-
Wilson, Steven Ray Haakon
(2021).
"Selected Talk: Direct Coupling of Organ-on-Chip with Electromembrane Extraction and Mass Spectrometry".
-
Moe, Harald; Lundanes, Elsa; Wilson, Steven Ray Haakon; Lund, Reidar & Røberg-Larsen, Hanne
(2021).
Releasing anitmicrobial peptides through liposomal nanocarriers to fight multidrug-resistant bacteria.
-
Nordli, Henriette; Røberg-Larsen, Hanne; Lundanes, Elsa & Wilson, Steven Ray Haakon
(2021).
Determination of sterols in liver organoids using liquid chromatography-mass spectrometry (LC-MS).
-
Nordli, Henriette; Aizenshtadt, Aleksandra; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne
(2021).
Oxysterols secreted from a Non-Alcoholic Fatty Liver Disease (NAFLD) induced liver organoids.
-
Wiborg, Elisa; Hermansen, Astrid; Olsen, Christine; Wilson, Steven Ray Haakon & Lundanes, Elsa
(2021).
Silica-based narrow inner diameter open tubular liquid
chromatography columns for use in proteomics.
Show summary
In the world of proteomics, which is the study of proteins and peptides, the samples are
usually small and/or with low analyte concentration. To analyse this type of samples it is
beneficial to use liquid chromatography (LC) columns with narrow inner diameter (ID)
coupled with an electrospray mass spectrometer (ESI-MS). The use of narrow columns in an
LC-ESI-MS system makes it possible to achieve high sensitivity [1, 2], due to less radial
dilution of the analytes [3]. Such narrow columns can be porous layer open tubular (PLOT)
columns, either organic polymer based [2] or silica-based [1, 4, 5].
A silica-based PLOT can be made by sol-gel synthesis [5]. In the present study, both 10 µm
and 5 µm ID silica-based PLOT columns have been prepared according to Hara et. al. [4, 5]
In this preparation method, a solution of tetramethyl orthosilicate (TMOS), polyethylenglycol
(PEG), urea and 0.01 M acetic acid is prepared and filled into pre-treated silica capillaries,
with 10 µm or 5 µm ID, either by a pressure bomb system or with a pump. The
polymerization into a porous layer structure (silica skeleton) takes place in a water bath at 25
℃, and a following heat treatment at 80 ℃ makes the mesopores in the silica skeleton. After
the porous layer has been formed, functionalization with C18 is preformed to obtain a
reversed phase PLOT column.
The columns made are used in a nano LC ESI-MS system for ultrasensitive analysis.
-
Bakketeig, Eris Aas; Olsen, Christine; Berg, Henriette Engen; Nyman, Tuula Anneli; Thiede, Bernd & Lundanes, Elsa
[Show all 8 contributors for this article]
(2021).
Optimization of concatenation of fractions in
comprehensive two dimensional liquid chromatography
bottom-up proteomics.
Show summary
Two dimensional liquid chromatography (2D-LC) has long been a commonly used technique
in bottom-up proteomics, and is as relevant today as ever [1, 2]. To maximize the number of
protein identifications, analytical chemists have strived to obtain 2D-LC systems with a high
degree of orthogonality, which is needed to reach the required high peak capacities. A system
utilizing reversed-phase chromatography (RP) in both column dimensions with a large
difference in pH has proven to yield a higher peak capacity for peptides, even with a lower
degree of orthogonality compared to other combinations [3].
The present study is based on Reubsaet, et al. [4]. The study is a comprehensive offline RPRP 2D-LC system with high pH (>9) in the first dimension and low pH (3) in the second
dimension. The method’s purpose is to maximise protein identifications from one trypsinated
protein sample. To achieve maximum identifications, Reubsaet incorporated the concept of
concatenation of fractions. In practice, concatenation is applied through collecting the column
output from the first dimension in eight numbered vials, where the 1st, 9th, 17th, … fractions
are collected in the first vial, the 2nd, 10th, 18th, … are collected in the second, etc. [4].
In the present study, the fractionation scheme is examined by changing the total number of
vials in which samples are concatenated, the number of fractions in each vial, and the
collection time for each fraction. As the method demands high precision and is very timeconsuming, automatization is practically essential. Automatization is achieved with the spider
fractionation scheme, proposed and shown by Kulak, et al.[5]. Here, a 10-port injector
connects the first dimension column to each sample vial, and switches between them
automatically, reducing the strain on the operator drastically, while removing human errors.
The optimization of the concatenation will lead to higher numbers of identified proteins in
proteomics.
-
Olsen, Christine; Lundanes, Elsa & Wilson, Steven Ray Haakon
(2021).
Disulfide bond reduction upfront liquid chromatography mass spectrometry for protein analysis.
Show summary
Microfluidic devices containing human cells or organoids (3D multicellular tissue derived in vitro from human pluripotent stem cells) constitute an exponentially growing technology for online analysis of cell responses and human organ functionality [1, 2]. Human pancreas-on-a-chip (PoC) is a relatively new technology, intended for the study of the endocrine tissue, the islets of Langerhans, which are responsible for regulation of the blood glucose levels [3]. These pancreatic islets consist of five major cell types, where the β-cells are responsible for the production of insulin [4]. For patients with type 1 diabetes, where the β-cells are destructed and unable to produce insulin, a common treatment is β-cell replacement therapy. Such therapy includes donor transplantation of either the entire pancreas or isolated islets.
Proteins, which are important compounds regarding quality of donor tissue or cells and the state of the isolated and purified islets may be studied in real-time analysis [3]. Normally, liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS) is employed to measure the proteins in complex cell samples. Prior to analysis, the proteins must be digested to peptides, as peptides are far more easily detected and identified with MS. Immobilized enzyme reactors are becoming more common for inclusion of online protein digestion, however, a remaining bottleneck to achieving complete online analysis is that time-consuming reduction and alkylation of the disulfide bonds need to be executed prior to analysis.
Therefore, a platform consisting of a photochemical system upfront an LC-MS system has been set up for achieving online reduction of the disulfide bonds [5]. Insulin has been used as a model protein as it consists of two relatively short chains, A and B, connected by two disulfide bonds. Additionally, there is an internal disulfide bond in chain A. The protein was dissolved in a solution of water and isopropanol (1+1, v/v) added 1% of acetone as the photoinitiator. As insulin was irradiated by UV-light at 254 nm, the MS revealed that intact insulin (present as m/z 1452 and m/z 1162) was broken into two chains (A chain with m/z 1191 and B chain with m/z 858), due to the reduction of the disulfide bonds. The set-up may be used for fast measurements of small proteins with post-translational modifications without protein digestion.
-
Røberg-Larsen, Hanne; Websdale, Alex; Solheim, Stian Kjønnås; Sande, Maria Therese; Hutchinson, Samantha A & Lundanes, Elsa
[Show all 8 contributors for this article]
(2020).
Fast LC-MS for determination of oxysterols in breast cancer tumours.
-
-
Wilson, Steven Ray Haakon & Gundersen, Lise-Lotte
(2020).
Fra Tippeligaen til Champions League.
Aftenposten (morgenutg. : trykt utg.).
ISSN 0804-3116.
-
Skottvoll, Frøydis Sved; Harrison, Sean; Hansen, Frederik; Pedersen-Bjergaard, Stig; Sullivan, Gareth & Bogen, Inger Lise
[Show all 8 contributors for this article]
(2020).
Liver mini-organ analyses using liquid chromatography mass spectrometry.
-
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.
-
Boger, Ida Caroline Sneis; Nerem, Elisabeth; Skottvoll, Frøydis Sved; Harrison, Sean; Sullivan, Gareth & Lundanes, Elsa
[Show all 8 contributors for this article]
(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.
-
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.
-
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.
-
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.
-
Flatval, Jonas; Berg, Henriette Engen; Lillerud, Karl Petter; Olsbye, Unni; Nielsen, Claus Jørgen & Wilson, Steven Ray Haakon
[Show all 7 contributors for this article]
(2020).
Retention of nitramines on UiO-66 ZrMOF materials and silica-based reversed phase materials
.
-
Andersen, Kari Anne; Flatval, Jonas; Mrša, Ago; Berg, Henriette Engen; Chavan, Sachin Maruti & Olsbye, Unni
[Show all 8 contributors for this article]
(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; Hvinden, Ingvild Comfort; Sachse, Daniel; Skaga, Erlend; Lundanes, Elsa & Sandberg, Cecilie
[Show all 9 contributors for this article]
(2020).
Observation of brain cancer cells’ molecular responses to chemotherapy
.
-
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?
-
Skogvold, Hanne Bendiksen; Østeby, Anja; Sandås, Elise Mørk; Rootwelt, Helge; Woldseth, Berit & Wilson, Steven Ray Haakon
[Show all 11 contributors for this article]
(2019).
Effects of storage time on the dried blood spot metabolome.
-
Hermansen, Astrid; Schüller, Maria; Skottvoll, Frøydis Sved; Hara, T; Desmet, Gert & Lundanes, Elsa
[Show all 7 contributors for this article]
(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.
-
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 Tranchida, Peter & Mondello, Luigi (Ed.),
Hyphenations of Capillary Chromatography with Mass Spectrometry.
Elsevier.
ISSN 9780128096383.
p. 319–358.
-
-
-
Berg, Henriette Engen; Skottvoll, Frøydis Sved; Bjørseth, Kamilla; Sæterdal, Kristina Erikstad; Brandtzaeg, Ole Kristian & Vehus, Tore
[Show all 11 contributors for this article]
(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.
-
Skjærvø, Øystein; Pilarova, Veronika; Khalikova, Maria; Reubsaet, Leon; Halvorsen, Trine Grønhaug & Gjelstad, Astrid
[Show all 10 contributors for this article]
(2018).
Those Who Can, Teach: Pharma Stars.
The Analytical Scientist.
ISSN 2051-4077.
1118,
p. 40–45.
-
Skogvold, Hanne Bendiksen; Sandås, Elise Mørk; Østeby, Anja; Rootwelt, Helge; Arnesen, Camilla Elene & Wilson, Steven Ray Haakon
[Show all 8 contributors for this article]
(2018).
Development and evaluation of an LC-Orbitrap MS method
for untargeted metabolomics of dried blood spots.
-
Røberg-Larsen, Hanne; Sande, Maria Therese; Hutchinson, Samantha A; Solheim, Stian Kjønnås; Lundanes, Elsa & Thorne, James L
[Show all 7 contributors for this article]
(2018).
Rapid determination of oxysterols in breast cancer tumors.
-
Skjærvø, Øystein; Pilarova, Veronika; Khalikova, Maria; Reubsaet, Leon; Halvorsen, Trine Grønhaug & Gjelstad, Astrid
[Show all 10 contributors for this article]
(2018).
Those Who Can, Teach: Pharma Stars.
The Analytical Scientist.
ISSN 2051-4077.
1118,
p. 40–45.
-
Brandtzaeg, Ole Kristian; Levernæs, Maren Christin Stillesby; Halvorsen, Trine Grønhaug; Reubsaet, Leon; Røen, Bent Tore & Vehus, Tore
[Show all 9 contributors for this article]
(2018).
Online sample processing systems for targeted nano liquid chromatography mass spectrometry bioanalysis.
-
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.
-
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.
-
Racz, Beatrix; Zawadzka, Malgorzata Elzbieta; Skytte Af Sätra, Jenny Marie; Ringvold, Amund; Rise, Frode & Lundanes, Elsa
[Show all 7 contributors for this article]
(2018).
Identification of “compound X” in goose aqueous humour.
-
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.
-
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.
-
Hvinden, Ingvild Comfort; Rise, Frode; Berg, Henriette Engen; Lundanes, Elsa & Wilson, Steven Ray Haakon
(2018).
Approaches to untargeted metabolomics of glioblastoma: NMR and MS.
-
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; Hutchinson, Sam Alex; Solheim, Stian; Wilson, Steven Ray Haakon; Lundanes, Elsa & Thorne, James L
(2018).
Oxysterols in breast cancer.
-
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.
-
Berg, Henriette Sjånes; Bjørseth, Kamilla; Sæterdal, Kristina Erikstad; Smetop, Tone; Skottvoll, Frøydis Sved & Vehus, Tore
[Show all 8 contributors for this article]
(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.
-
Wilson, Steven Ray Haakon
(2017).
Smedslunds hjertesukk : Jan Smedslund er for resignert på empiriens vegne.
Tidsskrift for Norsk Psykologforening.
ISSN 0332-6470.
54(5),
p. 488–489.
-
Sellathurai, Tharsana Mahendra; Elgstøen, Katja B. Prestø & Wilson, Steven Ray Haakon
(2022).
High precision metabolomics.
Kjemisk Institutt, Universitetet i Oslo.
-
Skottvoll, Frøydis Sved; Wilson, Steven Ray Haakon & Nilsen, Ola
(2022).
Liver organoids, mass spectrometry and separation science.
Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Oslo.
ISSN 1501-7710.
2022(2493).
Show summary
Organoids are predicted to change the future of medicine by acting as an in vitro 3D representation of human organ functionality. Organoids are grown from patient derived stem cells or adult tissue, which promise to enable new and faster drug discovery, disease modeling and personalized medicine under more realistic conditions. Organoids are still at the developmental stage and fundamental questions concerning the functionality of the developed organoids remain to be answered. Compared to other biosampling techniques, mass spectrometry (MS) has rarely been used in organoid analyses. This thesis leverages MS and separation science to analyze the biological properties of liver organoids, spanning from protein identification to small molecule drug metabolite detection. With the aim of developing selective, high throughput analyses and online biosampling integration, both conventional and cutting edge electromembrane extraction (EME) sample preparation approaches were explored.
Using bioanalytical tools including MS and separation science, we could establish that the liver organoids displayed liver organ functionality. The bioanalytical strategies we developed for liver organoid analyses contribute to greater insight into organoid response and functionality and could be important tools in future organoid development. Furthermore, online organoid integration using electromembrane extraction developed here could be used as a starting point towards developing future organ-on-a-chip systems integrated with mass spectrometry, which would be valuable in drug development, disease modeling, and personalized medicine.
-
Berg, Henriette Engen; Wilson, Steven Ray Haakon; Lundanes, Elsa; Thiede, Bernd & Vik-Mo, Einar Osland
(2022).
Exploring miniaturized chromatography formats and sample preparation methods for shotgun proteomics in limited biological samples.
Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Oslo.
ISSN 1501-7710.
2022(2522).
Show summary
A significant challenge in medical diagnostics is the development of simple but efficient tools for the detection/quantification of several biomarkers simultaneously using non-invasive sampling techniques. In this regard, the analysis of proteins (proteomics) is essential for understanding cellular processes and biomarker discovery. However, proteins vary greatly in terms of concentration levels and chemical properties in biological materials. Further, low sample sizes of modern biological models (e.g., patient-derived cell cultures, exosomes, and organoids) remain a big analytical challenge. The present work has focused on the brain cancer glioblastoma, which is in great need of increased knowledge and non-invasive sampling techniques. In addition, human organoids, which could act as a future in vitro model for disease modeling and personalized medicine, have been investigated.
We have used high-resolution mass spectrometry for protein identification, exploring a selection of miniaturized liquid chromatography formats (for separation) and sample preparation techniques. By implementing these techniques, we have been able to study exosomes, 2D/3D cell cultures, and organoids, identifying over 6300 proteins in a single run using less than 5 µg of protein. The work has provided important insight into the possibilities and challenges of several novel models. It represents a development toward deeper proteomic profiling focusing on maintaining a high protein yield and time efficiency.
-
-
Erlandsson, Tonje Monica; Wilson, Steven Ray Haakon; Skottvold, Frøydis Sved & Lundanes, Elsa
(2021).
Investigation of CYP activity in liver organoids by establishing a liquid chromatography-mass spectrometry method for measuring drug metabolism.
Universitetet i Oslo, Det matematisk-naturvitenskapelige fakultet.
-
Nordli, Henriette; Wilson, Steven Ray Haakon; Lundanes, Elsa & Røberg-Larsen, Hanne
(2021).
Towards method miniaturization for determination of sterols in liver organoids: an investigation of nano liquid chromatography-mass spectrometry (nLC-MS).
Universitetet i Oslo, Det matematisk-naturvitenskapelige fakultet.
-
Moe, Harald Røsand; Wilson, Steven Ray Haakon; Lundanes, Elsa; Røberg-Larsen, Hanne & Lund, Reidar
(2021).
Establishing a liquid chromatography-mass spectrometry method for the quantification of colistin A, a last line of defense against antibiotic resistance.
Matematisk-naturvitenskapelig fakultet, Universitetet i Oslo.
Show summary
Antibiotic resistance has become an increasingly pressing issue in recent times. With a dwindling discovery rate of new antibacterials, gram-negative bacteria threaten to return humanity to the pre-antibiotic era where mere paper cuts could result in fatal outcomes. The antimicrobial peptide colistin is our present-day last line of defense against gram-negative infections. Nevertheless, despite the recent surge in use and research on colistin, research on its biological effects and chemical behavior remains somewhat scarce. Colistin was initially abandoned due to numerous reports of neuro- and nephrotoxicity. Slow leakage from colistin-loaded liposomes may reduce this toxicity significantly. In this work, an LC-MS method for separating and quantifying colistin and polymyxin B1 was developed to measure colistin leakage from loaded liposomes over time. Isocratic elution provided adequate separation of polymyxin E1 and the internal standard, polymyxin B1, and measurements were done using a triple-quadrupole MS. Widely unreported and uninvestigated adsorption and degradation mechanisms of colistin were observed and addressed by adding a 100 mM ammonium formate buffer at pH 3 to solutions of colistin. The LC-MS method provides a reliable and accurate approach for measuring colistin at lower concentrations (< 10 µg/mL) in ammonium formate buffered solutions, and the future challenges and potential approaches for further research upon colistin are discussed. The LC-MS method described serves as a solid platform for further research on colistin, colistin toxicity negation, and more may be built upon.
-
-
Mrša, Ago & Wilson, Steven Ray Haakon
(2020).
Miniaturized liquid chromatography systems for small molecule analysis of organoids.
Kjemisk Institutt, Universitetet i Oslo.
-
Flatval, Jonas; Lundanes, Elsa; Wilson, Steven Ray Haakon; Olsbye, Unni; Nielsen, Claus Jørgen & Berg, Henriette Engen
(2020).
Retention of nitramines on silica-based reversed phase materials and UiO-66 MOF materials.
Kjemisk Institutt, Universitetet i Oslo.
Show summary
Capturing and storing of CO2 can reduce the emissions of CO2 in the atmosphere. However, such facilities may emit amines that can be transformed into the potentially carcinogenic nitramines dimethylnitramine (DMA-NO2), monoethanolnitramine (MEA-NO2) and monomethylnitramine (MMA-NO2) which may be released into the environment. Presently, methods for trace determination of these compounds are lacking. The goal of the study was to find a material that can give sufficient retention to provide an enrichment of the nitramines before liquid chromatography electrospray ionization-mass spectrometry determination. The retention of the nitramines was examined on various C18 materials and the metal-organic frameworks UiO-66 and UiO-66 1,4-benzenedicarboxylate-NH2 (UiO-66-NH2), which were packed in 100 µm inner diameter columns. Of the tested materials, UiO-66-NH2 showed the highest retention factors for the nitramines. UiO-66-NH2 with 0.1% formic acid adjusted to pH 7 as mobile phase gave the best compromise of retention factors for DMA-NO2 and MEA-NO2, as the intended liquid chromatography-mass spectrometry method was evaluated for DMA-NO2 and MEA-NO2 only. MMA-NO2 had the highest retention factor on UiO-66-NH2 with 0.05% dibutylamine in the mobile phase. In conclusion, the retention factors achieved were in general higher on the UiO-66 materials than on the C18 materials. However, the retention obtained is probably not large enough to give limit of quantification down to 4 ng/L, albeit, not tested, due to extraordinary circumstances.
-
Andersen, Kari Anne; Lundanes, Elsa; Berg, Henriette Engen; Wilson, Steven Ray Haakon; Chavan, Sachin Maruti & Lillerud, Karl Petter
[Show all 7 contributors for this article]
(2020).
UiO-66 metal-organic framework materials as stationary phases in liquid chromatography.
Kjemisk Institutt, Universitetet i Oslo.
Show summary
The metal-organic frameworks (MOFs) UiO-66 and UiO-66-NH2 are composed of zirconium oxide nodes (Zr6O4(OH)4(CO2)12)and benzene-1,4-dicarboxylate (BDC) linkers for UiO-66, and the amino-functionalised linker 2-amino-benzene-1,4-dicarboxylate (ABDC) for UiO-66-NH2. UiO-66 and UiO-66-NH2 have large surface areas and a defined pore size and are thus attractive materials for chromatographic separations. The structure of these MOFs also meansseveral retention mechanisms could influence the separation simultaneously, making the retention of functionalised organic molecules on these materials challenging to predict. Hence, the aim of this thesiswas to investigate the chromatography of selected compounds on UiO-66 and UiO-66-NH2 in liquid chromatography (LC), to learn more about the potential applications of these UiO materials.UiO-66 and UiO-66-NH2 particles were packed into 100 μm inner diameter (ID) capillaries, and a simple LC-UV system was used for testing. Both materials rapidly (<30 min) packed into columns of circa 10 cm and provided pressures < 300 bar when used in LC systems. The efficiency (plate height) of the UiO-66-and UiO-66-NH2-columnswerefound to be 18 μm and 199 μm, respectively, using uracil asa modelanalyte. However, phosphate-containing analytes were found to have severe tailing. With an aqueous mobile phase, the retention of a selection of small hydrophilic molecules was found to increase with decreasing organic component in the mobile phase. The flow rate was also found to affect retention factors, with larger retention factors for lower flowrates. The effect of temperature in the range 25–55°C appeared to follow the expected trend of decreasing retention with increasing temperature for the chosen model substances, benzene, ethylbenzene and butylbenzene.Pore volume accessibility studies wereinconclusive. However, observations made strongly suggest that benzene, ethylbenzene, propylbenzene, butylbenzene, naphthalene and phenanthrene were able to access the pores of UiO-66.In conclusion, the UiO-66 MOF materials have chromatographic properties different from thoseof the more common LC separation materials, notably in the ability to retain small polar molecules. However, more research is needed to better understand the retention mechanisms of the materials and to single out application areas for these materials in LC.
-
Boger, Ida Caroline Sneis; Wilson, Steven Ray Haakon; Skottvoll, Frøydis Sved; Lundanes, Elsa & Bogen, Inger Lise
(2020).
Determination of liver organoid- and spheroid induced drug metabolites using liquid chromatography-mass spectrometry.
Kjemisk Institutt, Universitetet i Oslo.
Show summary
The liver is the main metabolizing organ in the human body, and several in vitro models have been developed to resemble hepatic drug metabolism. However, commonly used in vitro models such as human liver microsomes (HLMs), S9 fraction, and hepatocytes lack the complexity of the corresponding in vivo tissue, which limits the in vivo resemblance. Liver organoids are three-dimensional tissue models typically derived from induced pluripotent stem cells (iPSCs) and are intended to recapitulate physiological functions of the human liver. Liver spheroids are similar 3D culture systems but lack the multicellularity that characterizes the organoids. Few studies have investigated drug metabolism in liver organoids- and spheroids. The aim of this study was, therefore, to explore liver organoid- and spheroid drug metabolism using liquid chromatography-mass spectrometry (LC-MS) as the liver organoids- and spheroids can be more representative to the in vivo situation. Additionally, an LC-MS method was to be established at the Department of Chemistry for drug metabolite detection. Drug incubated samples were analyzed using a validated LC-MS method at the Department of Forensic Sciences or by the LC-MS method established at the Department of Chemistry. Heroin was chosen as the model drug, and heroin metabolism studies were initially carried out in HLMs to establish a standardized conventional approach for later drug metabolism studies in organoids and spheroids. The concentrations of heroin and its phase I metabolites, 6-monoacetylmorphine (6-MAM) and morphine were measured over time (20 minutes). The heroin metabolism method was downscaled by decreasing the HLM concentration (from 2 mg/mL to 0.1 mg/mL) and the heroin concentration (from 1 μM to 0.1 μM). The downscaled method was applied to the S9 fraction and later executed with the addition of exogenous cofactors to look for the phase II metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). With the addition of cofactors, M3G could be quantified in the S9 fraction. Preliminary heroin metabolism studies in iPSC derived liver organoids showed that the organoids metabolized heroin (1 μM) to 6-MAM and morphine. The phase II metabolites M3G and M6G were also identified but below the limit of quantification (LOQ). Both the phase I- and phase II metabolites were detected above LOQ in liver spheroid samples after incubation in 10 M heroin. To sum up, the detection of heroin drug metabolites using LC-MS showed that the liver organoids- and spheroids had heroin metabolizing properties. Additionally, LC-MS proved to be a valuable tool for detection of liver organoid induced drug metabolites.
-
Schüller, Maria; Wilson, Steven Ray Haakon; Lundanes, Elsa & Skottvoll, Frøydis Sved
(2020).
A targeted proteomics approach for the investigation of drug-induced liver injury in hepatic organoids using nano liquid chromatography mass spectrometry.
Kjemisk Institutt, Universitetet i Oslo.
Show summary
Late-stage drug attrition and post-market withdrawals attributed to drug-induced liver injury (DILI) has been a prevailing concern for the pharmaceutical industry. The current gaps in preclinical safety assessment impede the termination of DILI drug candidates to earlier stages of drug development, with the consequence of possibly exposing patients to hepatotoxic agents. Recent advances in liver-emulating models, with the development of hepatic organoids, seem promising in filling these gaps. The purpose of this study was to look into opportunities for the advancement of the preclinical safety assessment of the hepatic system. This was approached by combining the liver-emulating power of hepatic organoids with the well-studied protein biomarker alanine aminotransferase isoform 1 (ALT1) to investigate their compatibility with nano liquid chromatography mass spectrometry (nanoLC-MS). A targeted proteomics approach for the absolute quantification of ALT1 was developed by a thorough assessment of signature peptide candidates and corresponding multiple reaction monitoring (MRM) transitions. NanoLC-MS platform optimization was performed to maximize detection sensitivity and reliability. From empirical nanoLC-MS platform optimization, the greatest improvement was achieved from collision energy optimization, with an increase of mean peptide peak area of 22%. ALT1 peptide assessment showed that the peptide LLVAGEGHTR with the MRM fragments y8+ and y7+ were best suited to infer the presence of ALT1 in absolute quantification. The finalized method was applied for the investigation of drug-induced liver injury in hepatic organoids, where acetaminophen was used as the model drug. This work has shown that ALT1 quantification with a nanoLC-MS platform is feasible and has great potential to support preclinical DILI detection. Further studies must be conducted to give reliable statements on the applicability of the method for drug-induced ALT1 release in hepatic organoids.
-
Hermansen, Astrid; Skottvoll, Frøydis Sved; Wilson, Steven Ray Haakon & Lundanes, Elsa
(2020).
Development and implementation of open tubular columns for high-sensitivity liquid chromatography-mass spectrometry proteomics of liver organoids.
Kjemisk Institutt, Universitetet i Oslo.
Show summary
The three-dimensional liver cellular cluster model, known as liver organoids, is a promising tool to better mimic human physiology for drug development. The composition and function of liver organoids arenot yet fully understood, calling for sensitive proteomic analysis due to limited sample size and availability. The use of narrow inner diameter (ID) liquid chromatography (LC) separation columns is beneficial for increased sensitivity. A relevant column format to explore is the porous layer open tubular (PLOT), which with narrow ID and low backpressure permit the use of longer columns for higher separation power.Proteomic analysis of organoids using PLOT at column format was yet to be explored.The goal of this work was to prepare silica-based PLOT columns functionalized with octadecyl (C18) with narrow ID (10 and 5 μm) suitable for sensitive proteomic analysis of liver organoids with mass spectrometry detection. Two approaches for the preparation of silica-based PLOT columns functionalized with C18 were explored and successfully prepared. Scanning electron micrographs could indicate that a porous layer was present with both preparation methods; however, one method had challenges regarding clogging during the preparation of the columns. A peak capacity of 127 was obtained with an in-house made PLOT column (10μm x 115.2 cm) when analysing 20 ng HeLa tryptic digest using a 240-minute linear gradient (2.7-15% acetonitrile in water + 0.1% formic acid).Proteins related to the liver metabolic pathways were identified from the analysis of liver organoid fractions using one in-house made PLOT column.Thus, indicating that silica-based C18 PLOT columns constitute a promising tool for the bioanalysis of limited samples.
-
Haugan, Hege Sofie; Elgstøen, Katja B. Prestø; Skogvold, Hanne Bendiksen & Wilson, Steven Ray Haakon
(2020).
Investigating the Lipid Coverage of the Dried Blood Spot Metabolome using Liquid Chromatography – Mass Spectrometry for Global Metabolomics.
Kjemisk Institutt, Universitetet i Oslo.
Show summary
Using global metabolomics for the diagnosis of inborn errors of metabolism canprovide greater insight into the diseased metabolic pathway, but it isimportant to understand thelimitations of the analytical techniquein terms of which metabolites are covered by it.In this project, the lipid coverage of a global metabolomics method usingliquid chromatography-electrospray ionization-mass spectrometryand dried blood spots wasinvestigated.The lipid coverage andpossible expansion of the coverage was investigated by extraction of commercially available lipid standards,metabolite standards,and dried blood spots,usinga selection ofsolvents in different ratioswith water: methanol, acetonitrile, 1-propanol, iso-propanol, 1-butanol, or methyl-tert-butyl-ether.The methods ability to detect physiological change in themetabolome and lipidome was also investigated.Quality control sampleswereimplemented to assessthe precision of the acquired data.Increasing the amount of organic solvent used in the extraction solution was found to increase lipid coverage, butitwasnot greatly affected by type of organic solventused. The method detectedchanges in the metabolic profile of seven healthy volunteers participating in a high intensityexercise. Several metabolites and lipids were identified and affected by the exercise.Pooled quality controls were implemented in the global metabolomics workflow and showed that the data acquired had high precision. A series of diluted pooled quality controls showed that there was a linear relationship between signal intensity and metabolite concentration, with R2>0.98.This method will bea usefulfor researching differences in metabolic profiles, but also as a supplementarydiagnostic toolfor inborn errors of metabolism.
-
Kogler, Stian & Wilson, Steven Ray Haakon
(2019).
On-line “Organ-in-a-Column” Liquid Chromatography Electrospray Ionization Mass Spectrometry for Drug Metabolism Studies.
Kjemisk institutt, Universitetet i Oslo.
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.
-
-
-
Olsen, Christine; Wilson, Steven Ray Haakon; Skottvoll, Frøydis Sved; Brandtzaeg, Ole Kristian; Rongved, Pål & Lundanes, Elsa
(2019).
Synthesis and immobilization of linked Wnt-signaling pathway inhibitor on organic monoliths as a proof-of-concept of a capti remedium ad monolitus reactor for online drug deconvolution.
Universitetet i Oslo.
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.
-
-
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.
Department of Chemistry, Faculty of Mathematics and Natural Sciences.
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.
-
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.
Department of Chemistry Faculty of Mathematics and Natural Sciences.
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.
-
Racz, Beatrix; Wilson, Steven Ray Haakon; Rise, Frode & Lundanes, Elsa
(2018).
Identification of a major UV absorbing compound in goose aqueous humour.
Kjemisk Institutt, Universitetet i Oslo.
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.
-
-
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.
Kjemisk Institutt, Universitetet i Oslo.
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.
-
-
-
-
-
-
-
