Faglige interesser
Analytisk kjemi
Massespektrometri
Nano væskekromatografi
Omvendt fase kromatografi
Biomolekyler
Organ on a chip
Mini-organer (organoider)
Undervisning
KJM2400 – Analytisk kjemi I
KJM3400 – Analytisk kjemi II - separasjonsmetoder
KJM1140 – Biokjemi 1 for kjemikere
Utmerkelser/formidling
Vinner av Kjemi Grand Prix, 2020
Finalist i Forsker Grand Prix, 2020
Teknisk Ukeblad, Tester ut legemidler på levende modeller av menneskeorganer, 2020
Bakgrunn
Bachelor of Science (2015, Biochemistry, UiO)
Master of Science (2017, Chemistry, UiO)
Publikasjoner
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Restan, Magnus Saed; Skottvoll, Frøydis Sved; Jensen, Henrik & Pedersen-Bjergaard, Stig
(2020).
Electromembrane extraction of sodium dodecyl sulfate from highly concentrated solutions.
The Analyst.
ISSN 0003-2654.
145,
s. 4957–4963.
doi:
10.1039/d0an00622j.
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Hvinden, Ingvild Comfort; Berg, Henriette Engen; Sachse, Daniel; Skaga, Erlend; Skottvoll, Frøydis Sved & Lundanes, Elsa
[Vis alle 10 forfattere av denne artikkelen]
(2019).
Nuclear Magnetic Resonance Spectroscopy to Identify Metabolite Biomarkers of Nonresponsiveness to Targeted Therapy in Glioblastoma Tumor Stem Cells.
Journal of Proteome Research.
ISSN 1535-3893.
18(5),
s. 2012–2020.
doi:
10.1021/acs.jproteome.8b00801.
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Se alle arbeider i Cristin
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Skottvoll, Frøydis Sved; Harrison, Sean; Hansen, Frederik; Pedersen-Bjergaard, Stig; Sullivan, Gareth & Bogen, Inger Lise
[Vis alle 8 forfattere av denne artikkelen]
(2020).
Liver mini-organ analyses using liquid chromatography mass spectrometry.
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Schüller, Maria; Skottvoll, Frøydis Sved; Lundanes, Elsa & Wilson, Steven Ray Haakon
(2020).
Bottom-up proteomics of biomarkers to investigate drug-induced hepatotoxicity in human liver organoids as part of preclinical development.
Vis sammendrag
Drug-induced liver injury (DILI) is an ongoing issue in the development of drugs. It is a major cause of
drug withdrawal during development, as well as being the leading cause for post-authorization
withdrawal. DILI is commonly discovered in later stages of clinical trials and poses a risk to trial subjects
involved in the study. European Medicines Agency (EMEA) provide guidelines for non-clinical
assessment of DILI, however regulatory authorities do not require this assessment for the approval of
the Investigational New Drug (IND) application for testing in human subjects. This is possibly due to
lack of research and validity of current DILI practices which, at worst, could hinder promising
therapeutics to enter the market. In recent years, the use of 3D liver cellular clusters, commonly known
as liver organoids, has alongside other advances, emerged as a promising tool to supplement the nonclinical assessment of drug candidates. Liver organoids closely resemble human liver physiology and
emulate basic liver function. In combination with well-established clinical protein biomarkers, they
might revolutionize the drug industry and strengthen patient safety. Organoids and organoid medium
are complex in nature and limited in size and availability. Implementing nanoLC for analytical method
development for liver organoid analysis will achieve improved sensitivity and therefore capture the
detection of minor fluctuations in protein biomarkers upon drug exposure. The goal of this work is to
develop a targeted proteomics method for the absolute quantification of alanine aminotransferase
(ALT) in organoid medium based on unique peptide MRM transitions with nanoLC-tandem MS. Pilot
experiments using human serum albumin (HSA) have been conducted to create a basic outline of
experiments which are reasonable to conduct when approaching the main goal. HSA is a readily
available and relatively affordable pure protein source, well suited for preliminary testing. The optimal
linear gradient for eluting HSA peptides within 48 min was 1% - 15% mobile phase component B (0.1%
formic acid and 10% water in acetonitrile). A positive correlation between SSRCalc values
(hydrophobicity factor) and elution time has been established and can be implemented in developing
the optimal gradient for peptides of ALT. The most abundant peptides have been optimized with
respect to collision energy (CE) using a scheduled MRM approach. Regression analysis was performed
to establish a relationship between CE and precursor m/z, which can be implemented in ALT analysis.
HSA was spiked into HeLa cell lysate to investigate matrix suppression effects, as well as to investigate
the effects of gradient length on ion suppression. Planned experiments will revolve around selecting
signature peptides for ALT, quantify ALT with suitable quantification methods and measure ALT levels
in organoid medium upon exposure to known hepatotoxins.
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Boger, Ida Caroline Sneis; Nerem, Elisabeth; Skottvoll, Frøydis Sved; Harrison, Sean; Sullivan, Gareth & Lundanes, Elsa
[Vis alle 8 forfattere av denne artikkelen]
(2020).
Determination of liver organoid-induced drug metabolites using liquid chromatography-mass spectrometry (LC-MS)”.
Vis sammendrag
Liver organoids are three-dimensional tissue models typically derived from adult and human induced pluripotent stem cells. They are intended to e.g. represent the physiological functions of a patient´s liver [1]. The liver is the main metabolizing organ in the human body [2]; thus, an important application of liver organoids is to map drug metabolism in vitro as liver organoids can be more patient-specific compared to traditional biomaterials, e.g. human liver microsomes (HLM). The aim of this study is to explore liver organoid drug metabolism in vitro using liquid chromatography-mass spectrometry (LC-MS). To establish a standardized conventional approach for metabolism studies for later comparison with organoids, heroin metabolism studies in HLMs and S9 fraction were carried out. Quantification of model substance heroin and its well-known metabolites, 6-Monoacetylmorphine (6-MAM) and morphine, was done using UHPLC-MS/MS. The heroin metabolism method was miniaturized to make it transferable to the small organoid samples. This was done by decreasing the microsome amount as much as possible while still observing heroin metabolism (HLM amount decreased from 0.2 mg to 0.01 mg; Heroin concentration was decreased from 10 µM to 0.1 µM). This miniaturized experiment was also transferred to S9 fraction Preliminary experiments with organoids showed that the organoids metabolize heroin to 6-MAM and morphine, as well as phase II biotransformation metabolites morphine-3-glucuronide and morphine-6-glucuronide. These results show that the studied organoids have metabolizing properties. Taken together, LC-MS can be a valuable tool for studying metabolism properties of organoids.
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Mrša, Ago; Skottvoll, Frøydis Sved; Harrison, Sean; Lundanes, Elsa; Sullivan, Gareth & Wilson, Steven Ray Haakon
(2020).
Miniaturized liquid chromatography systems for small molecule analysis of organoids.
Vis sammendrag
Liver organoids are three-dimensional tissue models (typically in the millimetre scale or below) derived from human-induced pluripotent stem cells [1]. They are intended to e.g. represent the physiological functions of a patient’s liver. The liver is the main metabolizing organ in the human body [2]; thus, an important application of liver organoids is to map drug metabolism in vitro, as liver organoids can be more patient-specific compared to traditional biomaterials. The aim of the study is to develop miniaturized liquid chromatography (LC) systems capable of analyzing very small samples, e.g secretion of liver organoids. Emphasis is placed on the determination of small molecules, i.e metabolites. Due to the small size of organoids, there are significant challenges in performing sensitive analysis of the organoid itself and it's minute secretion. Hence a nanoLC system has been explored, capable of sensitive analysis of model substance heroin and its well-known metabolites morphine and 6-Monoacetylmorphine (6-MAM). Using a 50 µm x 5 cm C18 pre-column and a 50 µm x 12 cm C18 analytical column, coupled up with a triple quadrupole mass spectrometer (TQMS) with electrospray ionization, a limit of detection of 0.7 fg was achieved for heroin. Further method development was done using commercially packed C18 columns typically used in proteomics. Separation and detection of heroin, morphine and 6-MaM were achieved in less than 8 minutes and with detection limits in the sub fg area. The method was successfully transferred to liver organoid samples that had been incubated with 1.5 µM heroin for 1 hour, cleaned using electromembrane extraction and further diluted 1:1000. Deuterated internal standards were implemented into the method for future work with organoid samples and the next step is to map the metabolism of liver organoids incubated within various time frames. Taken together, nanoLC-MS proves to be a viable tool for analysis of small molecules and the achievd results are promising for further work with organoid samples.
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Hermansen, Astrid; Skottvoll, Frøydis Sved; Olsen, Christine; Desmet, Gert; Wilson, Steven Ray Haakon & Lundanes, Elsa
(2020).
Preparation of porous layer open tubular columns for sensitive proteomics.
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Hermansen, Astrid; Schüller, Maria; Skottvoll, Frøydis Sved; Hara, T; Desmet, Gert & Lundanes, Elsa
[Vis alle 7 forfattere av denne artikkelen]
(2019).
Protein Analysis of Liver Oganoids with Minaturized LC-MS: Nano and PLOT Format.
Vis sammendrag
In recent years, 3D liver cellular clusters, commonly known as liver organoids, have
emerged as promising tools to replace 2D cell cultures and animal testing in the
development of drugs and the understanding of diseases. The composition and function of
liver organoids is yet not fully understood, calling for proteomic analysis. Organoids and
the medium in which they secrete into are complex in nature and limited in size and
availability; therefore, the use of narrow inner diameter separation columns is beneficial
to achieve better sensitivity. Conventional packed columns can have limitations regarding
sensitivity and separation power. Consequently, the employment of other column formats
can be explored. One such format is the porous layer open tubular (PLOT) column for
liquid chromatography (LC), which have low backpressures and smaller inner diameters,
permitting longer columns for expanding separation power. The goal of this work is to find
the optimal conditions for bottom-up proteomics of liver organoids and their media,
comparing reversed-phase LC with commercially packed columns and PLOT columns with
1 / 2
mass spectrometry (MS) detection. Initial analysis of liver organoids with commercially
packed columns and conventional run times revealed liver traits as phase 1 and phase 2
metabolism enzymes (aldehyde dehydrogenase mitochondrial, cytochrome P450 1B1 and
glutathione S-transferase Mu 3, UDP-glucuronosyltransferase 1, catechol Omethyltransferase), proteins related to nitrogen and glutamate- (glutamate
dehydrogenase) and amino acid metabolism (aspartate aminotransferase). Regarding 75
µm inner diameter/15 cm long packed columns, there was a significant difference (α =
0.05) in protein ID numbers when comparing 2 h with 4 and 8 h linear gradients from
2.7-15.3% ACN. The 4 h gradient did not significantly differ from an 8 h gradient, implying
that the protein ID numbers capacity was reached at 4 h. We have now proceeded to
explore the possibilities of PLOT LC-MS for organoid proteomics, using a 10 µm inner
diameter/56 cm long column.
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Berg, Henriette Engen; Skottvoll, Frøydis Sved; Bjørseth, Kamilla; Sæterdal, Kristina Erikstad; Brandtzaeg, Ole Kristian & Vehus, Tore
[Vis alle 11 forfattere av denne artikkelen]
(2018).
Development of nanoLC column technology for proteomic studies
.
Vis sammendrag
Background
NanoLC has emerged as a major technique in proteomics due to improved sensitivity, which enables analysis of small sample volumes and low concentrations. In our group (Bioanalytical chemistry, BACH) we focus on the development of column technology, including porous layer open tubular (PLOT) columns and self-packing of columns for proteomic applications.
Methods
The PLOT columns were used for both on-line digestion (enzyme reactors) and separations (10 µm ID, PS-DVB with ODS). Enzyme reactors (20 µm ID and multi-PLOT columns (~8 µm ID)) were immobilized with trypsin and Lys-C for protein digestion. We also compared self-packed columns of 50 µm ID (2.6 µm C18 core-shell particles) to a commercial column (75 µm ID). The columns were used for detection of CYP27a1 in MDA-MB-231 (breast cancer) cells, and biomarker candidates in MDA-MB-231 and glioblastoma multiforme (brain cancer) exosomes. For exosome isolation (cell culture medium), ultracentrifugation (“golden standard”) was compared to a Total Exosome Isolation Reagent from Thermo Fisher regarding protein yield and purity.
Results
Our PLOT columns were successfully employed for proteins, metabolites and peptides with attogram detection limits and peak capacities around 250 [1]. The enzyme reactors provided sufficient digestion in ~5 minutes compared to overnight digestions (common in off-line protocols) [2]. Furthermore, a quantitative method of the neurotoxin ricin was developed using the multi-PLOT to reduce manual sample handling [3] .
The self-packed columns obtained peak capacities comparable to the commercial nanoLC column for peptide separations [4]. The self-packed columns were used for the targeted detection of CYP27A1 (potential breast cancer biomarker) in MDA-MB-231 cells. In the recent exosome study, exosomes were present and biomarker candidates were identified. However, the characterization techniques are in our hands not satisfactory.
Conclusions
We have successfully developed sensitive nanoLC column formats for proteomic applications.
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Skottvoll, Frøydis Sved; Nilsen, Ola; Krauss, Stefan; Sullivan, Gareth & Wilson, Steven Ray Haakon
(2018).
Integrating highly miniaturized LC-MS systems with “Organ on a Chip”; the future of preclinical modelling
.
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Berg, Henriette Sjånes; Bjørseth, Kamilla; Sæterdal, Kristina Erikstad; Smetop, Tone; Skottvoll, Frøydis Sved & Vehus, Tore
[Vis alle 8 forfattere av denne artikkelen]
(2018).
Evaluation of isolation methods of glioma exosomes for biomarker discovery.
Vis sammendrag
Gliomas are the most common form of brain cancer, where the sub-group glioblastoma multiforme (GBM) is the most aggressive and the most common in adults [1]. For diagnostic, prognostic, and treatment monitoring, fast and reliable analytical methods for detection of glioblastoma tumors are essential. Non-invasive monitoring of circulating biomarkers in blood (liquid biopsy) is a desirable possibility [2]. Nano LC-MS can provide the high sensitivity, selectivity and low false positive rate needed for the proteomic analysis of exosomes, which are extracellular vesicles released from the tumor and into the bloodstream. We have optimized the packing of 50 µm capillaries (2.6 µm C18 core-shell particles) with peak capacities comparable to similar commercial nano-LC columns (75 µm ID) for peptide separations of minute samples (e.g. exosomes). These columns have previously been successfully used for the targeted detection of CYP27a (a potential breast cancer biomarker) in the MDA-MB-231 cell line.
In the present study, these in-house packed columns have been applied for proteins in exosomes isolated from GBM cell culture medium. The aim of this study was to compare isolation methods of GMB exosomes for biomarker discovery and study proteins related to GBM in exosomes. Ultracentrifugation (“golden standard” in exosome isolation) was compared to a Total Exosome Isolation Reagent from Invitrogen (Thermo Fisher Scientific) regarding sample volume, total protein amount, purity, and the presence of exosome markers and GBM biomarker candidates.
References
1. Molina, J.R., Y. Hayashi, C. Stephens, and M.M. Georgescu, Invasive Glioblastoma Cells Acquire Stemness and Increased Akt Activation. Neoplasia. 12 (2010) 453-U37.
2. Best, M.G., N. Sol, S. Zijl, J.C. Reijneveld, P. Wesseling, and T. Wurdinger, Liquid biopsies in patients with diffuse glioma. Acta Neuropathologica. 129 (2015) 849-865.
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Boger, Ida Caroline Sneis; Wilson, Steven Ray Haakon; Skottvoll, Frøydis Sved; Lundanes, Elsa & Bogen, Inger Lise
(2020).
Determination of liver organoid- and spheroid induced drug metabolites using liquid chromatography-mass spectrometry.
Kjemisk Institutt, Universitetet i Oslo.
Vis sammendrag
The liver is the main metabolizing organ in the human body, and several in vitro models have been developed to resemble hepatic drug metabolism. However, commonly used in vitro models such as human liver microsomes (HLMs), S9 fraction, and hepatocytes lack the complexity of the corresponding in vivo tissue, which limits the in vivo resemblance. Liver organoids are three-dimensional tissue models typically derived from induced pluripotent stem cells (iPSCs) and are intended to recapitulate physiological functions of the human liver. Liver spheroids are similar 3D culture systems but lack the multicellularity that characterizes the organoids. Few studies have investigated drug metabolism in liver organoids- and spheroids. The aim of this study was, therefore, to explore liver organoid- and spheroid drug metabolism using liquid chromatography-mass spectrometry (LC-MS) as the liver organoids- and spheroids can be more representative to the in vivo situation. Additionally, an LC-MS method was to be established at the Department of Chemistry for drug metabolite detection. Drug incubated samples were analyzed using a validated LC-MS method at the Department of Forensic Sciences or by the LC-MS method established at the Department of Chemistry. Heroin was chosen as the model drug, and heroin metabolism studies were initially carried out in HLMs to establish a standardized conventional approach for later drug metabolism studies in organoids and spheroids. The concentrations of heroin and its phase I metabolites, 6-monoacetylmorphine (6-MAM) and morphine were measured over time (20 minutes). The heroin metabolism method was downscaled by decreasing the HLM concentration (from 2 mg/mL to 0.1 mg/mL) and the heroin concentration (from 1 μM to 0.1 μM). The downscaled method was applied to the S9 fraction and later executed with the addition of exogenous cofactors to look for the phase II metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). With the addition of cofactors, M3G could be quantified in the S9 fraction. Preliminary heroin metabolism studies in iPSC derived liver organoids showed that the organoids metabolized heroin (1 μM) to 6-MAM and morphine. The phase II metabolites M3G and M6G were also identified but below the limit of quantification (LOQ). Both the phase I- and phase II metabolites were detected above LOQ in liver spheroid samples after incubation in 10 M heroin. To sum up, the detection of heroin drug metabolites using LC-MS showed that the liver organoids- and spheroids had heroin metabolizing properties. Additionally, LC-MS proved to be a valuable tool for detection of liver organoid induced drug metabolites.
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Schüller, Maria; Wilson, Steven Ray Haakon; Lundanes, Elsa & Skottvoll, Frøydis Sved
(2020).
A targeted proteomics approach for the investigation of drug-induced liver injury in hepatic organoids using nano liquid chromatography mass spectrometry.
Kjemisk Institutt, Universitetet i Oslo.
Vis sammendrag
Late-stage drug attrition and post-market withdrawals attributed to drug-induced liver injury (DILI) has been a prevailing concern for the pharmaceutical industry. The current gaps in preclinical safety assessment impede the termination of DILI drug candidates to earlier stages of drug development, with the consequence of possibly exposing patients to hepatotoxic agents. Recent advances in liver-emulating models, with the development of hepatic organoids, seem promising in filling these gaps. The purpose of this study was to look into opportunities for the advancement of the preclinical safety assessment of the hepatic system. This was approached by combining the liver-emulating power of hepatic organoids with the well-studied protein biomarker alanine aminotransferase isoform 1 (ALT1) to investigate their compatibility with nano liquid chromatography mass spectrometry (nanoLC-MS). A targeted proteomics approach for the absolute quantification of ALT1 was developed by a thorough assessment of signature peptide candidates and corresponding multiple reaction monitoring (MRM) transitions. NanoLC-MS platform optimization was performed to maximize detection sensitivity and reliability. From empirical nanoLC-MS platform optimization, the greatest improvement was achieved from collision energy optimization, with an increase of mean peptide peak area of 22%. ALT1 peptide assessment showed that the peptide LLVAGEGHTR with the MRM fragments y8+ and y7+ were best suited to infer the presence of ALT1 in absolute quantification. The finalized method was applied for the investigation of drug-induced liver injury in hepatic organoids, where acetaminophen was used as the model drug. This work has shown that ALT1 quantification with a nanoLC-MS platform is feasible and has great potential to support preclinical DILI detection. Further studies must be conducted to give reliable statements on the applicability of the method for drug-induced ALT1 release in hepatic organoids.
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Hermansen, Astrid; Skottvoll, Frøydis Sved; Wilson, Steven Ray Haakon & Lundanes, Elsa
(2020).
Development and implementation of open tubular columns for high-sensitivity liquid chromatography-mass spectrometry proteomics of liver organoids.
Kjemisk Institutt, Universitetet i Oslo.
Vis sammendrag
The three-dimensional liver cellular cluster model, known as liver organoids, is a promising tool to better mimic human physiology for drug development. The composition and function of liver organoids arenot yet fully understood, calling for sensitive proteomic analysis due to limited sample size and availability. The use of narrow inner diameter (ID) liquid chromatography (LC) separation columns is beneficial for increased sensitivity. A relevant column format to explore is the porous layer open tubular (PLOT), which with narrow ID and low backpressure permit the use of longer columns for higher separation power.Proteomic analysis of organoids using PLOT at column format was yet to be explored.The goal of this work was to prepare silica-based PLOT columns functionalized with octadecyl (C18) with narrow ID (10 and 5 μm) suitable for sensitive proteomic analysis of liver organoids with mass spectrometry detection. Two approaches for the preparation of silica-based PLOT columns functionalized with C18 were explored and successfully prepared. Scanning electron micrographs could indicate that a porous layer was present with both preparation methods; however, one method had challenges regarding clogging during the preparation of the columns. A peak capacity of 127 was obtained with an in-house made PLOT column (10μm x 115.2 cm) when analysing 20 ng HeLa tryptic digest using a 240-minute linear gradient (2.7-15% acetonitrile in water + 0.1% formic acid).Proteins related to the liver metabolic pathways were identified from the analysis of liver organoid fractions using one in-house made PLOT column.Thus, indicating that silica-based C18 PLOT columns constitute a promising tool for the bioanalysis of limited samples.
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Olsen, Christine; Wilson, Steven Ray Haakon; Skottvoll, Frøydis Sved; Brandtzaeg, Ole Kristian; Rongved, Pål & Lundanes, Elsa
(2019).
Synthesis and immobilization of linked Wnt-signaling pathway inhibitor on organic monoliths as a proof-of-concept of a capti remedium ad monolitus reactor for online drug deconvolution.
Universitetet i Oslo.
Vis sammendrag
A challenge in drug discovery is the identification of the drug target, called drug deconvolution. Additionally, off-target effects are considered as one of the reasons many developed drugs fail in the clinical trials. The goal of this work was to develop a solid support, displaying low secondary interactions, for immobilization of drugs (named by author as a CRAM reactor) suitable for incorporation online liquid chromatography mass spectrometry set-ups. The hypothesis was that selective purification on the online reactor would allow identification of low abundant drug targets as a consequence of reduced handling time, contamination and loss of the sample. As a proof-of-concept, an ethylene dimethacrylate-co-vinyl azlactone (EDMA-co-VDM) monolith, prepared in 180 µm inner diameter (ID) or 250 µm ID polyimide-coated fused silica capillaries, would be immobilized with Wnt-signaling pathway inhibitor 161. The 161-immobilized CRAM reactor would then attempt to selectively trap and release a low abundant protein target, tankyrase 2 (TNKS2). The EDMA-co-VDM monolith was successfully prepared in 250 µm ID capillaries. The Wnt-inhibitor 161 was rejected based on MS characterization and LDW639, a structural analogue of Wnt-inhibitor XAV939, was successfully synthesized by the author. To improve availability of LDW639 after immobilization, a linker was attached to LDW639 during synthesis. The linked LDW639 showed 50% inhibition of the Wnt-signaling pathway at a concentration of 11 µM after 24 hours incubation in cells. The EDMA-co-VDM monolith showed secondary interactions towards proteins, but the issues were resolved by quenching the reactive VDM monomer with either monoethanolamine (MEA) or an excess of linked LDW639. Immobilization of the linked LDW639 was found to be successful based on measured UV-Vis absorbance of solutions containing LDW639 was reduced by flushing a monolith, but not by monoliths already flushed with MEA (MEA monolith). The linked LDW639-immobilized CRAM reactors and the MEA monolith were not able to trap and release TNKS1/2 from human embryonic kidney 293 cells after cell lysis with a non-denaturing buffer. Showing that the identification of the drug target from complex matrices remained a challenge, even with tailored materials.
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Se alle arbeider i Cristin
Publisert 28. feb. 2018 11:59
- Sist endret 6. nov. 2020 10:02
