Frøydis Sved Skottvoll

• Hvinden, Ingvild Comfort; Berg, Henriette Engen; Sachse, Daniel; Skaga, Erlend; Skottvoll, Frøydis Sved; Lundanes, Elsa; Sandberg, Cecilie; Vik-Mo, Einar O.; Rise, Frode & Wilson, Steven Ray Haakon (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
• Lin, Ann; Skottvoll, Frøydis Sved; Rayner, Simon; Pedersen-Bjergaard, Stig; Sullivan, Gareth; Krauss, Stefan; Wilson, Steven Ray Haakon & Harrison, Sean (2019). 3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry. Electrophoresis.  ISSN 0173-0835.  41, s 56- 64 . doi: 10.1002/elps.201900170
• Olsen, Christine; Skottvoll, Frøydis Sved; Brandtzaeg, Ole Kristian; Schnaars, Christian; Rongved, Pål; Lundanes, Elsa & Wilson, Steven Ray Haakon (2019). Investigating Monoliths (Vinyl Azlactone-co-Ethylene Dimethacrylate) as a Support for Enzymes and Drugs, for Proteomics and Drug-Target Studies. Frontiers in Chemistry.  ISSN 2296-2646.  7 . doi: 10.3389/fchem.2019.00835
• Skottvoll, Frøydis Sved; Berg, Henriette Engen; Bjørseth, Kamilla; Lund, Kaja; Roos, Norbert; Bekhradnia, Sara; Thiede, Bernd; Sandberg, Cecilie; Vik-Mo, Einar O.; Røberg-Larsen, Hanne; Nyström, Bo; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). Ultracentrifugation versus kit exosome isolation: nanoLC-MS and other tools reveal similar performance biomarkers, but also contaminations. Future science OA.  ISSN 2056-5623.  5(1) . doi: 10.4155/fsoa-2018-0088 Fulltekst i vitenarkiv.

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• Boger, Ida Caroline Sneis; Nerem, Elisabeth; Skottvoll, Frøydis Sved; Harrison, Sean; Sullivan, Gareth; Lundanes, Elsa; Bogen, Inger Lise & Wilson, Steven Ray Haakon (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.

• 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. Vis sammendrag

  The newly introduces 3D liver cellular cluster model, known as liver organoids, is a promising tool to probe disease and human biology. The composition and function of liver organoids is not 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. The goal of this work is to prepare silica-based C18 PLOT columns with narrow ID (10 and 5 µm) suitable for peptide and intact protein separation and find the optimal conditions for proteomics of liver organoids and their media with mass spectrometry detection. Initial analysis of HeLa cell digest using a PLOT column (C18, 10 µm ID x 56 cm) made by Hara et al., has been performed. HeLa cell digest has also been analyzed using a commercially available packed column (C18, 75 µm x 15 cm) for comparison. Two approaches for the preparation of silica-based C18 PLOT columns with narrow ID were explored, based on methods established by Hara et al. and Vehus et al., respectively. We experienced challenges with regards to clogging of the fused silica capillaries during the formation of the porous silica layer when using the method established by Vehus et al., thus only the method of Hara et al. was further explored. Scanning electron micrographs indicate that a porous layer was present with both preparation methods.

• 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.

• 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.

• Skottvoll, Frøydis Sved; Harrison, Sean; Hansen, Frederik; Pedersen-Bjergaard, Stig; Sullivan, Gareth; Bogen, Inger Lise; Lundanes, Elsa & Wilson, Steven Ray Haakon (2020). Liver mini-organ analyses using liquid chromatography mass spectrometry. Vis sammendrag

  Organoids (self-organized multi-cellular tissue derived from e.g. patient-derived stem cells) are emerging in vitro 3D representations of human organ functionality, predicted to have a game-changing impact on drug discovery, disease modeling and personalized medicine [1]. Integrating the organoids in a microfluidic chip (i.e. organoids-on-a-chip) would allow for controllable perfusion of nutrients and manipulation of the organoids by the addition of e.g. drugs [2]. Coupling the organoid-on-a-chip online to liquid chromatography mass spectrometry (LC-MS) would enable real-time measurements of the biochemical processes that occur on-chip. However, limited sample sizes, complex matrices and the presence of low abundant analytes also create analytical challenges [3]. Moreover, the organoids are still at the developmental stage, thus fundamental questions on the functionality of the developed organoids remain to be answered. Liver organoid functionality was assessed by protein characterization of adult liver tissue and liver organoids using nanoLC-MS/MS. The proteins identified from the liver organoids covered 70-84% (1003 proteins) of the liver protein expression from adult liver tissue derived from five patients. The proteins that were shared between the adult liver tissue and liver organoids could also relate to central liver pathways (e.g. amino acid biosynthesis and glycolysis). The sample preparation technique electromembrane extraction (i.e. electrophoretic separation across an oil membrane, EME) has shown to have on-chip analytical potential [4] and was thus used in the study of heroin drug metabolism in liver organoids. Multi-well EME (100 µL solutions) allowed for simple and repeatable monitoring of heroin metabolism kinetics without interference from cell medium components (e.g. albumin). The EME extracts were also compatible with different analytical approaches (LC-MS, nanoLC-MS, capillary electrophoresis-UV). Hence, the hyphenation of EME with nanoLC-MS would be a natural next step. References 1. Method of the Year 2017: Organoids. Nature Methods. Vol. 15 (2018) 1-1. 2. Park, S.E., A. Georgescu, and D. Huh, Organoids-on-a-chip. Science. Vol. 364 (2019) 960. 3. Lin, A., F. Sved Skottvoll, S. Rayner, S. Pedersen-Bjergaard, G. Sullivan, S. Krauss, S. Ray Wilson, and S. Harrison, 3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry. ELECTROPHORESIS. Vol. 0 (2019). 4. Hansen, F.A., D. Sticker, J.P. Kutter, N.J. Petersen, and S. Pedersen-Bjergaard, Nanoliter-Scale Electromembrane Extraction and Enrichment in a Microfluidic Chip. Analytical Chemistry. Vol. 90 (2018) 9322-9329.

• Hermansen, Astrid; Schüller, Maria; Skottvoll, Frøydis Sved; Hara, T; Desmet, Gert; Lundanes, Elsa & Wilson, Steven Ray Haakon (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.

• Mrša, Ago; Boger, Ida Caroline Sneis; Nerem, Elisabeth; Skottvoll, Frøydis Sved; Harrison, Sean; Lundanes, Elsa; Sullivan, Gareth; Bogen, Inger Lise & Wilson, Steven Ray Haakon (2019). Studying organoid drug metabolism using UHPLC/nanoLC-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 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). 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. We are also exploring sensitive nano-LC-MS (traditionally used for proteomics) for studying organoid drug metabolism. Using a 50 µm (I.D) x 5 cm C18 pre-column and a 50 µm (I.D) x 12 cm C18 analytical column, coupled up with a triple quadrupole MS (QMS) using electrospray ionization (ESI), a LoD of 0.7 fg heroin was achieved. The LoD of the nano-LC-MS method gives a good foundation for future work with detection of trace liver-organoid induced drug metabolites. Taken together, LC-MS can be a valuable tool for studying metabolism properties of organoids. [1] Eisenstein, M., Organoids: the body builders, Nature Methods, (2018) 19. [2] Brandon, E. F., Raap, C. D., Meijerman, I., Beijnen, J. H., Schellens, J. H., An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons, Toxicology and applied pharmacology, (2003) 233-246

• 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. 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.

• Skottvoll, Frøydis Sved (2019). Miniaturizing LC-MS for high sensitivity clinical sample analyses. Vis sammendrag

  Clinical samples often exhibit limited sample sizes and analytes being present below the detection limit of the method, thus hampering the analytical assessment and consequently the research outcome. By narrowing the inner diameter of the separation column, the sample dilution is vastly reduced, adding to sensitivity. Hence in this study, miniaturized LC-MS has been widely used, allowing for sensitive proteomic measurements in limited samples from cell derived nanosized exosomes, patient derived glioblastoma cells and stem cell derived 3D organ models (i.e. organoids), subsequently providing with fundamental information on disease development, characteristics and responses on therapeutics.

• Skottvoll, Frøydis Sved; Boger, Ida Caroline Sneis; Harrison, Sean; Nerem, Elisabeth; Bogen, Inger Lise; Sullivan, Gareth & Wilson, Steven Ray Haakon (2019). LC-MS approaches to liver organoid analyses. Vis sammendrag

  Liquid chromatography coupled to mass spectrometry (LC-MS) is an analytical tool used to separate and measure the mass (m/z) and concentration of compounds in matrices like water, blood, and tumors. Drugs, metabolites and proteins are all measurable with LC-MS. Unfortunately, the sample is diluted during chromatography, hurting the sensitivity. Thus, the limited sample volumes and low abundant compounds of organ-on-a-chip and organoids demands miniaturization of LC-MS to enable measurements of high sensitivity. By narrowing the inner diameter (ID) of the separation column, the sample dilution is vastly reduced, adding to sensitivity, e.g. enabling analysis of limited sample volumes and low abundant compounds of the organ-on-a-chip and organoids. The bioanalytical group at the Department of Chemistry (UiO) is currently developing miniaturized systems for proteins and small molecules for assessing the properties of liver organoids. These efforts will be expanded to testing islet and adipose organoids, and will also be incorporated in to on-line systems, e.g. organ on a chip systems. We believe that LC-MS and organoid analysis will be an increasingly natural partnering.

• Berg, Henriette Engen; Skottvoll, Frøydis Sved; Bjørseth, Kamilla; Sæterdal, Kristina Erikstad; Brandtzaeg, Ole Kristian; Vehus, Tore; Hustoft, Hanne Kolsrud; Røberg-Larsen, Hanne; Thiede, Bernd; Lundanes, Elsa & Wilson, Steven Ray Haakon (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.

• Berg, Henriette Sjånes; Bjørseth, Kamilla; Sæterdal, Kristina Erikstad; Smetop, Tone; Skottvoll, Frøydis Sved; Vehus, Tore; Lundanes, Elsa & Wilson, Steven Ray Haakon (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.

• 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.
• Røberg-Larsen, Hanne; Skottvoll, Frøydis Sved; Solheim, Stian; Bjørseth, Kamilla; Thorne, James L; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Oxysterols in cancer.
• Skottvoll, Frøydis Sved; Berg, Henriette Engen; Lund, Kaja; Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon & Lundanes, Elsa (2017). Critical evaluation of isolation and characterization techniques of breast cancer exosomes.

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