our FRIPS experiments.
A gas chromatography/ mass spectrometric (GC/MS) method was developed as a candidate reference method for theaccurate determination of essential fatty acids (linoleic acid, α- and γ-linolenic acids) in food supplemental oil products. Sampleswere spiked with three internal standards (stearic acid-d35, 13C18-linoleic acid, and 13C18-α-linolenic acid). Samples were thensubject to saponification, derivatization for methylation, and extraction by organic solvent. For GC/MS measurement, an AgilentHP-88 column, designed for the separation of fatty acid methyl esters, was selected after comparing with other columns as it providedbetter separation for target analytes. Target analytes and internal standards were detected by selected ion monitoring ofmolecular ions of their methyl ester forms. The GC/MS method was applied for the measurement of three botanical oils in NISTSRM 3274 (borage oil, evening primrose oil, and flax oil), and measurement results agreed with the certified values. Measurementresults for target analytes which have corresponding isotope-labeled analogues as internal standard were calculated basedon isotope dilution mass spectrometry (IDMS) approach, and compared with results calculated by using the other two internalstandards. Results from the IDMS approach and the typical internal standard approach were in good agreement within their measurementuncertainties. It proves that the developed GC/MS method can provide similar metrological quality with IDMS methodsfor the measurement of fatty acids in natural oil samples if a proper fatty acid is used as an internal standard.
Various types of alkaloids observed in several herbal medicines were analyzed by electrospray ionization tandemmass spectrometry in positive ion mode. In the present study, MS/MS spectralpatterns were investigated for eight-types of alkaloids(aporpine, protoberberine, tetrahydroprotoberberine, benzylisoquinoline, protopine, phthalide, morpine, and bisbenzylisoquinoline). For aporpine- and protoberberine-type alkaloids, main fragmentations occurred at substituted groups on rigid ringstructures, not showing ring fusion. Interesting fragmentations due to iminolization and retro-Diels-Alder (RDA) reaction wereobserved in MS/MS spectra of protopine- and tetrahydroprotobereberine-type alkaloids. Also, several types of fragmentationssuch as inductive cleavage and α-cleavage, or bond cleavage between two ring structures were observed depending on theirstructural characteristics. These fragmentation patterns are expected to allow instant classification of the specific alkaloid type invarious MS/MS spectra of alkaloids.
A new interface for coupling CIEF and MS using a four-way cross has been developed in a single mechanical system. This new interface could be operated without the electric discontinuity and reinstallation of lines. Additionally, a bare fused silicacapillary was facilitated as a spray needle to produce electrospray and to guide catholyte or sheath liquid. Focusing for CIEFwas completed in a hanging droplet at the end of spray needle. This capillary spray needle also provided stable spray, enhancedthe ionization efficiency and increased sensitivity. Results with carbonic anhydrase I showed that focusing and spraying werewell completed with the new interface and the new spray needle.
Isotope amount ratios of lead in a bronze sample have been successfully determined using multicollector inductivelycoupled plasma mass spectrometry (MC-ICP-MS). Matrix separation conditions were tested and optimized using ion exchangechromatography with anion-exchange resin, AG1-X8, and sequential elution of the 0.5 M HBr and 7 M HNO3 to separate leadfrom very high contents of copper and tin in bronze matrix. Mercury was also removed efficiently in the optimized separationcondition. The instrumental isotope fractionation of lead in the MC-ICP-MS measurement was corrected by the external standardsample bracketing method using an external standard, NIST SRM 981 lead common isotope ratio standard followed by correctionof procedure blank to obtain reliable isotope ratios of lead. The isotope ratios, 206Pb/204Pb, 207Pb/204Pb, 208Pb/204Pb, and208Pb/206Pb, of lead were determined as 18.0802 ± 0.0114, 15.5799 ± 0.0099, 38.0853 ± 0.0241, and 2.1065 ± 0.0004, respectively,and the determined isotope ratios showed good agreement with the reference values of an international comparison for thesame sample within the stated uncertainties.
We performed computer simulations to improve transmission efficiencies of a dual ion funnel system implemented onan FT-ICR MS. We found that the low m/z range from 50 to 150 could be significantly improved by operating the two ion funnelsat different RF amplitudes. These new operational conditions could be applied to analyze metabolome samples, whichrequire high sensitivity in the m/z range from 50 to 1,000.