Browsing by Subject "mass spectrometry"

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  • Holma, Paula (Helsingfors universitet, 2011)
    Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.
  • Verma, Arvind; Laakso, Into; Seppanen-Laakso, Tuulikki; Huhtikangas, Aarre; Riekkola, Marja-Liisa (2007)
  • Berndt, Torsten; Scholz, Wiebke; Mentler, Bernhard; Fischer, Lukas; Herrmann, Hartmut; Kulmala, Markku; Hansel, Armin (2018)
    Hydrocarbons are emitted into the Earth's atmosphere in very large quantities by human and biogenic activities. Their atmospheric oxidation processes almost exclusively yield RO2 radicals as reactive intermediates whose atmospheric fate is not yet fully unraveled. Herein, we show that gas-phase reactions of two RO2 radicals produce accretion products composed of the carbon backbone of both reactants. The rates for accretion product formation are very high for RO2 radicals bearing functional groups, competing with those of the corresponding reactions with NO and HO2. This pathway, which has not yet been considered in the modelling of atmospheric processes, can be important, or even dominant, for the fate of RO2 radicals in all areas of the atmosphere. Moreover, the vapor pressure of the formed accretion products can be remarkably low, characterizing them as an effective source for the secondary organic aerosol.
  • Laakia, Jaakko (Finnish Meteorological Institute, 2017)
    Finnish Meteorological Institute Contributions 131
    This thesis covers two aspects of utilisation of advanced separation technology together with mass spectrometry: 1. Drift tube ion mobility spectrometry – mass spectrometry (IMS-MS) studies of the behaviour of ions in the gas phase and 2. Comprehensive two dimensional gas chromatography – time-offlight mass spectrometry (GC×GC-TOF-MS) studies for characterization of crude oil samples. In IMS studies, the focus was on the separation of isomeric compounds. For example, [M-H]- ions of 2,4-di-tert-butylphenol (2,4-DtBPh) and 2,6-di-tert-butylphenol (2,6-DtBPh) were separated. It was also observed that shielding of the charge site by the functional groups of a molecule has a large effect on the separation of the isomeric compounds. For example, amines with a shielded charge site were separated from those with a more open charge site, while some of the isomeric amines studied were not separated. Different kinds of adduct ions were observed for some of the analytes. Dioxygen adducts were seen for 2,4-DtBPh [M+O2]-, 2,6-di-tert-butylpyridine (2,6-DtBPyr) [M+O2]+· and 2,6-di-tert-butyl-4-methylpyridine (2,6-DtB-4MPyr) [M+O2]+·. The adduct formation increases the total mass of the analyte ion, and therefore, for example the 2,4-DtBPh [M+O2]- ion could be separated from its isomeric compound 2,6-DtBPh [M-H]-, which did not from the dioxygen adduct ion. In the case of 2,6-DtBPyr and 2,6-DtB-4MPyr, the [M]+ ions formed dioxygen adduct [M+O2]+· ions. The both ions, [M]+ and [M+O2]+·, shared the same drift time which was longer than their [M+H]+ ion species. This work demonstrates that measuring with IMS the mobility of different ion structures of the same molecule, especially dioxygen adducts, results in a better understanding of the role of adduct ions in the IMSseparation process. In GC×GC-TOF-MS studies, the focus was on detailed characterization of crude oil samples. For instance, oils from the Recôncavo Basin were classified to two different groups by using minor oil components. The GC×GC-TOF-MS data showed the correlation between 2D retention time and the number of carbons in a ring for several hydrocarbons as known from the literature. This information was used to achieve more structural information about eight new tetracyclic compounds, some of them similar to nor-steranes, detected during analysis. Some of these new compounds could be used as maturity indicators. This study demonstrated how GC×GC-TOF-MS can be used to separate geochemically interested isomers, identify minor geochemical differences between oils and achieve structural information about unknown biomarkers.
  • Kilpinen, Lotta; Tigistu-Sahle, Feven; Oja, Sofia; Greco, Dario; Parmar, Amarjit; Saavalainen, Päivi Marjaana; Nikkilä, Janne Tapio; Korhonen, Matti; Lehenkari, Petri; Käkelä, Reijo; Laitinen, Saara (2013)
  • Syrjä, Pernilla; Palviainen, Mari; Jokinen, Tarja; Kyöstilä, Kaisa; Lohi, Hannes; Roosje, Petra; Anderegg, Linda; Leeb, Tosso; Sukura, Antti; Eskelinen, Eeva-Liisa (2020)
    Lagotto Romagnolo breed dogs develop a progressive neurological disease with intracellular vacuolar storage when homozygous for a variant in the autophagy-related gene 4D (ATG4D). A lysosomal enzyme deficiency has not been proven in this disease, despite its overlapping morphology with lysosomal storage diseases. Instead, basal autophagy was altered in fibroblasts from affected dogs. The aim of this study was to clarify the origin of the limiting membrane of the accumulating vacuoles and determine whether altered basal autophagy affects the extracellular release of vesicles in cells from diseased dogs. When assessed by immunoelectron microscopy, the membrane of the cytoplasmic vacuoles in affected tissues contained ATG4D, markers for autolysosomes (microtubule-associated protein 1A/B light chain 3 and lysosome-associated membrane protein 2) and for recycling endosomes (transferrin receptor 2), indicating that the vacuoles are hybrid organelles between endocytic and autophagic pathways. Ultracentrifugation, nanoparticle tracking analysis, and mass spectrometry were used to analyze the vesicles released from cultured fibroblasts of affected and control dogs. The amount of extracellular vesicles (EVs) released from affected fibroblasts was significantly increased during basal conditions in comparison to controls. This difference disappeared during starvation. The basal EV proteome of affected cells was enriched with cytosolic, endoplasmic reticulum, and mitochondrial proteins. Heat shock proteins and chaperones, some of which are known substrates of basal autophagy, were identified among the proteins unique to EVs of affected cells. An increased release of extracellular vesicles may serve as a compensatory mechanism in disposal of intracellular proteins during dysfunctional basal autophagy in this spontaneous disease.
  • Kortteenniemi, Aaron; Ortega-Alonso, Alfredo; Javadi, Amir-Homayoun; Tolmunen, Tommi; Ali-Sisto, Toni; Kotilainen, Tuukka; Wikgren, Jan; Karhunen, Leila; Velagapudi, Vidya; Lehto, Soili M. (2020)
    Background Transcranial direct current stimulation (tDCS), a putative treatment for depression, has been proposed to affect peripheral metabolism. Metabolic products from brain tissue may also cross the blood-brain barrier, reflecting the conditions in the brain. However, there are no previous data regarding the effect of tDCS on circulating metabolites. Objective To determine whether five daily sessions of tDCS modulate peripheral metabolites in healthy adult men. Methods This double-blind, randomized controlled trial involved 79 healthy males (aged 20-40 years) divided into two groups, one receiving tDCS (2 mA) and the other sham stimulated. The anode was placed over the left dorsolateral prefrontal cortex and the cathode over the corresponding contralateral area. Venous blood samples were obtained before and after the first stimulation session, and after the fifth stimulation session. Serum levels of 102 metabolites were determined by mass spectrometry. The results were analysed with generalised estimating equations corrected for the family-wise error rate. In addition, we performed power calculations estimating sample sizes necessary for future research. Results TDCS-related variation in serum metabolite levels was extremely small and statistically non-significant. Power calculations indicated that for the observed variation to be deemed significant, samples sizes of up to 11,000 subjects per group would be required, depending on the metabolite of interest. Conclusion Our study found that five sessions of tDCS induced no major effects on peripheral metabolites among healthy men. These observations support the view of tDCS as a safe treatment that does not induce significant changes in the measured peripheral metabolites in healthy male subjects.
  • Kamyab, Elham; Goebeler, Norman; Kellermann, Matthias Y.; Rohde, Sven; Reverter, Miriam; Striebel, Maren; Schupp, Peter J. (2020)
    Sea cucumbers are bottom dwelling invertebrates, which are mostly found on subtropical and tropical sea grass beds, sandy reef flats, or reef slopes. Although constantly exposed to fouling communities in these habitats, many species are surprisingly free of invertebrate epibionts and microfouling algae such as diatoms. In our study, we investigated the anti-fouling (AF) activities of different crude extracts of tropical Indo-Pacific sea cucumber species against the fouling diatom Cylindrotheca closterium. Nine sea cucumber species from three genera (i.e., Holothuria, Bohadschia, Actinopyga) were selected and extracted to assess their AF activities. To verify whether the sea cucumber characteristic triterpene glycosides were responsible for the observed potent AF activities, we tested purified fractions enriched in saponins isolated from Bohadschia argus, representing one of the most active anti-fouling extracts. Saponins were quantified by vanillin-sulfuric acid colorimetric assays and identified by LC-MS and LC-MS/MS analyses. We were able to demonstrate that AF activities in sea cucumber extracts were species-specific, and growth inhibition as well as attachment of the diatom to surfaces is dependent on the saponin concentration (i.e., Actinopyga contained the highest quantities), as well as on the molecular composition and structure of the present saponins (i.e., Bivittoside D derivative was the most bioactive compound). In conclusion, the here performed AF assay represents a promising and fast method for selecting the most promising bioactive organism as well as for identifying novel compounds with potent AF activities for the discovery of potentially novel pharmacologically active natural products.
  • Knuuttila, Matias; Hämäläinen, Esa; Poutanen, Matti (2019)
    Recent development of gas chromatography and liquid chromatography-tandem mass spectrometry (GC-MS/MS, LC-MS/MS) has provided novel tools to define sex steroid concentrations. These new methods overcome several of the problems associated with immunoassays for sex steroids. With the novel MS-based applications we are now able to measure small concentrations of the steroid hormones reliably and with high accuracy in both body fluids and tissue homogenates. The sensitivity of the tandem mass spectrometry assays allows us also for the first time to reliably measure picomolar or even femtomolar concentrations of estrogens and androgens. Furthermore, due to a high sensitivity and specificity of MS technology, we are also able to measure low concentrations of steroid hormones of interest in the presence of pharmacological concentration of other steroids and structurally closely related compounds. Both of these features are essential for multiple preclinical models for prostate cancer. The MS assays are also valuable for the simultaneous measurement of multiple steroids and their metabolites in small sample volumes in serum and tissue biopsies of prostate cancer patients before and after drug interventions. As a result, novel information about steroid hormone synthesis and metabolic pathways in prostate cancer has been obtained. In our recent studies, we have extensively applied a GC-MS/MS method to study androgen biosynthesis and metabolism in VCaP prostate cancer xenografts in mice. In the present review, we shortly summarize some of the benefits of the GC-MS/MS and novel LC-MS/MS assays, and provide examples of their use in defining novel mechanisms of androgen action in prostate cancer.
  • Backman, Nina (Helsingfors universitet, 2011)
    Screening of drugs of abuse has to combine sensitivity, selectivity and repeatability. The conventional screening methods include immunoassay screening followed by a more sensitive confirmation method. The aim of the study was to develop a simple, yet sensitive sample preparation method for screening of benzodiazepines and amphetamine derivatives in urine samples with silicon micropillar array electrospray ionization chip (µPESI) coupled to mass spectrometric analysis. Another aim was to evaluate the suitability of µPESI in biological sample analysis. Ideally, the developed method would provide an alternative to immunoassay screening method in forensic urine analysis. The sample preparation methods were separately optimized for benzodiazepines and amphetamine derivatives. Methods used included solid- phase extraction with Oasis HLB cartridge and C18-phase containing ZipTip®-pipette tip, liquid-liquid extraction, and dilution and filtering without prior extraction. Optimization focused, however, on ZipTip®-extraction. The compounds were spiked in blank urine to their cut-off levels, 200 ng/ml for benzodiazepines and 300 ng/ml for amphetamine derivatives. For benzodiazepines, every extraction phase was optimized. The sample pH was adjusted to 5, the ZipTip® phase was conditioned with acetonitrile and washed with a mixture of water (pH 5) and acetonitrile (10 % v/v) and the sample was eluted with a mixture of acetonitrile, formic acid and water (95:1:4 v/v/v). For amphetamine derivatives, pH values of sample and solvents were optimized. The sample pH was adjusted to 10, the ZipTip® phase was conditioned with a mixture of water and ammoniumbicarbonate (pH 10, 1:1 v/v), washed with a mixture of water and acetonitrile (1:5 v/v) and the sample was eluted with methanol. The optimized methods were tested with authentic urine samples obtained from Yhtyneet Medix Laboratories and compared to the results of quantitative GC/MS analysis. Benzodiazepine samples were hydrolyzed prior to extraction to improve recovery. All samples were measured with Q-TOF Micro apparatus and hydrolyzed benzodiazepine samples additionally with microTOF apparatus in Yhtyneet Medix Laboratories. Based on the results the developed method needs more optimization to function properly. The main problems were lack of reproducibility and poor sample ionization. Manual sample preparation and adding to the chip sample introduction spot increased variation. Authentic benzodiazepine samples gave false negative and authentic amphetamine derivative samples false positive results. False negatives may be due to the lack of sensitivity and false positives due to the contamination of sample cone, chips or solvents.
  • Kareinen, Ilona; Baumann, Marc; Su Duy Nguyen,; Maaninka, Katariina; Anisimov, Andrey; Tozuka, Minoru; Jauhiainen, Matti; Lee-Rueckert, Miriam; Kovanen, Petri T. (2018)
    ApoA-I, the main structural and functional protein of HDL particles, is cardioprotective, but also highly sensitive to proteolytic cleavage. Here, we investigated the effect of cardiac mast cell activation and ensuing chymase secretion on apoA-I degradation using isolated rat hearts in the Langendorff perfusion system. Cardiac mast cells were activated by injection of compound 48/80 into the coronary circulation or by low-flow myocardial ischemia, after which lipid-free apoA-I was injected and collected in the coronary effluent for cleavage analysis. Mast cell activation by 48/80 resulted in apoA-I cleavage at sites Tyr(192) and Phe(229), but hypoxic activation at Tyr(192) only. In vitro, the proteolytic end-product of apoA-I with either rat or human chymase was the Tyr(192)-truncated fragment. This fragment, when compared with intact apoA-I, showed reduced ability to promote migration of cultured human coronary artery endothelial cells in a wound-healing assay. We propose that C-terminal truncation of apoA-I by chymase released from cardiac mast cells during ischemia impairs the ability of apoA-I to heal damaged endothelium in the ischemic myocardium.
  • Böcker, Sebastian; Mäkinen, Veli (IEEE/ACM, 2008)
    Mass spectrometry has become one of the most popular analysis techniques in Proteomics and Systems Biology. With the creation of larger datasets, the automated recalibration of mass spectra becomes important to ensure that very peak in the sample spectrum is correctly assigned to some peptide and protein. Algorithms for recalibrating mass spectra have to be robust with respect to wrongly assigned peaks, as well as efficient due to the amount of mass spectrometry data. The recalibration of mass spectra leads us to the problem of finding an optimal matching between mass spectra under measurement errors. We have developed two deterministic methods that allow robust computation of such a matching: The first approach uses a computational geometry interpretation of the problem, and tries to find two parallel lines with constant distance that stab a maximal number of points in the plane. The second approach is based on finding a maximal common approximate subsequence, and improves existing algorithms by one order of magnitude exploiting the sequential nature of the matching problem. We compare our results to a computational geometry algorithm using a topological line-sweep.
  • Sulima, Anna; Savijoki, Kirsi; Bien, Justyna; Nareaho, Anu; Salamatin, Ruslan; Conn, David Bruce; Mlocicki, Daniel (2018)
    Cestodiases are common parasitic diseases of animals and humans. As cestodes have complex lifecycles, hexacanth larvae, metacestodes (including cysticercoids), and adults produce proteins allowing them to establish invasion and to survive in the hostile environment of the host. Hymenolepis diminuta is the most commonly used model cestode in experimental parasitology. The aims of the present study were to perform a comparative proteomic analysis of two consecutive developmental stages of H. diminuta (cysticercoid and adult) and to distinguish proteins which might be characteristic for each of the stages from those shared by both stages. Somatic proteins of H. diminuta were isolated from 6-week-old cysticercoids and adult tapeworms. Cysticercoids were obtained from experimentally infected beetles, Tenebrio molitor, whereas adult worms were collected from experimentally infected rats. Proteins were separated by GeLC-MS/MS (one dimensional gel electrophoresis coupled with liquid chromatography and tandem mass spectrometry). Additionally protein samples were digested in-liquid and identified by LC-MS/MS. The identified proteins were classified according to molecular function, cellular components and biological processes. Our study showed a number of differences and similarities in the protein profiles of cysticercoids and adults; 233 cysticercoid and 182 adult proteins were identified. From these proteins, 131 were present only in the cysticercoid and 80 only in the adult stage samples. Both developmental stages shared 102 proteins; among which six represented immunomodulators and one is a potential drug target. In-liquid digestion and LC-MS/MS complemented and confirmed some of the GeLC-MS/MS identifications. Possible roles and functions of proteins identified with both proteomic approaches are discussed.
  • Kiamehr, Mostafa; Heiskanen, Laura; Laufer, Thomas; Duesterloh, Aneta; Kahraman, Mustafa; Kakela, Reijo; Laaksonen, Reijo; Aalto-Setala, Katriina (2019)
    Aim: Primary human hepatocytes (PHHs) undergo dedifferentiation upon the two-dimensional (2D) culture, which particularly hinders their utility in long-term in vitro studies. Lipids, as a major class of biomolecules, play crucial roles in cellular energy storage, structure, and signaling. Here, for the first time, we mapped the alterations in the lipid profile of the dedifferentiating PHHs and studied the possible role of lipids in the loss of the phenotype of PHHs. Simultaneously, differentially expressed miRNAs associated with changes in the lipids and fatty acids (FAs) of the dedifferentiating PHHs were investigated. Methods: PHHs were cultured in monolayer and their phenotype was monitored morphologically, genetically, and biochemically for five days. The lipid and miRNA profile of the PHHs were analyzed by mass spectrometry and Agilent microarray, respectively. In addition, 24 key genes involved in the metabolism of lipids and FAs were investigated by qPCR. Results: The typical morphology of PHHs was lost from day 3 onward. Additionally, ALB and CYP genes were downregulated in the cultured PHHs. Lipidomics revealed a clear increase in the saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) containing lipids, but a decrease in the polyunsaturated fatty acids (PUFA) containing lipids during the dedifferentiation of PHHs. In line with this, FASN, SCD, ELOVL1, ELOVL3, and ELOVL7 were upregulated but ELOVL2 was downregulated in the dedifferentiated PHHs. Furthermore, differentially expressed miRNAs were identified, and the constantly upregulated miR-27a and miR-21, and downregulated miR-30 may have regulated the synthesis, accumulation and secretion of PHH lipids during the dedifferentiation. Conclusion: Our results showed major alterations in the molecular lipid species profiles, lipid-metabolizing enzyme expression as wells as miRNA profiles of the PHHs during their prolonged culture, which in concert could play important roles in the PHHs' loss of phenotype. These findings promote the understanding from the dedifferentiation process and could help in developing optimal culture conditions, which better meet the needs of the PHHs and support their original phenotype.
  • Shishido, Tania Keiko; Popin, Rafael Vicentini; Jokela, Jouni; Wahlsten, Matti; Fiore, Marli Fatima; Fewer, David P.; Herfindal, Lars; Sivonen, Kaarina (2020)
    Cyanobacteria are photosynthetic organisms that produce a large diversity of natural products with interesting bioactivities for biotechnological and pharmaceutical applications. Cyanobacterial extracts exhibit toxicity towards other microorganisms and cancer cells and, therefore, represent a source of potentially novel natural products for drug discovery. We tested 62 cyanobacterial strains isolated from various Brazilian biomes for antileukemic and antimicrobial activities. Extracts from 39 strains induced selective apoptosis in acute myeloid leukemia (AML) cancer cell lines. Five of these extracts also exhibited antifungal and antibacterial activities. Chemical and dereplication analyses revealed the production of nine known natural products. Natural products possibly responsible for the observed bioactivities and five unknown, chemically related chlorinated compounds present only in Brazilian cyanobacteria were illustrated in a molecular network. Our results provide new information on the vast biosynthetic potential of cyanobacteria isolated from Brazilian environments.
  • Lindfors, Pia (Helsingfors universitet, 2010)
    The most important part in bioanalysis is the sample cleanup process which is usually the most laborious and time consuming part of the analysis and very susceptible to errors. A functional bioanalysis has to be quick, easily automated, sensitive, selective and stable. It also needs to be suitable for high throughput analysis. Desorption atmospheric pressure photoionization (DAPPI) is a novel direct desorption/ionization technique for mass spectrometry that enables direct analysis of solids from surfaces or liquid samples from a suitable sample plate often without any sample preparation. The suitability of DAPPI-MS for biological samples was investigated by measuring the limits of detection for selected opioids and benzodiazepines and screening them from authentic urine samples. Limits of detection were measured for standard solutions and spiked urine. Opioids and benzodiazepines were analyzed from post mortem urine samples with an optimized DAPPI-MS method. Post mortem urine samples were analyzed with and without sample preparation. Sample preparation improved the sensitivity of the method remarkably. About 50 % of the analytes were detected without sample preparation and almost 100 % after sample cleanup. It is however difficult to estimate the suitability of DAPPI-MS as a screening method because not all analyte concentrations of the urine samples were known. Therefore we cannot be certain weither the results obtained without sample preparation are caused by the suppression of the urine matrix or if the concentrations of the analytes are below the limits of detection. The reliability of the method can further be improved by investigating the metabolites of the analytes and improving the system towards automation. On grounds of this research DAPPI-MS should be used cautiously as a screening method for urine samples without sample preparation and with only high enough analyte concentrations. DAPPI-MS shows promise as a screening method for opioids and benzodiazepines from urine when the sample cleanup is used before the analysis.
  • Aalto, Henni (Helsingfors universitet, 2011)
    Lipids are fat soluble compounds that are derived from living tissues. Lipids have many important physiological functions. Developing methods for efficient lipid analysis is important since lipids can function as biomarkers in diseases. Additionally these methods can be used for the discovery of the biological processes of disease development. Lipids comprise of molecules with different polarity and structure. Several mass spectrometric ionization methods have been used in the analysis of lipids but they usually require sample preparation prior to the analysis. Desorption electrospray ionization-mass spectrometry (DESI-MS) and desorption photoionization-mass spectrometry (DAPPI-MS) are novel ionization methods that allow sample analysis straight from the matrix, such as tissue, usually without any sample preparation. DESI-MS has already been used in the analysis of different lipids, but DAPPI-MS has only been used in the analysis of steroids. The ionization of a range of lipid compounds (phospholipids, triglycerides, fat soluble vitamins, fatty acids, and steroids) by DAPPI-MS and DESI-MS was studied. Analysis conditions were optimized for all the different lipid classes with both DAPPI and DESI using standard samples. Some lipids were also analysed straight from pharmaceutical preparations. There were differences in the suitabilities of DAPPI-MS and DESI-MS for the ionization of different lipid classes. DAPPI-MS worked well for the ionization of nonpolar lipids like triglycerides, vitamins and fatty acids, but the phospholipids fragmented in the DAPPI-MS process and showed no molecular ion. Previous studies have shown that DESI-MS works well in the ionization of phospholipids, and this study showed that it works reasonably well for other lipid groups as well, with the exception of some of the nonpolar lipids. New knowledge was acquired especially about the suitability of DAPPI-MS for the analysis of different lipids. Based on the results it can be said that DAPPI-MS works equally well or better than DESI-MS in the ionization of most lipid classes. The DAPPI method should still be further developed so that phospholipids, which are very important lipids in human physiology, could be analysed by DAPPI-MS. As lipids were not analysed straight from a tissue sample, there are no conclusions about the suitability of DAPPI-MS for the analysis of lipids straight from tissue samples.
  • Scifo, Enzo; Szwajda, Agnieszka; Debski, Janusz; Uusi-Rauva, Kristiina; Kesti, Tapio; Dadlez, Michal; Gingras, Anne-Claude; Tyynelä, Jaana; Baumann, Marc H.; Jalanko, Anu; Lalowski, Maciej (2013)
  • Vorrink, Sabine U.; Ullah, Shahid; Schmidt, Staffan; Nandania, Jatin; Velagapudi, Vidya; Beck, Olof; Ingelman-Sundberg, Magnus; Lauschke, Volker M. (2017)
    Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.
  • Bickert, Andreas; Ginkel, Christina; Kol, Matthijs; vom Dorp, Katharina; Jastrow, Holger; Degen, Joachim; Jacobs, Rene L.; Vance, Dennis E.; Winterhager, Elke; Jiang, Xian-Cheng; Doermann, Peter; Somerharju, Pentti; Holthuis, Joost C. M.; Willecke, Klaus (2015)
    Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS) 2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.