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Røberg-Larsen, Hanne; Kømurcu, Kristina Sæterdal; Kogler, Stian; Olsen, Christine; Skottvoll, Frøydis Sved & Aizenshtadt, Aleksandra
[Show all 8 contributors for this article]
(2023).
Organoids, Organ-on-a-chip and Mass Spectrometry.
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Midtøy, Lise; Kømurcu, Kristina Sæterdal; Kogler, Stian; wilhelmsen, ingrid; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon
(2023).
Sensitive targeted proteomic analysis of cholesterol metabolism enzymes in hepatic stellate cells - going towards single cell analysis .
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kvalvik, Eva; Kogler, Stian; Kømurcu, Kristina Sæterdal; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne
(2023).
Screening for biomarkers for non-alcoholic fatty liver disease using organ-in-a-column technology.
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Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L.; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne
(2022).
Oxysterols are secreted from Non-Alcoholic Fatty Liver Disease (NAFLD) induced organoids.
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midtøy, Lise; Kømurcu, Kristina Sæterdal; Kogler, Stian; wilhelmsen, Ingrid; Røberg-Larsen, Hanne & Wilson, Steven Ray Haakon
(2022).
Sensitive targeted proteomic analysis of cholesterol metabolism enzymes in hepatic stellate cells - going towards single cell analysis .
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Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne
(2022).
Measuring oxysterols secreted from liver organoids by LC-MS for the understanding of Non-alcoholic fatty liver disease
.
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Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne
(2022).
Oxysterols are secreted from Non-alcoholic Fatty Liver disease induced organoids.
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Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Krauss, Stefan Johannes Karl; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne
(2022).
Measuring oxysterols secreted from liver organoids by LC-MS for the understanding of Non-alcoholic fatty liver disease.
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Kømurcu, Kristina Sæterdal; Aizenshtadt, Aleksandra; Thorne, James L; Wilson, Steven Ray Haakon & Røberg-Larsen, Hanne
(2022).
Measuring oxysterols using LC-MS for the understanding of non alcoholic fatty liver disease (NAFLD).
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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.
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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.
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Røberg-Larsen, Hanne; Lund, Kaja; Sæterdal, Kristina Erikstad; Solheim, Stian; Vehus, Tore & Solberg, Nina
[Show all 9 contributors for this article]
(2016).
Capillary LC mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes.
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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.
Universitetet i Oslo.
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.