| 1. |
- Erngren, Ida, et al.
(författare)
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Adduct formation in electrospray ionisation-mass spectrometry with hydrophilic interaction liquid chromatography is strongly affected by the inorganic ion concentration of the samples
- 2019
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Ingår i: Journal of Chromatography A. - : ELSEVIER SCIENCE BV. - 0021-9673 .- 1873-3778. ; 1600, s. 174-182
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Tidskriftsartikel (refereegranskat)abstract
- Hydrophilic interaction liquid chromatography (HILIC)/electrospray ionisation-mass spectrometry (ESI-MS) has gained interest for the analysis of polar analytes in bioanalytical applications in recent years. However, ESI-MS is prone to adduct formation of analytes. In contrast to reversed phase chromatography, small inorganic ions have retention in HILIC, i.e. analytes and inorganic ions may co-elute, which could influence the adduct formation. In the present paper, it was demonstrated that the co-elution of sodium ions or potassium ions and analytes in HILIC/ESI-MS affect the adduct formation and that different concentrations of sodium ions and potassium ions in biological samples could have an impact on the quantitative response of the respective adducts as well as the quantitative response of the protonated adduct. The co-elution also lead to cluster formation of analytes and sodium formate or potassium formate, causing extremely complicated spectra. In analytical applications using HILIC/ESI-MS where internal standards are rarely used or not properly matched, great care needs to be taken to ensure minimal variation of inorganic ion concentration between samples. Moreover, the use of alkali metal ion adducts as quantitative target ions in relative quantitative applications should be made with caution if proper internal standards are not used.
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| 2. |
- Lindell Jonsson, Eva, et al.
(författare)
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Exploring Radiation Response in Two Head and Neck Squamous Carcinoma Cell Lines Through Metabolic Profiling
- 2019
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Ingår i: Frontiers in Oncology. - : Frontiers Media SA. - 2234-943X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Head and neck squamous cell carcinoma (HNSCC) is the sixth most common form of cancer worldwide. Radiotherapy, with or without surgery, represents the major approach to curative treatment. However, not all tumors are equally sensitive to irradiation. It is therefore of interest to apply newer system biology approaches (e.g., metabolic profiling) in squamous cancer cells with different radiosensitivities in order to provide new insights on the mechanisms of radiation response. In this study, two cultured HNSCC cell lines from the same donor, UM-SCC-74A and UM-SCC-74B, were first genotyped using Short Tandem Repeat (STR), and assessed for radiation response by the means of clonogenic survival and growth inhibition assays. Thereafter, cells were cultured, irradiated and collected for subsequent metabolic profiling analyses using liquid chromatography-mass spectrometry (LC-MS). STR verified the similarity of UM-SCC-74A and UM-SCC-74B cells, and three independent assays proved UM-SCC-74B to be clearly more radioresistant than UM-SCC-74A. The LC-MS metabolic profiling demonstrated significant differences in the intracellular metabolome of the two cell lines before irradiation, as well as significant alterations after irradiation. The most important differences between the two cell lines before irradiation were connected to nicotinic acid and nicotinamide metabolism and purine metabolism. In the more radiosensitive UM-SCC-74A cells, the most significant alterations after irradiation were linked to tryptophan metabolism. In the more radioresistant UM-SCC-74B cells, the major alterations after irradiation were connected to nicotinic acid and nicotinamide metabolism, purine metabolism, the methionine cycle as well as the serine, and glycine metabolism. The data suggest that the more radioresistant cell line UM-SCC-74B altered the metabolism to control redox-status, manage DNA-repair, and change DNA methylation after irradiation. This provides new insights on the mechanisms of radiation response, which may aid future identification of biomarkers associated with radioresistance of cancer cells.
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| 3. |
- Pirttilä, Kristian, et al.
(författare)
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An LCMS-based untargeted metabolomics protocol for cochlear perilymph : highlighting metabolic effects of hydrogen gas on the inner ear of noise exposed Guinea pigs
- 2019
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Ingår i: Metabolomics. - : Springer Science and Business Media LLC. - 1573-3882 .- 1573-3890. ; 15:10
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Tidskriftsartikel (refereegranskat)abstract
- Introduction Noise-induced hearing loss (NIHL) is an increasing problem in society and accounts for a third of all cases of acquired hearing loss. NIHL is caused by formation of reactive oxygen species (ROS) in the cochlea causing oxidative stress. Hydrogen gas (H-2) can alleviate the damage caused by oxidative stress and can be easily administered through inhalation.Objectives To present a protocol for untargeted metabolomics of guinea pig perilymph and investigate the effect of H-2 administration on the perilymph metabolome of noise exposed guinea pigs.Methods The left ear of guinea pigs were exposed to hazardous impulse noise only (Noise, n = 10), noise and H-2 (Noise + H2, n = 10), only H-2 (H2, n = 4), or untreated (Control, n = 2). Scala tympani perilymph was sampled from the cochlea of both ears. The polar component of the perilymph metabolome was analyzed using a HILIC-UHPLC-Q-TOF-MS-based untargeted metabolomics protocol. Multivariate data analysis (MVDA) was performed separately for the exposed- and unexposed ear.Results MVDA allowed separation of groups Noise and Noise + H2 in both the exposed and unexposed ear and yielded 15 metabolites with differentiating relative abundances. Seven were found in both exposed and unexposed ear data and included two osmoprotectants. Eight metabolites were unique to the unexposed ear and included a number of short-chain acylcarnitines.Conclusions A HILIC-UHPLC-Q-TOF-MS-based protocol for untargeted metabolomics of perilymph is presented and shown to be fit-for-purpose. We found a clear difference in the perilymph metabolome of noise exposed guinea pigs with and without H-2 treatment.
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| 4. |
- Rodríguez-Gómez, José A, et al.
(författare)
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Microglia : Agents of the CNS Pro-Inflammatory Response.
- 2020
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 9:7
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Tidskriftsartikel (refereegranskat)abstract
- The pro-inflammatory immune response driven by microglia is a key contributor to the pathogenesis of several neurodegenerative diseases. Though the research of microglia spans over a century, the last two decades have increased our understanding exponentially. Here, we discuss the phenotypic transformation from homeostatic microglia towards reactive microglia, initiated by specific ligand binding to pattern recognition receptors including toll-like receptor-4 (TLR4) or triggering receptors expressed on myeloid cells-2 (TREM2), as well as pro-inflammatory signaling pathways triggered such as the caspase-mediated immune response. Additionally, new research disciplines such as epigenetics and immunometabolism have provided us with a more holistic view of how changes in DNA methylation, microRNAs, and the metabolome may influence the pro-inflammatory response. This review aimed to discuss our current knowledge of pro-inflammatory microglia from different angles, including recent research highlights such as the role of exosomes in spreading neuroinflammation and emerging techniques in microglia research including positron emission tomography (PET) scanning and the use of human microglia generated from induced pluripotent stem cells (iPSCs). Finally, we also discuss current thoughts on the impact of pro-inflammatory microglia in neurodegenerative diseases.
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| 5. |
- Aftab, Obaid, 1984-, et al.
(författare)
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NMR spectroscopy based metabolic profiling of drug induced changes in vitro can discriminate between pharmacological classes
- 2014
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Ingår i: Journal of chemical information and modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 54:11, s. 3251-3258
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Tidskriftsartikel (refereegranskat)abstract
- Drug induced changes in mammalian cell line models have already been extensively profiled at the systemic mRNA level and subsequently used to suggest mechanisms of action for new substances as well as to support drug repurposing, i.e. identifying new potential indications for drugs already licensed for other pharmacotherapy settings. The seminal work in this field, which includes a large database and computational algorithms for pattern matching, is known as the “Connectivity Map” (CMap). The potential of similar exercises at the metabolite level is, however, still largely unexplored. Only recently the first high throughput metabolomic assay pilot study was published, involving screening of metabolic response to a set of 56 kinase inhibitors in a 96-well format. Here we report results from a separately developed metabolic profiling assay, which leverages 1H NMR spectroscopy to the quantification of metabolic changes in the HCT116 colorectal cancer cell line, in response to each of 26 compounds. These agents are distributed across 12 different pharmacological classes covering a broad spectrum of bioactivity. Differential metabolic profiles, inferred from multivariate spectral analysis of 18 spectral bins, allowed clustering of most tested drugs according to their respective pharmacological class. A more advanced supervised analysis, involving one multivariate scattering matrix per pharmacological class and using only 3 spectral bins (three metabolites), showed even more distinct pharmacology-related cluster formations. In conclusion, this kind of relatively fast and inexpensive profiling seems to provide a promising alternative to that afforded by mRNA expression analysis, which is relatively slow and costly. As also indicated by the present pilot study, the resulting metabolic profiles do not seem to provide as information rich signatures as those obtained using systemic mRNA profiling, but the methodology holds strong promise for significant refinement.
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| 6. |
- Elmsjö, Albert, et al.
(författare)
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Method selectivity evaluation using the co-feature ratio in LC/MS metabolomics : Comparison of HILIC stationary phase performance for the analysis of plasma, urine and cell extracts.
- 2018
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1568, s. 49-56
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Tidskriftsartikel (refereegranskat)abstract
- Evaluation of the chromatographic separation in metabolomics studies has primarily been done using preselected sets of standards or by counting the number of detected features. An alternative approach is to calculate each feature's co-feature ratio, which is a combined selectivity measurement for the separation (i.e. extent of co-elution) and the MS-signal (i.e. adduct formation and in-source fragmentation). The aim of this study was to demonstrate how the selectivity of different HILIC stationary phases can be evaluated using the co-feature ratio approach. The study was based on three sample types; plasma, urine and cell extracts. Samples were analyzed on an UHPLC-ESI-Q-ToF system using an amide, a bare silica and a sulfobetaine stationary phase. For each feature, a co-feature ratio was calculated and used for multivariate analysis of the selectivity differences between the three stationary phases. Unsupervised PCA models indicated that the co-feature ratios were highly dependent on type of stationary phase. For several metabolites a 15-30 fold difference in the co-feature ratio were observed between the stationary phases. Observed selectivity differences related primarily to the retention patterns of unwanted matrix components such as inorganic salts (detected as salt clusters), glycerophospholipids, and polyethylene glycols. These matrix components affected the signal intensity of co-eluting metabolites by interfering with the ionization efficiency and/or their adduct formation. Furthermore, the retention pattern of these matrix components had huge influence on the number of detected features. The co-feature ratio approach has successfully been applied for evaluation of the selectivity performance of three HILIC stationary phases. The co-feature ratio could therefore be used in metabolomics for developing selective methods fit for their purpose, thereby avoiding generic analytical approaches, which are often biased, as type and amount of interfering matrix components are metabolome dependent.
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| 7. |
- Elmsjö, Albert, et al.
(författare)
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NMR-based metabolic profiling in healthy individuals overfed different types of fat : links to changes in liver fat accumulation and lean tissue mass.
- 2015
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Ingår i: Nutrition & Diabetes. - : Springer Science and Business Media LLC. - 2044-4052. ; 5:19, s. e182-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Overeating different dietary fatty acids influence the amount of liver fat stored during weight gain, however, the mechanisms responsible are unclear. We aimed to identify non-lipid metabolites that may differentiate between saturated (SFA) and polyunsaturated fatty acid (PUFA) overfeeding using a non-targeted metabolomic approach. We also investigated the possible relationships between plasma metabolites and body fat accumulation.METHODS: In a randomized study (LIPOGAIN study), n=39 healthy individuals were overfed with muffins containing SFA or PUFA. Plasma samples were precipitated with cold acetonitrile and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Pattern recognition techniques were used to overview the data, identify variables contributing to group classification and to correlate metabolites with fat accumulation.RESULTS: We previously reported that SFA causes a greater accumulation of liver fat, visceral fat and total body fat, whereas lean tissue levels increases less compared with PUFA, despite comparable weight gain. In this study, lactate and acetate were identified as important contributors to group classification between SFA and PUFA (P<0.05). Furthermore, the fat depots (total body fat, visceral adipose tissue and liver fat) and lean tissue correlated (P(corr)>0.5) all with two or more metabolites (for example, branched amino acids, alanine, acetate and lactate). The metabolite composition differed in a manner that may indicate higher insulin sensitivity after a diet with PUFA compared with SFA, but this needs to be confirmed in future studies.CONCLUSION: A non-lipid metabolic profiling approach only identified a few metabolites that differentiated between SFA and PUFA overfeeding. Whether these metabolite changes are involved in depot-specific fat storage and increased lean tissue mass during overeating needs further investigation.
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| 8. |
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| 9. |
- Elmsjö, Albert, et al.
(författare)
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The co-feature ratio, a novel method for the measurement of chromatographic and signal selectivity in LC-MS-based metabolomics.
- 2017
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Ingår i: Analytica Chimica Acta. - : Elsevier BV. - 0003-2670 .- 1873-4324. ; 956, s. 40-47
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Tidskriftsartikel (refereegranskat)abstract
- Evaluation of analytical procedures, especially in regards to measuring chromatographic and signal selectivity, is highly challenging in untargeted metabolomics. The aim of this study was to suggest a new straightforward approach for a systematic examination of chromatographic and signal selectivity in LC-MS-based metabolomics. By calculating the ratio between each feature and its co-eluting features (the co-features), a measurement of the chromatographic selectivity (i.e. extent of co-elution) as well as the signal selectivity (e.g. amount of adduct formation) of each feature could be acquired, the co-feature ratio. This approach was used to examine possible differences in chromatographic and signal selectivity present in samples exposed to three different sample preparation procedures. The capability of the co-feature ratio was evaluated both in a classical targeted setting using isotope labelled standards as well as without standards in an untargeted setting. For the targeted analysis, several metabolites showed a skewed quantitative signal due to poor chromatographic selectivity and/or poor signal selectivity. Moreover, evaluation of the untargeted approach through multivariate analysis of the co-feature ratios demonstrated the possibility to screen for metabolites displaying poor chromatographic and/or signal selectivity characteristics. We conclude that the co-feature ratio can be a useful tool in the development and evaluation of analytical procedures in LC-MS-based metabolomics investigations. Increased selectivity through proper choice of analytical procedures may decrease the false positive and false negative discovery rate and thereby increase the validity of any metabolomic investigation.
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| 10. |
- Engskog, Mikael K. R., et al.
(författare)
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A dual role for the lex2 locus : identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019
- 2009
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Ingår i: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 344:5, s. 632-641
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Tidskriftsartikel (refereegranskat)abstract
- Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-L-glycero-D-manno-heptosyl inner-core moiety (L-alpha-D-Hepp-(1 -> 2)-[PEtn -> 6]-L-alpha-D-Hepp-(1 -> 3)-[beta-D-Glclp-(1 -> 4)]-L-alpha-D-Hepp-(1 -> 5)-alpha-Kdop) to which addition of beta-D-Glcp to O-4 of Glcl in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a beta-D-Galp is linked to O-4 of Glcl. In order to test the hypothesis that the 1ex2 locus is involved in the expression Of beta-D-Galp-(1 -> 4-beta-D-Glcp-(1 -> - from Hepl, 1ex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-cleacylated LPS and core oligosaccharide material (OS), as well as ESIMS" on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only beta-D-Glcp extensions from Hepl. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a beta-D-Galp to O-4 of Glcl in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product.
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