Electrospray Ionization Quadrupole Mass Spectrometry Research Articles

Lipidomics reveal aryl hydrocarbon receptor (Ahr)-regulated lipid metabolic pathway in alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis

1. Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry (UPLC-ESI-QTOF MS)-based lipidomics was employed to elucidate new mechanism of alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis in mice.2. Multiple lipid components significantly increased in ANIT-induced intrahepatic cholestasis, including PC 16:0, 20:4, PC 16:0, 22:6, PC 16:0, 18:2, LPC 18:2, PC 18:2, LPC 18:1, PC 18:1, 14:0, SM 18:1, 16:0, oleoylcarnitine and palmitoylcarnitine. This alteration of lipid profile was induced by the changed expression of genes choline kinase (Chk) a, sphingomyelin phosphodiesterase (SMPD) and stearoyl-coenzyme A desaturase 1 (SCD1).3. Knockout of aryl hydrocarbon receptor (Ahr) in mice can significantly reverse ANIT-induced intrahepatic cholestasis, as indicated by lowered ALT, AST and ALP activity, and liver histology. Aryl hydrocarbon receptor knockout significantly reversed ANIT-induced lipid metabolism alteration through regulating the expression of Chka.4. In conclusion, this study demonstrated ANIT-induced lipid metabolism disruption might be the potential pathogenesis of ANIT-induced intrahepatic cholestasis in mice.

Role of the lipid-regulated NF-κB/IL-6/STAT3 axis in alpha-naphthyl isothiocyanate-induced liver injury

Alpha-naphthyl isothiocyanate (ANIT)-induced liver damage is regarded as a useful model to study drug-induced cholestatic hepatitis. Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry (UPLC-ESI-QTOF MS)-based metabolomics revealed clues to the mechanism of ANIT-induced liver injury, which facilitates the elucidation of drug-induced liver toxicity. 1-Stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 18:0) and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 18:1) were significantly increased in serum from ANIT-treated mice, and this increase resulted from altered expression of genes encoding the lipid metabolism enzymes Chka and Scd1. ANIT also increased NF-κB/IL-6/STAT3 signaling, and in vitro luciferase reporter gene assays revealed that LPC 18:0 and LPC 18:1 can activate NF-κB in a concentration-dependent manner. Activation of PPARα through feeding mice a Wy-14,643-containing diet (0.1%) reduced ANIT-induced liver injury, as indicated by lowered ALT and AST levels, and liver histology. In conclusion, the present study demonstrated a role for the lipid-regulated NF-κB/IL-6/STAT3 axis in ANIT-induced hepatotoxicity, and that PPARα may be a potential therapeutic target for the prevention of drug-induced cholestatic liver injury.

Identification of saponins from sugar beet (Beta vulgaris) by low and high-resolution HPLC-MS/MS.

We profiled triterpene saponins from the roots of sugar beet Beta vulgaris L. cultivars Huzar and Boryna using reversed-phase liquid chromatography combined with negative-ion electrospray ionization quadrupole mass spectrometry. We tentatively identified 26 triterpene saponins, including 17 that had not been detected previously in this plant species and 7 saponins that were tentatively identified as new compounds. All observed compounds were glycosides of five different aglycones, of which gypsogenin and norhederagenin are reported for the first time in sugar beet. Thirteen of the saponins detected in sugar beet roots were substituted with dioxolane-type (4 saponins) or acetal-type (9 saponins) dicarboxylic acids. Among the 26 detected saponins, we identified 2 groups of isomers distinguished using high-resolution mass measurements that were detected only in the Huzar cultivar of sugar beet.

A simple and rapid method to identify and quantitatively analyze triterpenoid saponins in Ardisia crenata using ultrafast liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry

Ardisia plant species have been used in traditional medicines, and their bioactive constituents of 13,28-epoxy triterpenoid saponins have excellent biological activities for new drug development. In this study, a fast and simple method based on ultrafast liquid chromatography coupled to electrospray ionization mass spectrometry (UFLC–MS) was developed to simultaneously identify and quantitatively analyze triterpenoid saponins in Ardisia crenata extracts. In total, 22 triterpenoid saponins, including two new compounds, were identified from A. crenata. The method exhibited good linearity, precision and recovery for the quantitative analysis of eight marker saponins. A relative quantitative method was also developed using one major saponin (ardisiacrispin B) as the standard to break through the choke-point of the lack of standards in phytochemical analysis. The method was successfully applied to quantitatively analyze saponins in commercially available plant samples. This study describes the first systematic analysis of 13,28-epoxy-oleanane-type triterpenoid saponins in the genus Ardisia using LC-ESI–MS. The results can provide the chemical support for further biological studies, phytochemotaxonomical studies and quality control of triterpenoid saponins in medicinal plants of the genus Ardisia.

Lipidomics Reveals a Link between CYP1B1 and SCD1 in Promoting Obesity

Cytochrome P450 1B1 (CYP1B1) is involved in the metabolism of xenobiotic compounds and endogenous metabolites. Disruption of Cyp1b1 in mice results in suppression of high-fat diet (HFD)-induced obesity and an extensive change in hepatic energy regulation despite minimal constitutive expression of CYP1B1 in hepatocytes. Lack of CYP1B1 is correlated with altered lipid metabolism, especially lysophosphatidylcholines, contributing to protection against obesity. Ultraperformance liquid chromatography coupled to electrospray ionization quadrupole mass spectrometry (UPLC-ESI-QTOFMS)-based metabolomics revealed lysophosphatidylcholine 18:0 (LPC 18:0) as a biomarker positively related to HFD-induced obesity. The increased serum LPC 18:0 in wild-type mice is reduced in Cyp1b1-null mice on a HFD, which is reversed in CYP1B1-humanized mice. CYP1B1-humanized mice show higher diet-induced obesity compared with Cyp1b1-null mice, suggesting that human CYP1B1 shows a similar response to HFD as mouse Cyp1b1. In addition, hepatic stearoyl-CoA desaturase 1 (SCD1) expression was decreased in Cyp1b1-null mice, and the attenuated diet-induced obesity and lower serum LPC 18:0 in the Cyp1b1-null mice is elevated after SCD1 overexpression, suggesting that SCD1 is correlated with CYP1B1-induced obesity. These studies establish a biochemical link between cytochromes P450, lipids, and metabolic disorders and suggest that inhibition of CYP1B1 could be target for antiobesity drugs.

Metabolomics Reveals That Tumor Xenografts Induce Liver Dysfunction

Metabolomics, based on ultraperformance liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry, was used to explore metabolic signatures of tumor growth in mice. Urine samples were collected from control mice and mice injected with squamous cell carcinoma (SCCVII) tumor cells. When tumors reached ∼2 cm, all mice were killed and blood and liver samples collected. The urine metabolites hexanoylglycine, nicotinamide 1-oxide, and 11β,20α-dihydroxy-3-oxopregn-4-en-21-oic acid were elevated in tumor-bearing mice, as was asymmetric dimethylarginine, a biomarker for oxidative stress. Interestingly, SCCVII tumor growth resulted in hepatomegaly, reduced albumin/globulin ratios, and elevated serum triglycerides, suggesting liver dysfunction. Alterations in liver metabolites between SCCVII-tumor-bearing and control mice confirmed the presence of liver injury. Hepatic mRNA analysis indicated that inflammatory cytokines, tumor necrosis factor α, and transforming growth factor β were enhanced in SCCVII-tumor-bearing mice, and the expression of cytochromes P450 was decreased in tumor-bearing mice. Further, genes involved in fatty acid oxidation were decreased, suggesting impaired fatty acid oxidation in SCCVII-tumor-bearing mice. Additionally, activated phospholipid metabolism and a disrupted tricarboxylic acid cycle were observed in SCCVII-tumor-bearing mice. These data suggest that tumor growth imposes a global inflammatory response that results in liver dysfunction and underscore the use of metabolomics to temporally examine these changes and potentially use metabolite changes to monitor tumor treatment response.

High‐expression EGFR/cell membrane chromatography‐online‐high‐performance liquid chromatography/mass spectrometry: rapid screening of EGFR antagonists from Semen Strychni

Traditional methods for screening active compounds from complex system such as traditional Chinese medicines are relatively cumbersome and time-consuming. In order to improve this situation, we established an online analytical method for screening, separation and identification EGFR antagonists from traditional Chinese medicines, which is described in this study. Cells with high EGFR expression levels were used to prepare the cell membrane stationary phase for the EGFR cell membrane chromatography model with the purpose of screening active compounds. Separation of the retention fractions was achieved by the high-performance liquid chromatography, and identification was conducted via electrospray ionization quadrupole mass spectrometry. The inhibitory effects of active compounds on EGFR cell growth were also demonstrated in vitro. The screening results showed that vauquline and strychnine from Semen Strychni could be active components acting on EGFR similarly to gefitinib as a control drug. Results from biological trials showed that vauquline and strychnine inhibited cell proliferation of HEK293/EGFR cells, and inhibited Erk phosphorylation, and can effectively reduce expression of downstream signaling molecules. This EGFR cell membrane chromatography-online-high-performance liquid chromatography/tandem mass spectrometry method can be applied for rapid screening, separation and identification of EGFR antagonists from traditional Chinese medicines and should be useful for drug discovery with natural medicinal herbs.

Determination of Carboxylic Acids from Plant Root Exudates by Ion Exclusion Chromatography with ESI-MS

Ion-exclusion chromatography (IEC) coupled with electrospray ionization quadrupole mass spectrometry (ESI-MS) has been used for the analysis of carboxylic acids. The use of ESI-MS provides increased specificity and sensitivity compared to existing detection methods. This paper applies IEC-ESI-MS to the analysis of carboxylic acids in commonly found root exudates and shows that the separation of nine carboxylic acids (pyruvic, oxalic, lactic, malonic, maleic, succinic, tartaric, aconitic and citric acids) can be achieved by IEC within 8 min. The ESI provided reasonable signals from negative ions, [M−H]− in the negative ionization mode. Linear plots of peak area versus concentration were obtained in the range 50–25,000 μg L−1 for MS detection under optimized MS conditions. The detection limits of target organic acids, based upon signal to noise ratio (S/N = 3), ranged from 10 to 30 μg L−1. The reproducibility of peak areas was <2.5% (n = 5). The proposed method was used for the confirmation and quantification of carboxylic acids in nutrient solutions containing root exudates.

Discrimination of recombinant and pituitary-derived bovine and porcine growth hormones by peptide mass mapping.

Somatotropins, which are used in cattle for growth and lactating performances, are difficult to reliably detect because no direct method exists. Reversed-phase high-performance liquid chromatography (RP-HLC) coupled to electrospray ionization quadrupole mass spectrometry (ESI/MS) has been developed to separate and characterize the N-terminal peptides resulting from tryptic cleavage of natural and recombinant growth hormones from different species (bovine, porcine, and human) and suppliers. Conditions for tryptic digestion were optimized. This technique was found to be optimal to cleave efficiently the N-terminal peptide of the proteins without releasing too much noise from the matrix. Characterization of the peptides through ESI(+)-MS allowed natural and recombinant growth hormones from bovine and porcine species with N-terminal amino acid sequences differing from one amino acid residue to be discriminated. However, the studied human growth hormones had similar primary sequences that did not permit any discrimination between recombinant and natural forms, thus confirming the known identity of these hormones. Protein digestions with pepsin and chymotrypsin were also compared but were not conclusive due to the too small N-terminal peptides released after proteolysis.

Accurate mass measurement by electrospray ionization quadrupole mass spectrometry: detection of variants differing by

Mass spectrometry has a basic limitation when human hemoglobin variants are analyzed, because it cannot resolve two globin chains that differ in mass by <6 Da. Several common beta-chain variants differ by 1 Da from normal and, hence, when present in heterozygotes, are not resolved from the normal beta-chain. Normal and variant chains appear together in the spectrum as a single entity, whose mass is the abundance weighted mean of the two chains. Here we show that such heterozygotes can be detected in 500-fold diluted blood by accurately measuring the mass of the beta-chain using an electrospray ionization quadrupole instrument and the alpha-chain for internal mass calibration. A statistical analysis of the normal beta-chain mass (n = 86) showed that the standard deviation (SD) of the mean was <+/-0.05 Da (<+/-3.2 ppm). Hence, at the 95% confidence level (+/-2 SD), an abnormal alpha- or beta-chain differing by 1 Da from normal should be detectable in a heterozygote provided its abundance is >10% of total alpha- or beta-chains, respectively. Variants whose masses lay between 1 and 4 Da from normal were detected in 19 heterozygotes. Moreover, the proportion of each variant estimated from the mass change correlated with the proportion determined by cation-exchange HPLC. Variants were assigned to the alpha- or beta-chain by combining the sign of the mass change with the polarity change inferred from electrophoretic data. This procedure could be used for screening clinically significant hemoglobin variants.

Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry.

Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.