Steven Ray Haakon Wilson

(Recent, selected publications)

"18.8 Tesla NMR spectroscopy: an effective platform for studying glioblastoma cells´ response to a cancer drug". Ingvild Comfort Hvinden, Henriette Engen Berg, Daniel Sachse, Erlend Skaga, Elsa Lundanes, Cecilie J. Sandberg, Einar O. Vik-Mo, Frode Rise, Steven Ray Wilson J. Proteome. Res. 2019, DOI: 10.1021/acs.jproteome.8b00801.

"Online peptide epitope fishing – nanoLC-MS of protein biomarkers". Maren C. S. Levernæs, Ole Kristian Brandtzaeg, Léon Reubsaet, Elsa Lundanes, Trine G. Halvorsen, Steven Ray Wilson, Anal. Chem. 2018, 90 (23), pp 13860–13866

"A multi-channeled open tubular enzyme reactor on-line coupled with mass spectrometry for detecting ricin". Ole Kristian Brandtzaeg, Bent-Tore Roen, Siri Enger, Elsa Lundanes, Steven Ray Wilson. Anal. Chem. 2017 89 (17), 8667-8673

"Mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes". Roberg-Larsen H, Lund K, Seterdal KE, Solheim S, Vehus T, Solberg N, Krauss S, Lundanes E, Wilson SR. J. Steroid. Biochem. Mol. Biol. 2017 169, 22-28

"Proteomics tools reveal startlingly high amounts of oxytocin in plasma and serum". Brandtzaeg OK, Johnsen E, Roberg-Larsen H, Seip KF, MacLean EL, Gesquiere LR, Leknes S, Lundanes E, Wilson SR. Sci Rep 2016. 6, Article number: 31693.

• Tsjokajev, Ahmad; Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon; Anne Berit Dyve, Lingelem; Skotland, Tore; Sandvig, Kirsten & Lundanes, Elsa (2020). Mass spectrometry-based measurements of cyclic adenosine monophosphate in cells, simplified using reversed phase liquid chromatography with a polar characterized stationary phase. Journal of chromatography. B.  ISSN 1570-0232.  1160:122384, s 1- 6 . doi: 10.1016/j.jchromb.2020.122384
• Bergh, Marianne Skov-Skov; Bogen, Inger Lise; Wohlfarth, Ariane; Wilson, Steven Ray Haakon & Øiestad, Åse Marit Leere (2019). Distinguishing Between Cyclopropylfentanyl and Crotonylfentanyl by Methods Commonly Available in the Forensic Laboratory. Therapeutic Drug Monitoring.  ISSN 0163-4356.  41(4), s 519- 527 . doi: 10.1097/FTD.0000000000000617 Full text in Research Archive.
• 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 Full text in Research Archive.
• MacLean, Evan L.; Wilson, Steven Ray Haakon; Martin, W. Lance; Davis, John; Nazarloo, Hossein P. & Carter, C. Sue (2019). Challenges for measuring oxytocin: The blind men and the elephant?. Psychoneuroendocrinology.  ISSN 0306-4530.  107, s 225- 231 . doi: 10.1016/j.psyneuen.2019.05.018 Full text in Research Archive.
• 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
• Solheim, Stian; Hutchinson, Samantha A; Lundanes, Elsa; Wilson, Steven Ray Haakon; Thorne, James L & Røberg-Larsen, Hanne (2019). Fast liquid chromatography-mass spectrometry reveals side chain oxysterol heterogeneity in breast cancer tumour samples. Journal of Steroid Biochemistry and Molecular Biology.  ISSN 0960-0760.  192 . doi: 10.1016/j.jsbmb.2019.02.004 Full text in Research Archive.
• Wilson, Steven Ray Haakon; Olsen, Christine & Lundanes, Elsa (2019). Nano liquid chromatography columns. The Analyst.  ISSN 0003-2654.  144(24), s 7090- 7104 . doi: 10.1039/c9an01473j Full text in Research Archive.
• Bergh, Marianne Skov-Skov; Bogen, Inger Lise; Wilson, Steven Ray Haakon & Øiestad, Åse Marit Leere (2018). Addressing the Fentanyl Analogue Epidemic by Multiplex UHPLC-MS/MS Analysis of Whole Blood. Therapeutic Drug Monitoring.  ISSN 0163-4356.  40(6), s 738- 748 . doi: 10.1097/FTD.0000000000000564
• Dias, Irundika H.K.; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2018). Chromatography of oxysterols. Biochimie.  ISSN 0300-9084. . doi: 10.1016/j.biochi.2018.05.004
• Levernæs, Maren Christin Stillesby; Brandtzaeg, Ole Kristian; Amundsen, Sunniva Furre; Reubsaet, Leon; Lundanes, Elsa; Halvorsen, Trine Grønhaug & Wilson, Steven Ray Haakon (2018). Selective Fishing for Peptides with Antibody Immobilized Acrylate Monoliths, Coupled Online with NanoLC-MS. Analytical Chemistry.  ISSN 0003-2700.  90, s 13860 . doi: 10.1021/acs.analchem.8b00935
• 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 Full text in Research Archive.
• Berg, Henriette Sjånes; Sæterdal, Kristina Erikstad; Smetop, Tone; Rozenvalds, Rūdolfs; Brandtzaeg, Ole Kristian; Vehus, Tore; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Self-packed core shell nano liquid chromatography columns and silica-based monolithic trap columns for targeted proteomics. Journal of Chromatography A.  ISSN 0021-9673.  1498, s 111- 119 . doi: 10.1016/j.chroma.2017.03.043
• Brandtzaeg, Ole Kristian; Røen, Bent Tore; Enger, Siri; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Multichannel open tubular enzyme reactor online coupled with mass spectrometry for detecting ricin. Analytical Chemistry.  ISSN 0003-2700.  89(17), s 8667- 8673 . doi: 10.1021/acs.analchem.7b02590
• Comninos, Alexander N; Wall, Matthew B; Demetriou, Lysia; Shah, Amar J; Clarke, Sophie A; Narayanaswamy, Shakunthala; Nesbitt, Alexander; Izzi-Engbeaya, Chioma; Prague, Julia K; Abbara, Ali; Ratnasabapathy, Risheka; Salem, Victoria; Nijher, Gurjinder M; Jayasena, Channa N; Tanner, Mark; Bassett, Paul; Mehta, Amrish; Rabiner, Eugenii A; Hönigsperger, Christoph; da Silva, Meire Ribeiro; Brandtzaeg, Ole Kristian; Lundanes, Elsa; Wilson, Steven Ray Haakon; Brown, Rachel C; Thomas, Sarah A; Bloom, Stephen R & Dhillo, Waljit S (2017). Kisspeptin modulates sexual and emotional brain processing in humans. Journal of Clinical Investigation.  ISSN 0021-9738.  127(2), s 709- 719 . doi: 10.1172/JCI89519
• da Silva, Meire Ribeiro; Brandtzaeg, Ole Kristian; Vehus, Tore; Lancas, Fernando Mauro; Wilson, Steven Ray Haakon & Lundanes, Elsa (2017). An automated and self-cleaning nano liquid chromatography mass spectrometry platform featuring an open tubular multi-hole crystal fiber solid phase extraction column and an open tubular separation column. Journal of Chromatography A.  ISSN 0021-9673.  1518, s 104- 110 . doi: 10.1016/j.chroma.2017.08.071
• Gregus, Michael; Røberg-Larsen, Hanne; Lundanes, Elsa; Foret, Frantisek; Kubán, Petr & Wilson, Steven Ray Haakon (2017). Non-aqueous capillary electrophoretic separation of cholesterol and 25-hydroxycholesterol after derivatization with Girard P reagent. Chemistry and Physics of Lipids.  ISSN 0009-3084.  207, s 87- 91 . doi: 10.1016/j.chemphyslip.2017.05.014
• Gundersen, Cathrine Brecke; Zhu, Liang; Lindahl, Sofia; Wang, Shiyu; Wilson, Steven Ray Haakon & Lundanes, Elsa (2017). LC-MS/MS Method for Simultaneous Determination of Monoethanol- and Dimethylnitramine in Aqueous Soil Extracts. Chromatographia.  ISSN 0009-5893.  80(9), s 1475- 1481 . doi: 10.1007/s10337-017-3355-6
• Røberg-Larsen, Hanne; Abele, Silvija; Demir, Deniz; Dzabijeva, Diana; Amundsen, Sunniva Furre; Wilson, Steven Ray Haakon; Bartkevics, Vadims & Lundanes, Elsa (2017). Rugged Large Volume Injection for Sensitive Capillary LC-MS Environmental Monitoring. Frontiers in Chemistry.  ISSN 2296-2646.  AUG(2017) . doi: 10.3389/fchem.2017.00062 Full text in Research Archive.
• Røberg-Larsen, Hanne; Lund, Kaja; Sæterdal, Kristina Erikstad; Solheim, Stian; Vehus, Tore; Solberg, Nina; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes. Journal of Steroid Biochemistry and Molecular Biology.  ISSN 0960-0760.  169, s 22- 28 . doi: 10.1016/j.jsbmb.2016.02.006
• Skjærvø, Øystein; Brandtzaeg, Ole Kristian; Lausund, Kristian Blindheim; Pabst, Oliver; Martinsen, Ørjan Grøttem; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Exploring bioimpendance instrumentation for the characterization of open tubular liquid chromatography columns. Journal of Chromatography A.  ISSN 0021-9673.  1534, s 195- 200 . doi: 10.1016/j.chroma.2017.12.060
• Ulanova, Lilia; Pinheiro, Marina; Vibe, Carina Beatrice; Nunes, Clàudia; Misaghian, Dorna; Wilson, Steven Ray Haakon; Zhu, Kaizheng; Fenaroli, Federico; Winther-Larsen, Hanne Cecilie; Reis, Saletta & Griffiths, Gareth (2017). Treatment of Francisella infections via PLGA-and lipid-based nanoparticle delivery of antibiotics in a zebrafish model. Diseases of Aquatic Organisms.  ISSN 0177-5103.  125(1), s 19- 29 . doi: 10.3354/dao03129
• Vehus, Tore; Waaler, Jo; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Combining HIC, SEC, and IEX with Fluorescence Polarization for Drug Target Discovery. LC GC North America.  ISSN 1527-5949.  30(5), s 232- 239 Full text in Research Archive.
• Åstrand, Ove Alexander Høgmoen; Viktorsson, Elvar Örn; Kristensen, Aleksander Lim; Ekeberg, Dag; Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon; Gabrielsen, Mari; Sylte, Ingebrigt; Rustan, Arild; Thoresen, G. Hege; Rongved, Pål & Kase, Eili Tranheim (2017). Synthesis, in vitro and in vivo biological evaluation of new oxysterols as modulators of the liver X receptors. Journal of Steroid Biochemistry and Molecular Biology.  ISSN 0960-0760.  165, s 323- 330 . doi: 10.1016/j.jsbmb.2016.07.010 Full text in Research Archive.
• Vibe, Carina Beatrice; Fenaroli, Federico; Pires, David; Wilson, Steven Ray Haakon; Bogoeva, Vanya; Kalluru, Raja Sab; Speth, Martin; Anes, Elsa; Griffiths, Gareth & Hildahl, Jon Paul (2016). Thioridazine in PLGA nanoparticles reduces toxicity and improves rifampicin therapy against mycobacterial infection in zebrafish. Nanotoxicology.  ISSN 1743-5390.  10(6), s 680- 688 . doi: 10.3109/17435390.2015.1107146
• Brandtzaeg, Ole Kristian; Johnsen, Elin Follaug; Røberg-Larsen, Hanne; Seip, Knut Fredrik; MacLean, Evan L.; Gesquiere, Laurence R.; Leknes, Siri; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Proteomics tools reveal startlingly high amounts of oxytocin in plasma and serum. Scientific Reports.  ISSN 2045-2322.  6:31693 . doi: 10.1038/srep31693 Full text in Research Archive.
• Doowod, Rua Kareem; Adusumalli, Ravi; Tykesson, Emil; Johnsen, Elin Follaug; Lundanes, Elsa; Prydz, Kristian & Wilson, Steven Ray Haakon (2016). Determination of 3′-phosphoadenosine-5′-phosphosulfate in cells and Golgi fractions using hydrophilic interaction liquid chromatography–mass spectrometry. Journal of Chromatography A.  ISSN 0021-9673. . doi: 10.1016/j.chroma.2016.10.001
• Johnsen, Elin Follaug; Brandtzaeg, Ole Kristian; Vehus, Tore; Røberg-Larsen, Hanne; Bogoeva, Vanya; Ademi, Ornela; Hildahl, Jon Paul; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). A critical evaluation of Amicon Ultra centrifugal filters for separating proteins, drugs and nanoparticles in biosamples. Journal of Pharmaceutical and Biomedical Analysis.  ISSN 0731-7085.  120, s 106- 111 . doi: 10.1016/j.jpba.2015.12.010

View all works in Cristin

• Andersen, Kari Anne; Flatval, Jonas; Mrša, Ago; Berg, Henriette Engen; Chavan, Sachin Maruti; Olsbye, Unni; Wilson, Steven Ray Haakon & Lundanes, Elsa (2020). Metal organic framework material UiO-66 as a stationary phase in liquid chromatography. Show summary

  Metal organic framework materials (MOFs) are interesting candidates for applications in multiple disciplines, where chromatography is one of the fields where possible applications might exist. The porous structure and large surface area are key features that make MOFs potential candidates for future stationary phases. In the present study, the MOF UiO-66 has been investigated as a stationary phase in a nano-liquid chromatography (LC) system. UiO-66 is composed of zirconium oxide ion nodes and benzene dicarboxylate (BDC) linkers which form a face-centred cubic structure with octahedral pores (opening 6 Å, cavity 11 Å) and tetrahedral pores (opening 6 Å, cavity 7.5 Å) in a 1+2 ratio. Figure adapted from Cliffe et al., Nature communications volume 5 (2014)1. Columns with a 100 µm inner diameter have been packed in-house and tested in a simple LC-UV system for several test substances. The UiO-66 particles pack rapidly and yield columns with low back pressure. We observe reversed phase selectivity, but our experimental data also suggest other interaction mechanisms, phosphate group adsorption to zirconium oxide nodes among them.

• Berg, Henriette Engen; Halldórsson, Skarphéðinn; Thiede, Bernd; Vik-Mo, Einar O.; Lundanes, Elsa & Wilson, Steven Ray Haakon (2020). Micro pillar array columns for comprehensive proteomics. Show summary

  Pillar array nano liquid chromatography columns can potentially replace packed nano liquid chromatography (nLC) columns for analyses of complex samples due to high chromatographic performance and low backpressure. In our study, pillar array columns are used for the detection of potential biomarkers in Glioblastoma Multiforme (GBM), which is the most aggressive form of brain cancer in adults. Pillar array columns consist of several micropillars etched into silicon wafers using lithographic micromachining. The interest of these columns in nLC format has increased as an option to packed nLC columns as the chromatographic efficiency could outperform nLC columns packed with sub-2 µm particles while maintaining the sensitivity [1]. In contrast to packed columns, where eddy dispersion is the main source of band broadening due to irregular flow paths between the particles, the perfectly ordered pillars in pillar array columns eliminate the eddy dispersion to a minimum. The low backpressure of pillar array columns compared to that of packed columns also permits the use of exceptionally long columns, increasing the chromatographic performance further. Using pillar array columns, plate numbers over 1 million and reduced plate heights (h) even <1 µm have been achieved [2-4]. High chromatographic performance is especially important in comprehensive proteomics. Pre-fractionation (using e.g. gel electrophoresis) is often added in proteomic protocols to increase the throughput [5]. However, performing pre-fractionation can be time-consuming in addition to the highly increased instrument time required. Recently, a micro-Chip pillar array nLC column (µPACTM) compatible with standard nLC instrumentation has been commercialized (Figure 1) [6]. Our question was; could the use of µPAC nLC columns reduce the need for pre-fractionation in comprehensive proteomics of GBM biopsies?

• Berg, Henriette Engen; Hvinden, Ingvild Comfort; Sachse, Daniel; Skaga, Erlend; Lundanes, Elsa; Sandberg, Cecilie; Vik-Mo, Einar O.; Rise, Frode & Wilson, Steven Ray Haakon (2020). Observation of brain cancer cells’ molecular responses to chemotherapy.
• 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)”. Show summary

  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.

• Flatval, Jonas; Berg, Henriette Engen; Lillerud, Karl Petter; Olsbye, Unni; Nielsen, Claus Jørgen; Wilson, Steven Ray Haakon & Lundanes, Elsa (2020). Retention of nitramines on UiO-66 ZrMOF materials and silica-based reversed phase materials.
• 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. Show summary

  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. Show summary

  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.

• Olsen, Christine; Scholz, Hanne; Wilson, Steven Ray Haakon & Lundanes, Elsa (2020). Towards online reduction and protein digestion of secreted proteins from pancreatic islets of Langerhans related to diabetes mellitus type I. Show summary

  What is diabetes mellitus type 1, and how can liquid chromatography assist in the search for a cure? The human pancreas is involved in control of metabolism and energy consumption by both an exocrine and an endocrine function. In the exocrine tissue, a multitude of digestive enzymes are produced and secreted into the small intestine to assist in breaking down proteins, fats and carbohydrates. The endocrine tissue, islets of Langerhans (5% of the total pancreatic mass) are responsible for regulation of the blood glucose levels. The vital metabolic hormone insulin is synthesized in one of five major cell types in the islets, called β-cells [1]. However, there are two types of chronic diseases related to insulin deficiency; an autoimmune disease causes selectively destruction of the β-cells in the islets or the body cannot effectively use the produced insulin, called diabetes mellitus type 1 and type 2, respectively. Diabetes mellitus type 1 or type 2 have been diagnosed in 250 000 Norwegians (approx. 4.7% of the Norwegian population), where 28 000 have been diagnosed with type 1 diabetes mellitus (0.5%) [2]. The most common treatment is exogenous insulin therapy. When insulin was discovered in 1920s the mortality of diabetes type 1 was reduced from a guaranteed fatal diagnosis to a manageable condition with a normal lifespan within the 1950s. However, secondary complications due to diabetic nephropathy, retinopathy and neuropathy led to a renewed interest in β-cell replacement therapy. Progress in β-cell replacement with either pancreas or islet transplantation has led to impressive 1- and 5-year insulin independence rates of approximately 85% and 50 %, respectively [3]. To assist in further progress in β-cell replacement therapy, liquid chromatography with mass spectrometry detection (LC-MS) of the secreted proteins from the pancreas and the islets will aid in the understanding of the treatment of diabetes mellitus type 1. In this work, we seek to enable fully online determination of proteins (in a bottom-up approach) by combining reduction and protein digestion upstream LC-MS (Figure 1). Online protein digestion has been successfully executed with immobilized enzyme reactors (IMERs) in different formats [4-6], but most of the samples have been reduced and alkylated prior to online digestion. We hope to enable real-time determination of proteins with disulfide bonds (such as insulin) by incorporation of a photochemical system for rapid reduction of disulfide bonds [7].

• 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. Show summary

  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. Show summary

  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. Show summary

  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.

• Kogler, Stian & Wilson, Steven Ray Haakon (2019). On-line “Organ-in-a-Column” Liquid Chromatography Electrospray Ionization Mass Spectrometry for Drug Metabolism Studies. Show summary

  Organoids and organ-on-a-chip (OoC) are human-induced pluripotent stem cell-derived miniaturized organs that recapitulate organ specific functions. They open up a plethora of opportunities to change the way we test drugs, research diseases, and treat patients. In this context, liquid chromatography (LC) and mass spectrometry (MS) will have to be used, e.g. for monitoring drug metabolism, toxicity screens and organ function. On-line coupling of these technologies would allow for automatized, high-throughput systems. By downsizing the LC-MS part, low abundant analytes can also be determined. In this study, an on-line sampling and analysis system for OoC-technology has been developed. The system features hepatic organoids in a flow-through tube with continuous medium flow (organ-in-a-column, OiC), a two-loop valve system for sampling, reverse phase liquid chromatography (LC) analysis, and electrospray ionization mass spectrometry (ESI-MS). Phase 1 metabolism of heroin to its metabolite morphine was successfully measured on-line with the described system. Live/dead double staining revealed the potential of long-term cultivation of organoids in the system. In parallel, tin oxide coated fused silica nanoelectrospray ionization (nanoESI) emitters suitable for the most narrow bore chromatographic columns have been developed with HF etching and an atomic layer deposition (ALD) approach. The emitters have been tested against commercially available stainless steel (SS) emitters by injections of heroin and showed similar signal-to-noise ratios. These emitters will find a use in a future miniaturized version of the system described herein. In summary, a proof-of-concept system for on-line measurement of heroin metabolites has successfully been developed for a simple OoC-like device. NanoESI emitters for the most narrow columns have also been developed with a conductive ALD-coating of SnO2.

• 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. Show summary

  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. Show summary

  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.

• Riddervold, Jan Thomas & Wilson, Steven Ray Haakon (2019). Immunocapture of oxytocin followed by liquid chromatography-mass-spectrometric detection.
• Sande, Maria Therese; Wilson, Steven Ray Haakon; Lundanes, Elsa & Røberg-Larsen, Hanne (2019). Enzymatic hydrolysis with cholesterol esterase in the presence of surfactant improves efficiency for sample preparation of side-chain oxysterols in biosamples.
• Skogvold, Hanne Bendiksen; Østeby, Anja; Sandås, Elise Mørk; Rootwelt, Helge; Woldseth, Berit; Wilson, Steven Ray Haakon; Ferrante, Linda; Opdal, Siri Hauge; Rognum, Torleiv Ole; Rønning, Per Ola & Elgstøen, Katja B. Prestø (2019). Effects of storage time on the dried blood spot metabolome.
• 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. Show summary

  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.

• Wilson, Steven Ray Haakon; Berg, Henriette Engen; Røberg-Larsen, Hanne & Lundanes, Elsa (2019). Hyphenations of one-dimensional capillary liquid chromatography with mass spectrometry: state-of-the-art applications, In Peter Tranchida & Luigi Mondello (ed.),  Hyphenations of Capillary Chromatography with Mass Spectrometry.  Elsevier.  ISBN 9780128096383.  Kapittel 3.3.  s 319 - 358
• Amundsen, Sunniva Furre; Wilson, Steven Ray Haakon; Berg, Henriette Engen; Røberg-Larsen, Hanne & Lundanes, Elsa (2018). Online entrapment of epitope-containing peptides from integrins using immobilized antibodies.
• Ben Hassine, Esma; Lundanes, Elsa; Wilson, Steven Ray Haakon; Vehus, Tore; Brandtzaeg, Ole Kristian & Berg, Henriette Engen (2018). Capillary poly(styrene-co-octadecene-co-divinylbenzene) monolithic trap columns for bioanalytical analysis. Show summary

  The increasing demand for faster and more efficient separations for biological samples have driven organic polymer-based monolithic columns back into the spotlight. The nature of the monolithic structure allows the use of higher flow rates without increasing the system backpressure. They are also easier to contain inside the column body, opposed to particle packed columns, which are held in place by frits. These are some of the advantages that monolithic columns offer compared to particle packed columns. Poly(styrene-co-octadecene-co-divinylbenzene) (PS-OD-DVB) monolithic columns have been prepared and characterized using the neuropeptide oxytocin, “the love hormone” as test compound. The columns, 50 µm inner diameter x 100 mm in length, were to be used as trap columns in a miniaturized liquid chromatography-mass spectrometry (LC-MS) column switching system for bottom-up proteomics analysis. The monolithic PS-OD-DVB columns were prepared in situ (inside the fused silica capillary), using a polymerization mixture consisting of an initiator (lauroyl peroxide), a crosslinker (divinylbenzene), monomers (styrene and 1-octadecene), a good porogen (N,N-dimethylformamide (DMF)) and a bad porogen (1-decanol). Polymerization time, polymerization temperature, initiator concentration, and monomer to porogen ratios were investigated in order to obtain a monolithic structure with a high surface area and good permeability. The best PS-OD-DVB monolithic column was prepared using a mixture of 1.9% lauroyl peroxide (weight percentage of monomer amount), 14.2% divinylbenzene, 15.2% monomers, 11.9% DMF and 57.5% 1-decanol. The column was polymerized for one hour at 73°C and gave a plate height of 62 µm, a backpressure of 23 bar at a flow rate of 500 nL/min with a mobile phase consisting of 20% B (acetonitrile (ACN), water, formic acid (FA), 80/20/0.1, v/v/v) and 80% A (water with 0.1% (v/v) FA) and a retention time repeatability of 2.6% (relative standard deviation). The repeatability of the columns made by various individuals was satisfactory. However, when preparing twenty-one columns by the same individual, the repeatability revealed to be questionable. Hence, further improvements in the polymerization procedure do seem necessary and might be able to enhance the repeatability of the columns.

• 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. Show summary

  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. Show summary

  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.

• Brandtzaeg, Ole Kristian; Levernæs, Maren Christin Stillesby; Halvorsen, Trine Grønhaug; Reubsaet, Leon; Røen, Bent Tore; Vehus, Tore; da Silva, Meire Ribeiro; Røberg-Larsen, Hanne; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). Online sample processing systems for targeted nano liquid chromatography mass spectrometry bioanalysis.
• Brandtzaeg, Ole Kristian; Røen, Bent Tore; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). Qualitative detection of ricin using a multichannel enzyme reactor coupled online with nano liquid chromatography with mass spectrometry detection.
• Brandtzaeg, Ole Kristian; Wilson, Steven Ray Haakon; Lundanes, Elsa; Reubsaet, Leon; Krauss, Stefan & Martinsen, Ørjan Grøttem (2018). Online sample Preparation Platforms for Targeted Nano Liquid Chromatography Mass Spectrometry Bioanalysis. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. 1968.
• Hassine, Esma Ben; Berg, Henriette Engen; Brandtzaeg, Ole Kristian; Vehus, Tore; Wilson, Steven Ray Haakon & Lundanes, Elsa (2018). Capillary poly(styrene-co-octadecene-co-divinylbenzene) monolithic columns.
• Hvinden, Ingvild Comfort; Rise, Frode; Berg, Henriette Engen; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). Approaches to untargeted metabolomics of glioblastoma: NMR and MS.
• Hvinden, Ingvild Comfort; Wilson, Steven Ray Haakon; Rise, Frode; Berg, Henriette Engen & Lundanes, Elsa (2018). Nuclear magnetic resonance spectroscopy based metabolomics discovers biomarkers of glioblastoma drug response. Show summary

  Glioblastoma is the most common and aggressive form of brain cancer. Even with comprehensive treatment regimes, patients face an expected survival of only 15 months. Currently, methods for assessing treatment response are lacking; it is difficult to accurately determine the efficacy of a treatment. The goal of the present study was to contribute to treatment assessment by scouting for metabolic biomarkers occurring in response to exposure to chemotherapeutic agents temozolomide (TMZ) and sepantronium bromide (YM155). Untargeted metabolomics of lysate from cultured glioblastoma cells was carried out with liquid state proton nuclear magnetic resonance (NMR) spectroscopy at resonance frequency 800 MHz. Spectral data were analyzed with two different multivariate statistical methods: principal component analysis (PCA) and partial least squares (PLS) regression. For YM155, two biomarker candidates were found: citric and lactic acid. Citric acid appeared to increase most in samples from cell lines less sensitive to YM155. Lactic acid decreased in all cell lines and was considered a more general biomarker of treatment exposure. TMZ-treated samples were not distinguishable from control samples, most likely due to too short exposure time (24 hours). Analyses with nano hydrophilic interaction liquid chromatography coupled with mass spectrometry (MS) corroborated the findings by NMR spectroscopy and statistical analyses. Both citric acid and lactic acid are biomarker candidates, but a more detailed understanding of their fluctuations in glioblastoma during treatment is needed. Nevertheless, they represent genuine candidates and should be considered for further in vivo magnetic resonance spectroscopy (MRS) studies. In the future, the biomarkers could be monitored with MRS, allowing a more unambiguous and personalized assessment of response to treatment in individual patients.

• Kular, Ramneet Kaur; Wilson, Steven Ray Haakon; Røberg-Larsen, Hanne & Lundanes, Elsa (2018). Towards ultrafast liquid chromatography of 3’-phosphoadenosine-5’-phosphosulfate using a short hydrophilic interaction chromatography column.
• Kular, Ramneet Kaur; Wilson, Steven Ray Haakon; Røberg-Larsen, Hanne & Lundanes, Elsa (2018). Towards ultrafast liquid chromatography of 3’-phosphoadenosine-5’-phosphosulfate using zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry.
• Olsen, Christine; Brandtzaeg, Ole Kristian; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). On-line trapping of proteins with monolith-bound drugs, as a tool in drug target discovery.
• Pabst, Oliver; Martinsen, Ørjan Grøttem; Tronstad, Christian & Wilson, Steven Ray Haakon (2018). Electrical properties of human skin: From linear recordings of exogenous electrodermal activity to non-linear memristor measurements. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. 1971.
• Racz, Beatrix; Wilson, Steven Ray Haakon; Rise, Frode & Lundanes, Elsa (2018). Identification of a major UV absorbing compound in goose aqueous humour. Show summary

  About 5% of solar radiation comes from the ultraviolet (UV) region. It carries high energy and can do serious damage to living organisms. The eye of animals and humans is a critically exposed area and contains UV absorbing compounds like proteins, tryptophan, tyrosine, ascorbic acid and uric acid in the aqueous humour (AH) for protection. In addition, an unknown component (compound X) which causes red-shift in the UV absorbance spectrum at 254 nm has been observed in heavily exposed species like goose, which migrates at 10 000 m altitude. X is a major absorber. UV measurements confirmed the presence of a compound in geese which has higher absorption at 254 nm compared to other species. Compound X also has fluorescence activity that resembled indole-functionality. Aqueous humour samples from different species (goose, chicken, turkey) were measured by nuclear magnetic resonance spectroscopy (NMR). The proton NMR spectra did not show any specific high abundant compound in the goose eye at the aromatic region compared to the other species, thus NMR could not reveal what made the difference between in UV absorbance. Mass spectrometry with atmospheric pressure photoionization (APPI) has earlier showed that the molar mass of the compound X could be 149 g/mole, which suggests that compound X might be the indole: 5,6-dihydroxyindole (DHI). DHI has a central role in the biosynthesis of melanin which takes place in melanocyte cells. DHI tends to easily be polymerized and one of the results is a notable change in its solubility. It was not possible to obtain a suitable external standard which did not undergo auto-polymerization, mirroring the observations with the isolated compound.To further test the hypothesis of DHI being compound X, reversed phase (RP) high performance liquid chromatography (HPLC) and hydrophilic interaction liquid chromatography (HILIC) were performed. Ascorbic acid co-eluted with the most abundant peak (compound X) in RP-HPLC. In HILIC the most abundant peak was not ascorbic acid. Hence, compound X is not ascorbic acid, and subsequent experiments showed that it was neither the UV absorbing compounds tryptophan, tyrosine or uric acid. After using several analytical tools, the hypothesis that compound X is 5,6-dihydroxyindole was strengthened. However an external standard is still necessary to provide accurate information.

• Racz, Beatrix; Zawadzka, Malgorzata Elzbieta; Skytte Af Sätra, Jenny Marie; Ringvold, Amund; Rise, Frode; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). Identification of “compound X” in goose aqueous humour.
• Røberg-Larsen, Hanne; Hutchinson, Sam Alex; Solheim, Stian; Wilson, Steven Ray Haakon; Lundanes, Elsa & Thorne, James L (2018). Oxysterols in breast cancer.
• Røberg-Larsen, Hanne; Sande, Maria Therese; Hutchinson, Samantha A; Solheim, Stian Kjønnås; Lundanes, Elsa; Thorne, James L & Wilson, Steven Ray Haakon (2018). Rapid determination of oxysterols in breast cancer tumors.
• Sandås, Elise Mørk; Elgstøen, Katja B. Prestø; Østeby, Anja; Skogvold, Hanne Bendiksen & Wilson, Steven Ray Haakon (2018). Liquid chromatography - Orbitrap mass spectrometry is a useful tool in untargeted metabolomics analysis of dried blood spots in clinical chemistry. Show summary

  There is a need for comprehensive analysis of the dried blood spot (DBS) metabolome to study both new and known inborn errors of metabolism (IEM). The purpose of this study was to complete and evaluate an untargeted metabolomics method using liquid chromatography – electrospray ionization – Q Exactive Orbitrap mass spectrometry (MS), for analysis of one punch (3.2 mm diameter corresponding to about 3 µL whole blood) of a DBS. The criteria for evaluation and measurements were inspired by stringency applied by the World Anti-Doping Agency. In this regard, the instrument repeatability and assay reproducibility were satisfactory with relative standard deviation (% RSD) of peak areas below 10 % and retention times below1 %, using a pooled control sample, injected three times each day during sample analysis for 11 days. Compared to perimeter punches, analyzing center punches improved the repeatability. An increased organic solvent amount in the reconstitution solution, and reduced m/z range of the MS (focusing more on each m/z increases sensitivity) increased the number of hydrophobic and low-abundant compounds detected, respectively. The quantification of most of the method evaluation compounds (acylcarnitines and amino acids) was satisfactory, with linear correlation of R2 above 0.99 in signal vs. concentration plots, using 1-4 punches (about 3, 6, 9 and 12 µL whole blood) of the DBS, but some polar compounds were affected by matrix effects. Tandem MS data was acquired to secure better identification. Combining the method with bioinformatics, one could identify changes between samples taken after free diet, overnight fasting (12 hours) and prolonged fasting (36 hours), using only 3 µL blood on paper. The method will be used to detect differences in the metabolome of patient and healthy samples in research and in future diagnostics.

• Skjærvø, Øystein; Pilarova, Veronika; Khalikova, Maria; Reubsaet, Leon; Halvorsen, Trine Grønhaug; Gjelstad, Astrid; Øiestad, Elisabeth Leere; Lundanes, Elsa; Wilson, Steven Ray Haakon & Pedersen-Bjergaard, Stig (2018). Those Who Can, Teach: Pharma Stars. The Analytical Scientist.  ISSN 2051-4077.  1118, s 40- 45
• Skjærvø, Øystein; Pilarova, Veronika; Khalikova, Maria; Reubsaet, Leon; Halvorsen, Trine Grønhaug; Gjelstad, Astrid; Øiestad, Elisabeth Leere; Lundanes, Elsa; Wilson, Steven Ray Haakon & Pedersen-Bjergaard, Stig (2018). Those Who Can, Teach: Pharma Stars. The Analytical Scientist.  ISSN 2051-4077.  1118, s 40- 45
• Skogvold, Hanne Bendiksen; Sandås, Elise Mørk; Østeby, Anja; Rootwelt, Helge; Arnesen, Camilla Elene; Wilson, Steven Ray Haakon; Rønning, Per Ola & Elgstøen, Katja B. Prestø (2018). Development and evaluation of an LC-Orbitrap MS method for untargeted metabolomics of dried blood spots.
• 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.
• Skytte Af Sätra, Jenny Marie; Nielsen, Claus Jørgen; Van Bavel, Albert; Lundanes, Elsa & Wilson, Steven Ray Haakon (2018). Developing procedures for trace analysis of small amines in water, using LC-MS and GC-MS.
• Solheim, Stian Kjønnås; Lundanes, Elsa; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne (2018). Exploring selectivity and sensitivity for oxysterol measurements using liquid chromatography-mass spectrometry (LC-MS).
• Vehus, Tore; Wilson, Steven Ray Haakon; Lundanes, Elsa; Krauss, Stefan; Halvorsen, Trine Grønhaug & Morth, Jens Preben (2018). Nano Liquid Chromatography-Tandem Mass Spectrometry Platforms for Determination of Low Abundant Wnt/Beta-Catenin Proteins in Cancer Research. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. 1962.
• Arnesen, Camilla Elene; Wilson, Steven Ray Haakon; Elgstøen, Katja B. Prestø & Østeby, Anja (2017). Optimization of liquid chromatographic parameters for untargeted metabolomics of dried blood spots.
• Bjørseth, Kamilla; Wilson, Steven Ray Haakon; Brandtzaeg, Ole Kristian & Lundanes, Elsa (2017). Evaluation of exosome isolation techniques for hydrophilic interaction liquid chromatography-mass spectrometry based metabolomics.
• Demir, Deniz; Abele, Silvija; Røberg-Larsen, Hanne; Wilson, Steven Ray Haakon & Lundanes, Elsa (2017). Development of an ultrasensitive method for multiresidue screening of pharmaceutical products in environmental water samples.
• Demir, Deniz; Lundanes, Elsa; Wilson, Steven Ray Haakon; Abele, Silvija & Røberg-Larsen, Hanne (2017). Automatic capillary liquid chromatography tandem mass spectrometry method for pharmaceutical products in environmental water samples.
• Johnsen, Elin Follaug; Lundanes, Elsa; Wilson, Steven Ray Haakon & Greibrokk, Tyge (2017). High performance analytical Tools for small molecules in biosamples. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. 1803.
• Røberg-Larsen, Hanne; Horne, Hildegunn; Holme, Ane Moe; Holm, Maia Blomhoff; Roland, Marie Cecilie; Michelsen, Trond; Wilson, Steven Ray Haakon & Henriksen, Tore (2017). Human in vivo studies of placenta physiology: oxysterols and metabolomic profiling.
• Røberg-Larsen, Hanne; Skottvoll, Frøydis Sved; Solheim, Stian; Bjørseth, Kamilla; Thorne, James L; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Oxysterols in cancer.
• Skogvold, Hanne Bendiksen; Elgstøen, Katja B. Prestø; Østeby, Anja & Wilson, Steven Ray Haakon (2017). Laboratory diagnostics: Maximizing sensitivity of a Q-Exactive Orbitrap mass spectrometer for untargeted metabolomics of dried blood spots.
• 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.
• Solheim, Stian; Røberg-Larsen, Hanne; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Optimizing selectivity and sensitivity for oxysterol measurements using liquid chromatography-mass spectrometry (LC-MS).
• Sved Skottvoll, Frøydis; Lund, Kaja; Røberg-Larsen, Hanne; Berg, Henriette Sjånes; Thiede, Bernd; Lundanes, Elsa & Wilson, Steven Ray Haakon (2017). Diagnostic potential of exosomes: The purification issue.
• Wilson, Steven Ray Haakon (2017). Smedslunds hjertesukk : Jan Smedslund er for resignert på empiriens vegne. Tidsskrift for Norsk Psykologforening.  ISSN 0332-6470.  54(5), s 488- 489
• Abele, Silvija; Røberg-Larsen, Hanne; Dzabijeva, Diana; Demir, Deniz; Amundsen, Sunniva F.; Wilson, Steven Ray Haakon; Bartkevics, Vadims & Lundanes, Elsa (2016). Simultaneous determination of pharmaceutical product residues in water by HPLC-MS/MS using online SPE.
• Berg, Henriette Engen; Vehus, Tore; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Optimization of in-house packed nano-LC columns for peptide separations in clinical medicine. Show summary

  Early detection of biomarkers for diseases, such as cancer, is one of the most challenging and important goals in clinical medicine [1]. In modern protein analysis, mass spectrometry (MS) with electrospray ionization (ESI) has increased in popularity (compared to typical immunoassay methods) because of the sensitivity, universality, and low false positive rate. However, the MS is dependent of chromatographic separation of biological samples to lower the complexity (reduced number of analytes for the MS to handle per time unit). In this regard, the goal is an effective separation with high peak capacity (for gradient elution) and sensitivity to be able to detect possible biomarkers in or low concentrations and/or low sample volumes. Nano-LC columns can provide high sensitivity because of the low inner diameter (ID) and compatibility with ESI-MS, but the scarcity of commercial <75 µm ID packed columns limits the separation principles available. In pre-clinical research, self-packing of nano-LC columns can increase the flexibility in assessing (novel) separation materials to a low cost. In the present study, the packing process has been optimized to obtain sensitive and efficient packed columns (50 µm ID) for peptide separations with comparable performance to commercial nano-LC columns. In addition, PicoFrit columns with integrated emitter from New Objective (Woburn, MA, USA) will be examined to evaluate the potential reduction in extra-column band broadening.

• Berg, Henriette Sjånes; Lundanes, Elsa; Wilson, Steven Ray Haakon & Vehus, Tore (2016). Optimization of in-house packing of nano liquid chromatography columns - How to pack low-cost nano liquid chromatography columns?. Show summary

  Liquid chromatography-mass spectrometry (LC-MS) approaches for measurement of biomarkers are increasing in popularity due to the selectivity and sensitivity. To match the detection limits of the immunoassay approaches (such as enzyme-linked immunosorbent assay (ELISA) and Western Blot (WB)), LC columns with narrow inner diameter are preferable. The goal of this work was to establish a simple and low cost slurry packing procedure to obtain 50 µm inner diameter (ID) x 150 mm particle packed nanoLC columns with comparable performance to a 75 µm ID x 150 mm commercial nanoLC column. Slurry packing with 2.6 µm solid-core reversed phase particles was performed with an in-house made pressure bomb system (< 200 bar). Column efficiency was measured as peak capacity (number of peaks separated in a gradient window, PC), calculated using peptides from tryptic digest of human serum albumin (HSA). To maintain sedimentation of particles, heat or magnetic stirring was needed. 80/20 % acetonitrile (ACN)/water as slurry solvent was found to give both the fastest packing and the most efficient columns measured as PC (89 ± 11 at 10 % of the peak height for columns packed with magnetic stirring). Nevertheless, the column performance was quite similar for all slurry solvents and packing approaches. “Pico Frit” column housings from New Objective and two frit-making procedures (polymerization and sintering of silica particles) for standard column housings were compared, but none outperformed the others. The PC of the commercial column packed with the same material as the in-house packed columns, was approximately 30 % higher than the best performing in-house packed column (102 vs 136). However, the retention time repeatability between replicates was equal to the commercial column for most columns packed with 80/20 % ACN/water. In conclusion, this work presents a fast and low cost packing procedure of nanoLC columns with similar performance to commercial nanoLC columns.

• Berg, Henriette Sjånes; Vehus, Tore; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Optimization of in-house packed nano-LC columns for peptide separations.
• Brandtzæg, Ole Kristian Merkesvik; Johnsen, Elin Follaug; Røberg-Larsen, Hanne; Leknes, Siri; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Robust nano liquid chromatography mass spectrometry system for targeted determination of oxytocin in human serum/plasma..
• Dowood, Rua Kareem; Wilson, Steven Ray Haakon; Lundanes, Elsa & Johansen, Elin (2016). Determination of 3'-phosphoadenosine-5'-phosphosulfate in cells using hydrophilic interaction liquid chromatography and mass spectrometry. Show summary

  3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the universal donor of sulfate to sulfotransferases, a large group of enzymes that transfer sulfate to various molecules in the sulfation pathway. Methods that have been previously used to determine this important metabolite lack in specificity. The aim of the project was to develop a method for determining PAPS in cells and cellular fractions, using hydrophilic interaction liquid chromatography and mass spectrometry. The developed method gave a retention time under 10 minutes, acceptable chromatographic efficiency and satisfactory repeatability in measurements. The method was able to separate PAPS from ATP and ADP, which could interfere with PAPS signal in the MS as they share mass spectrometric features. In addition, a simple sample preparation procedure was developed using ultra centrifugal filters. The method was evaluated regarding linearity, carry-over, within- and between-day precision etc., and PAPS levels was estimated in MDCK cell lines and their Golgi fractions, also following treatment with sodium chlorate (a PAPS synthesis inhibitor). The combination of the ultra centrifugal filtration, as a sample preparation step, ZIC-pHILIC and ESI-MS detection was found to be a suitable technique for the analysis of PAPS.

• Røberg-Larsen, Hanne; Lund, Kaja; Sved Skottvoll, Frøydis; Gregus, Michael; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). 27-hydroxycholesterol in cancer exosomes – potential new separation method?.
• Røberg-Larsen, Hanne; Lund, Kaja; Sæterdal, Kristina Erikstad; Solheim, Stian; Vehus, Tore; Solberg, Nina; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Capillary LC mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes.
• Røberg-Larsen, Hanne; Lundanes, Elsa; Wilson, Steven Ray Haakon & Krauss, Stefan (2016). High sensitivity measurements of side-chain oxysterols using liquid chromatography mass spectrometry. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. ?.
• Skjærvø, Øystein; Brandtzæg, Ole Kristian Merkesvik; Martinsen, Ørjan Grøttem; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Characterization of polymer layer open tubular columns for liquid chromatography and open tubular enzyme reactors for on-line sample preparation.
• Skjærvø, Øystein; Wilson, Steven Ray Haakon; Lundanes, Elsa; Brandtzaeg, Ole Kristian & Martinsen, Ørjan Grøttem (2016). Characterization of open tubular enzyme reactors and polymer layer open tubular columns for liquid chromatography. Show summary

  The structure, thickness and characteristics of liquid chromatography (LC) column materials are central factors regarding their performance. However, miniaturized capillaries (sub 20.0 μm inner diameter, typically used for small sample analysis) are difficult to characterize. The main goal of this work was to develop a simple, yet accurate, electrochemical impedance spectroscopy (EIS) method for characterization of the polymer layers of narrow open tubular (OT) columns. In situ prepared polymerized capillaries (poly(styrene-co-divinylbenzene) and poly(2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone) OT capillaries were characterized. The developed EIS method proved simplicity in terms of providing layer thickness determinations compared to determination by scanning electron microscope (SEM). In addition, screening of the polymer layer homogeneity throughout the capillaries was simplified by EIS. The porosity of the polymer layer OT columns was investigated by EIS, SEM and sample loading capacity measurements (using LC). Under pore-blocking conditions, none of the polymerized capillaries could be shown to have porosity, so it was assumed that a thin polymer layer is beneficial for both types of capillaries in order to provide a high surface area, i.e. more available functional groups. EIS methods have not been published for capillary characterization by date. However, the method showed promising results and has great potential for future applications regarding characterization and preparation optimization of polymerized capillaries.

• Smetop, Tone; Lundanes, Elsa; Vehus, Tore & Wilson, Steven Ray Haakon (2016). Silica-based monolithic pre-columns in miniaturized liquid chromatography. Show summary

  In miniaturized liquid chromatography (LC) an online clean up system and/or pre-concentration step, is essential to increase large loading capacity, loading speed could be used so the analysis is faster, and it is more robust (i.e. sample cleanup of salts and excess reagents. In this study the goal was to make efficient and robust reversed phase (RP) silica-based monolithic pre-columns with an inner diameter (ID) of ≤50 µm to be used in a switching system with an online pre-column. The silica skeleton was found to be the trickiest part to make in these columns, because they easily shrink and crack. Different procedures were investigated and a procedure was found to give the thinnest skeleton (0.4 µm) without shrinking/cracking. The solutions found to give the most homogenous skeleton was obtained with this procedure: Pre-treated and silanized fused silica capillaries were filled with a polymerization solution consisting of 4.5 mL tetramethoxysilane (TMOS), 1.06 g polyethylene glycol (PEG) (Mn = 10 000) and 10 mL aqueous acetic acid (0.01 M), which was allowed to react at 40 oC for 24 h. The capillary was subsequently filled with ammonium hydroxide (0.1 M) and allowed to react at 120 oC for 3h, before the skeleton was dried at 330 oC for 24 h. The reason for absence of shrinking/cracking was the final heating step at 330 oC. A “thiol-ene” click reaction was found to be an easy way to link the stationary phase (SP) to the skeleton. First vinylfunctionalization of the skeleton was carried out with vinyltrimethoxysilane (VTMS) followed by a click reaction with octanethiol, that gave a RP column with C8 as SP. Columns with plate height down to 70 µm were made, tested with a simple LC-UV system. One of the columns was successfully implemented in an LC-Mass Spectrometry (MS) system as a pre-column in combination with a 50 µm x 15 cm Accucore (2.6 µm) RP analytical column. A peak capacity of 79 was obtained, compared to a system with a commercial packed pre-column that gave a peak capacity of 47(measured at half peak height). Many procedures presented in papers were investigated and they are not good enough to reproduce a successful silica- based monolithic column.

• Smetop, Tone; Vehus, Tore; Wilson, Steven Ray Haakon & Lundanes, Elsa (2016). Silica-based monolithic pre-columns in miniaturized liquid chromatography.
• Sæterdal, Kristina E.; Vehus, Tore; Røberg-Larsen, Hanne; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). CYP27A1 – its relation to oxysterols and cancer, and analytical challenges.
• Sæterdal, Kristina Erikstad; Vehus, Tore; Røberg-Larsen, Hanne; Krauss, Stefan; Lundanes, Elsa & Wilson, Steven Ray Haakon (2016). Determination of CYP27a1 in biological samples using nano liquid chromatography mass spectrometry. Show summary

  The cytochrome P450 enzyme, CYP27a1, converts cholesterol into 27-hydroxycholesterol, and both the metabolite and the enzyme are associated with breast cancer, promoting proliferation and metastasis. CYP27A1 is enriched in cancer tissues, and the amount of CYP27a1 correlates to tumor grade. Hence, CYP27a1 is a potential biomarker, and a method for determination and quantification is needed. Antibody assays shows limited performance, and therefore the goal of this work was to develop a nano liquid chromatography mass spectrometry (LC-MS) method for determination of CYP27a1. A sample preparation workflow for CYP27a1 in cell samples was investigated. CYP27a1 was identified by nano LC-MS in MDA-MB-231 breast cancer cells by fractionating cell lysates using gel electrophoresis. Enrichment of CYP27a1 in cell samples using immunoprecipitation, an antibody based method for protein extraction, did not provide adequate identification. However the former method works when looking at other proteins, and this confirms that there are limitations in using antibody based methods for determination and quantification of CYP27a1.

View all works in Cristin