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Jankovskaja, Skaidre
Publications (10 of 11) Show all publications
Tassidis, H., Jankovskaja, S., Awad, K., Ohlsson, L., Gjörloff Wingren, A. & Gustafsson, A. (2024). Investigation of tryptophan to kynurenine degradation in response to interferon-γ in melanoma cell lines. Biochemistry and Biophysics Reports, 37, Article ID 101612.
Open this publication in new window or tab >>Investigation of tryptophan to kynurenine degradation in response to interferon-γ in melanoma cell lines
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2024 (English)In: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 37, article id 101612Article in journal (Refereed) Published
Abstract [en]

Background and aim: Melanoma is a fatal form of skin cancer that carries a grave prognosis if the cancer cells spread and form metastases. The Kynurenine (Kyn) pathway is activated by the enzyme indoleamine 2,3-dioxygenase 1 (IDO-1) and has been shown to have a role in tumour progression. We have previously shown that interferon-γ (IFN-γ) acts as an inducer of tryptophan (Trp) degradation to Kyn in keratinocytes of the basal layer in a 3D epidermis model. Before extending our reconstructed human epidermis model to not only contain keratinocytes but also fibroblasts and melanocytes/melanoma cells, we have in this study set out to investigate possible differences between primary adult melanocytes and six melanoma cell lines regarding the expression of the immune checkpoint inhibitors IDO-1 and programmed death ligand 1 (PD-L1) together with Kyn production.

Methods: The melanocytes and melanoma cells were stimulated with 1–20 ng/ml of IFN-γ and the levels of Trp to Kyn degradation were monitored with high-performance liquid chromatography (HPLC). To analyze the viability of the cell types after IFN-γ treatment, an MTT assay was performed. mRNA quantity of IDO-1, PD-L1 and IFN-γ receptor (IFN-GR1) was analyzed with qPCR.

Results: After 24 h, only the metastatic cell line WM-266-4 was affected by all concentrations of IFN-γ, whereas at 48 h, the higher IFN-γ concentrations gave a more pronounced effect on the viability in all cell types. Trp was detected at various levels in the culture medium from all cell types before and after IFN-γ treatment. The degradation to Kyn was detected in primary melanocytes, Mel Juso, and Mel Ho cell lines after 24 h of treatment and low levels of IFN-γ. However, the higher concentration of IFN-γ, 20 ng/ml, induced Kyn to various degrees in all cell types after 24 h. The change in mRNA quantity of IDO-1 and PD-L1 was similar in all cell types.

Conclusion: To conclude, no significant difference in upregulation of the immune checkpoint inhibitors PD-L1 and IDO-1 was seen between primary tumour and metastatic melanoma. IFN-γ stimulation of melanocytes and different stages of melanoma cell lines resulted in an increased Kyn/Trp ratio in the more aggressive melanoma cells when a high concentration was used (20 ng/ml) but when a lower concentration of IFN-γ (5 ng/ml) was used an increased Kyn/Trp ratio were detected in media from primary melanocytes and early-stage melanoma.

Place, publisher, year, edition, pages
Elsevier, 2024
Keywords
IDO-1, Interferon-γ, Kynurenine, Melanocytes, melanoma, Programmed death ligand 1, Tryptophan
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:mau:diva-64868 (URN)10.1016/j.bbrep.2023.101612 (DOI)001146252700001 ()38188364 (PubMedID)2-s2.0-85180557691 (Scopus ID)
Available from: 2024-01-08 Created: 2024-01-08 Last updated: 2024-02-27Bibliographically approved
Morin, M., Jankovskaja, S., Ruzgas, T., Henricson, J., Anderson, C. D., Brinte, A., . . . Björklund, S. (2022). Hydrogels and Cubic Liquid Crystals for Non-Invasive Sampling of Low-Molecular-Weight Biomarkers-An Explorative In Vivo Study. Pharmaceutics, 14(2), Article ID 313.
Open this publication in new window or tab >>Hydrogels and Cubic Liquid Crystals for Non-Invasive Sampling of Low-Molecular-Weight Biomarkers-An Explorative In Vivo Study
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2022 (English)In: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 14, no 2, article id 313Article in journal (Refereed) Published
Abstract [en]

The molecular composition of human skin is altered due to diseases, which can be utilized for non-invasive sampling of biomarkers and disease diagnostics. For this to succeed, it is crucial to identify a sampling formulation with high extraction efficiency and reproducibility. Highly hydrated skin is expected to be optimal for increased diffusion of low-molecular-weight biomarkers, enabling efficient extraction as well as enhanced reproducibility as full hydration represents a well-defined endpoint. Here, the aim was to explore water-based formulations with high water activities, ensuring satisfactory skin hydration, for non-invasive sampling of four analytes that may serve as potential biomarkers, namely tryptophan, tyrosine, phenylalanine, and kynurenine. The included formulations consisted of two hydrogels (chitosan and agarose) and two different liquid crystalline cubic phases based on the polar lipid glycerol monooleate, which were all topically applied for 2 h on 35 healthy subjects in vivo. The skin status of all sampling sites was assessed by electrical impedance spectroscopy and transepidermal water loss, enabling explorative correlations between biophysical properties and analyte abundancies. Taken together, all formulations resulted in the successful and reproducible collection of the investigated biomarkers. Still, the cubic phases had an extraction capacity that was approximately two times higher compared to the hydrogels.

Place, publisher, year, edition, pages
MDPI, 2022
Keywords
low-molecular-weight biomarker, tryptophan, kynurenine, tyrosine, phenylalanine, tryptophan-to-kynurenine ratio, skin barrier integrity, stratum corneum, natural moisturizing factor, electrical impedance spectroscopy, transepidermal water loss
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:mau:diva-50952 (URN)10.3390/pharmaceutics14020313 (DOI)000765124800001 ()35214046 (PubMedID)2-s2.0-85124460011 (Scopus ID)
Available from: 2022-04-06 Created: 2022-04-06 Last updated: 2024-02-05Bibliographically approved
Jankovskaja, S., Morin, M., Gustafsson, A., Anderson, C. D., Lehoczki, B., Engblom, J., . . . Ruzgas, T. (2022). Non-Invasive, Topical Sampling of Potential, Low-Molecular Weight, Skin Cancer Biomarkers: A Study on Healthy Volunteers.. Analytical Chemistry, 94(15), 5856-5865
Open this publication in new window or tab >>Non-Invasive, Topical Sampling of Potential, Low-Molecular Weight, Skin Cancer Biomarkers: A Study on Healthy Volunteers.
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2022 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 94, no 15, p. 5856-5865Article in journal (Refereed) Published
Abstract [en]

Monitoring of low-molecular weight cancer biomarkers, such as tryptophan (Trp) and its derivative kynurenine (Kyn), might be advantageous to non-invasive skin cancer detection. Thus, we assessed several approaches of topical sampling of Trp and Kyn, in relation to phenylalanine (Phe) and tyrosine (Tyr), on the volar forearm of six healthy volunteers. The sampling was performed with three hydrogels (made of agarose or/and chitosan), hydrated starch films, cotton swabs, and tape stripping. The biomarkers were successfully sampled by all approaches, but the amount of collected Kyn was low, 20 ± 10 pmol/cm2. Kyn quantification was below LOQ, and thus, it was detected only in 20% of topical samples. To mitigate variability problems of absolute amounts of sampled amino acids, Tyr/Trp, Phe/Trp, and Phe/Tyr ratios were assessed, proving reduced inter-individual variation from 79 to 45% and intra-individual variation from 42 to 21%. Strong positive correlation was found between Phe and Trp, pointing to the Phe/Trp ratio (being in the 1.0–2.0 range, at 95% confidence) being least dependent on sampling materials, approaches, and sweating. This study leads to conclusion that due to the difficulty in quantifying less abundant Kyn, and thus the Trp/Kyn ratio, the Phe/Trp ratio might be a possible, alternative biomarker for detecting skin cancers.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2022
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:mau:diva-51301 (URN)10.1021/acs.analchem.1c05470 (DOI)000792814500018 ()35394278 (PubMedID)2-s2.0-85128387453 (Scopus ID)
Available from: 2022-05-04 Created: 2022-05-04 Last updated: 2023-11-29Bibliographically approved
Morin, M., Björklund, S., Jankovskaja, S., Moore, K., Delgado-Charro, M. B., Ruzgas, T., . . . Engblom, J. (2022). Reverse Iontophoretic Extraction of Skin Cancer-Related Biomarkers. Pharmaceutics, 14(1), Article ID 79.
Open this publication in new window or tab >>Reverse Iontophoretic Extraction of Skin Cancer-Related Biomarkers
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2022 (English)In: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 14, no 1, article id 79Article in journal (Refereed) Published
Abstract [en]

Non-invasive methods for early diagnosis of skin cancer are highly valued. One possible approach is to monitor relevant biomarkers such as tryptophan (Trp) and kynurenine (Kyn), on the skin surface. The primary aim of this in vitro investigation was, therefore, to examine whether reverse iontophoresis (RI) can enhance the extraction of Trp and Kyn, and to demonstrate how the Trp/Kyn ratio acquired from the skin surface reflects that in the epidermal tissue. The study also explored whether the pH of the receiver medium impacted on extraction efficiency, and assessed the suitability of a bicontinuous cubic liquid crystal as an alternative to a simple buffer solution for this purpose. RI substantially enhanced the extraction of Trp and Kyn, in particular towards the cathode. The Trp/Kyn ratio obtained on the surface matched that in the viable skin. Increasing the receiver solution pH from 4 to 9 improved extraction of both analytes, but did not significantly change the Trp/Kyn ratio. RI extraction of Trp and Kyn into the cubic liquid crystal was comparable to that achieved with simple aqueous receiver solutions. We conclude that RI offers a potential for non-invasive sampling of low-molecular weight biomarkers and further investigations in vivo are therefore warranted.

Place, publisher, year, edition, pages
MDPI, 2022
Keywords
tryptophan, kynurenine, tryptophan-to-kynurenine ratio, cancer-related biomarkers, non-invasive extraction, bicontinuous cubic liquid crystals
National Category
Pharmaceutical Chemistry
Identifiers
urn:nbn:se:mau:diva-50524 (URN)10.3390/pharmaceutics14010079 (DOI)000758502900001 ()35056976 (PubMedID)2-s2.0-85122161916 (Scopus ID)
Available from: 2022-03-09 Created: 2022-03-09 Last updated: 2024-02-05Bibliographically approved
Hoang, V. C., Shafaat, A., Jankovskaja, S., Gomes, V. G. & Ruzgas, T. (2021). Franz cells for facile biosensor evaluation: A case of HRP/SWCNT-based hydrogen peroxide detection via amperometric and wireless modes. Biosensors & bioelectronics, 191, Article ID 113420.
Open this publication in new window or tab >>Franz cells for facile biosensor evaluation: A case of HRP/SWCNT-based hydrogen peroxide detection via amperometric and wireless modes
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2021 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 191, article id 113420Article in journal (Refereed) Published
Abstract [en]

Reducing animal use in biosensor research requires broader use of in vitro methods. In this work, we present a novel application of Franz cells suitable for biosensor development and evaluation in vitro. The work describes how Franz cell can be equipped with electrodes enabling characterization of biosensors in close proximity to skin. As an example of a sensor, hydrogen peroxide biosensor was prepared based on horseradish peroxidase (HRP)/single-walled carbon nanotube (SWCNT)-modified textile. The electrode exhibited lower detection limit of 0.3 μM and sensitivity of 184 μA mM−1 cm−2. The ability of this biosensor to monitor H2O2 penetration through skin and dialysis membranes was evaluated in Franz cell setup in amperometric and wireless modes. The results also show that catalase activity present in skin is a considerable problem for epidermal sensing of H2O2. This work highlights opportunities and obstacles that can be addressed by assessment of biosensors in Franz cell setup before progressing to their testing in animals and humans.

Place, publisher, year, edition, pages
Elsevier, 2021
Keywords
Epidermal sensing, Franz cell, Hydrogen peroxide biosensor, Skin membrane, Animals, Biosensors, Electrodes, Oxidation, Amperometric, H$-2$/O$-2$, Horse-radish peroxidase, Hydrogen peroxide biosensors, Hydrogen peroxide detections, In-vitro, Single-walled carbon, Hydrogen peroxide
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:mau:diva-44657 (URN)10.1016/j.bios.2021.113420 (DOI)000685993400006 ()34182432 (PubMedID)2-s2.0-85108599982 (Scopus ID)
Available from: 2021-07-07 Created: 2021-07-07 Last updated: 2023-10-31Bibliographically approved
Jankovskaja, S. (2021). Non-invasive monitoring of low molecular weight biomarkers relevant to skin inflammation and cancer. (Doctoral dissertation). Malmö: Malmö universitet
Open this publication in new window or tab >>Non-invasive monitoring of low molecular weight biomarkers relevant to skin inflammation and cancer
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Development of skin inflammation and cancer in viable epidermis and dermis involve slow molecular weight (LMW) metabolites. We hypothesize that these LMW compounds can be collected on the surface of the skin and used for non-invasive diagnostics of skin disorders. Keeping in mind that substantial transdermal penetrationis achieved only for molecules of < 500 Da, we focused on topical monitoring of LMW biomarkers. In this thesis we investigated non-invasive, topical methods for monitoring LMW biomarkers by relevant in vitro and in vivo experiments. The LMW biomarkers were:

- reactive oxygen species (ROS), specifically, hydrogen peroxide, H2O2

- amino acids and their derivatives, i.e., tryptophan (Trp), kynurenine (Kyn; a Trpderivative), phenylalanine (Phe), and tyrosine (Tyr; a Phe derivative).

Initially, we have carried out in vitro experiments using dermatomed porcine skin and cell cultures. We characterized permeability of the biomarkers through skin and assessed methods of their monitoring. By using Prussian white particles, deposited on porcine skin, we demonstrated that hydrophilic biomarkers, such as H2O2, permeate the skin mainly through hair follicle pathways (Paper I). In paper II, we have showed that the enzymes transforming Trp to the inflammation and cancer biomarker Kyn, are expressed in the basal layer of epidermis. The magnitude of changes of the Trp/Kyn ratio in the cell culture model was assessed. In paper III, we have characterized Trp and Kyn permeability through skin in vitro, concluding that their permeabilities through stratum corneum are comparable. By in vivo experiments outlined in Paper IV, we have demonstrated the feasibility of topical, non-invasive sampling of Trp and Kyn, in relation to other amino acids. Kyn detection was compromised by its low abundance on the skin. In paper V, we performed a proof-of-concept study in vivo and confirmed that non-invasive sampling of Trp and amino acids of similar abundance, such as Phe and Tyr, is more robust. We concluded that Phe/Trp ratio might be equally good biomarker of skin disorders as a predicted Trp/Kyn ratio. Summarizing, the results of this thesis provide basic knowledge for deeper clinical studies of non-invasive, topical sampling of hydrophilic LMW biomarkers of skin inflammation and cancer.

Place, publisher, year, edition, pages
Malmö: Malmö universitet, 2021. p. 66
Series
Malmö University Health and Society Dissertations, ISSN 1653-5383 ; 2021:9
Keywords
Tryptophan, Kynurenine, Phenylalanine, Tyrosine, Low molecular Weight Biomarkers, Non-Invasive, Skin Surface Sampling, Skin Permeability, Hydrogels, Prussian White
National Category
Health Sciences
Research subject
Health and society
Identifiers
urn:nbn:se:mau:diva-46859 (URN)10.24834/isbn.9789178772230 (DOI)978-91-7877-222-3 (ISBN)978-91-7877-223-0 (ISBN)
Public defence
2021-11-29, Live stream and on location at HS assembly hall, Jan Waldenströms gata 25, ö, 09:00
Opponent
Supervisors
Note

Paper III published in dissertation as manuscript with title Non-invasive, Topical Sampling of Potential Skin Cancer Biomarkers,Kynurenine and Tryptophan: Study on Healthy Volunteers

Available from: 2021-11-15 Created: 2021-11-15 Last updated: 2022-11-09Bibliographically approved
Jankovskaja, S., Engblom, J., Rezeli, M., Marko-Varga, G., Ruzgas, T. & Björklund, S. (2021). Non-invasive skin sampling of tryptophan/kynurenine ratio in vitro towards a skin cancer biomarker. Scientific Reports, 11(1), Article ID 678.
Open this publication in new window or tab >>Non-invasive skin sampling of tryptophan/kynurenine ratio in vitro towards a skin cancer biomarker
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2021 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 11, no 1, article id 678Article in journal (Refereed) Published
Abstract [en]

The tryptophan to kynurenine ratio (Trp/Kyn) has been proposed as a cancer biomarker. Non-invasive topical sampling of Trp/Kyn can therefore serve as a promising concept for skin cancer diagnostics. By performing in vitro pig skin permeability studies, we conclude that non-invasive topical sampling of Trp and Kyn is feasible. We explore the influence of different experimental conditions, which are relevant for the clinical in vivo setting, such as pH variations, sampling time, and microbial degradation of Trp and Kyn. The permeabilities of Trp and Kyn are overall similar. However, the permeated Trp/Kyn ratio is generally higher than unity due to endogenous Trp, which should be taken into account to obtain a non-biased Trp/Kyn ratio accurately reflecting systemic concentrations. Additionally, prolonged sampling time is associated with bacterial Trp and Kyn degradation and should be considered in a clinical setting. Finally, the experimental results are supported by the four permeation pathways model, predicting that the hydrophilic Trp and Kyn molecules mainly permeate through lipid defects (i.e., the porous pathway). However, the hydrophobic indole ring of Trp is suggested to result in a small but noticeable relative increase of Trp diffusion via pathways across the SC lipid lamellae, while the shunt pathway is proposed to slightly favor permeation of Kyn relative to Trp.

Place, publisher, year, edition, pages
Nature Publishing Group, 2021
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:mau:diva-39748 (URN)10.1038/s41598-020-79903-w (DOI)000621920400026 ()33436784 (PubMedID)2-s2.0-85099386099 (Scopus ID)
Available from: 2021-01-25 Created: 2021-01-25 Last updated: 2024-02-05Bibliographically approved
Gustafsson, A., Prgomet, Z., Jankovskaja, S., Ruzgas, T., Engblom, J., Ohlsson, L. & Gjörloff Wingren, A. (2020). Effect of IFN-γ on the kynurenine/tryptophan ratio in monolayer-cultured keratinocytes and a 3D reconstructed human epidermis model. Journal of dermatological science (Amsterdam), 99(3), 177-184, Article ID S0923-1811(20)30234-6.
Open this publication in new window or tab >>Effect of IFN-γ on the kynurenine/tryptophan ratio in monolayer-cultured keratinocytes and a 3D reconstructed human epidermis model
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2020 (English)In: Journal of dermatological science (Amsterdam), ISSN 0923-1811, E-ISSN 1873-569X, Vol. 99, no 3, p. 177-184, article id S0923-1811(20)30234-6Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Interferon-gamma (IFN-γ) represents a potent inducer for keratinocyte inflammatory and immune activation in vitro. Since tryptophan (trp) conversion to kynurenine (kyn) is involved in inflammation, the topical kyn/trp ratio may serve as a biomarker of skin inflammation. However, the trp metabolism in keratinocytes exposed to IFN-γ is not yet fully understood.

OBJECTIVE: The aim of this study was to establish a human epidermis model in order to quantify cytokine and kyn/trp secretion from IFN-γ stimulated cells and tissues. Moreover, to compare the cell response of 2D-cultured keratinocytes and the 3D epidermis model.

METHODS: Polycarbonate filters were used on which primary keratinocytes could attach and stratify in order to form the typical layers of reconstructed human epidermis (RHE). After IFN-γ treatment, secretion of kyn/trp was measured by high performance liquid chromatography. Gene and protein expression of indoleamine 2,3-dioxygenase 1 (IDO) was analyzed with real-time PCR and immunohistochemistry. The secretion of cytokines was quantified with ELISA.

RESULTS: Trp catabolism to kyn was significantly increased (P < 0.01) in the 2D culture in response to IFN-γ treatment. Before kyn secretion, IDO was strongly upregulated (P < 0.001). IFN-γ treatment also increased the secretion of IL-6 and IL-8 from the keratinocytes. In the RHE, IDO was upregulated by IFN-γ, and kyn secretion could be detected. Interestingly, IDO expression was only present in the basal cells of the RHE.

CONCLUSION: Our results suggest that IFN-γ acts as an inducer of trp degradation preferentially in undifferentiated keratinocytes, indicated by the IDO expression in the basal layer of the RHE.

Place, publisher, year, edition, pages
Japanese Society for Investigative Dermatology, 2020
Keywords
IDO, Kynurenine, Pro-inflammatory cytokines, Reconstructed human epidermis, Tryptophan
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:mau:diva-18014 (URN)10.1016/j.jdermsci.2020.07.005 (DOI)000582365800005 ()32782183 (PubMedID)2-s2.0-85089257839 (Scopus ID)
Available from: 2020-08-18 Created: 2020-08-18 Last updated: 2024-02-05Bibliographically approved
Jankovskaja, S., Labrousse, A., Prevaud, L., Holmqvist, B., Brinte, A., Engblom, J., . . . Ruzgas, T. (2020). Visualisation of H2O2 penetration through skin indicates importance to develop pathway-specific epidermal sensing. Microchimica Acta, 187(12), Article ID 656.
Open this publication in new window or tab >>Visualisation of H2O2 penetration through skin indicates importance to develop pathway-specific epidermal sensing
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2020 (English)In: Microchimica Acta, ISSN 0026-3672, E-ISSN 1436-5073, Vol. 187, no 12, article id 656Article in journal (Refereed) Published
Abstract [en]

Elevated amounts of reactive oxygen species (ROS) including hydrogen peroxide (H2O2) are observed in the epidermis in different skin disorders. Thus, epidermal sensing of H2O2 should be useful to monitor the progression of skin pathologies. We have evaluated epidermal sensing of H2O2 in vitro, by visualising H2O2 permeation through the skin. Skin membranes were mounted in Franz cells, and a suspension of Prussian white microparticles was deposited on the stratum corneum face of the skin. Upon H2O2 permeation, Prussian white was oxidised to Prussian blue, resulting in a pattern of blue dots. Comparison of skin surface images with the dot patterns revealed that about 74% of the blue dots were associated with hair shafts. The degree of the Prussian white to Prussian blue conversion strongly correlated with the reciprocal resistance of the skin membranes. Together, the results demonstrate that hair follicles are the major pathways of H2O2 transdermal penetration. The study recommends that the development of H2O2 monitoring on skin should aim for pathway-specific epidermal sensing, allowing micrometre resolution to detect and quantify this ROS biomarker at hair follicles. Graphical abstract

Place, publisher, year, edition, pages
Springer, 2020
Keywords
Epidermal sensing, Hydrogen peroxide, Prussian blue, Hair follicles, Skin penetration
National Category
Dermatology and Venereal Diseases
Identifiers
urn:nbn:se:mau:diva-37075 (URN)10.1007/s00604-020-04633-9 (DOI)000589701400001 ()33188446 (PubMedID)2-s2.0-85095947121 (Scopus ID)
Available from: 2020-12-03 Created: 2020-12-03 Last updated: 2024-06-17Bibliographically approved
Jankovskaja, S., Kamiie, J., Rezeli, M., Gustavsson, L., Sugihara, Y., Miliotis, T., . . . Marko-Varga, G. (2018). Optimization of sample preparation for transporter protein quantification in tissues by LC-MS/MS (ed.). Journal of Pharmaceutical and Biomedical Analysis, 164, 9-15
Open this publication in new window or tab >>Optimization of sample preparation for transporter protein quantification in tissues by LC-MS/MS
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2018 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 164, p. 9-15Article in journal (Refereed)
Abstract [en]

BACKGROUND: Reproducible quantification of drug transporter protein expression in tissues is important for predicting transporter mediated drug disposition. Many mass-spectrometry based transporter protein quantification methods result in high variability of the estimated transporter quantities. Therefore, we aimed to evaluate and optimize mass spectrometry-based quantification method for drug transporter proteins in tissues. MATERIALS AND METHODS: Plasma membrane (PM) proteins from mouse tissues were isolated by applying three extraction protocols: commercial plasma membrane extraction kit, tissue homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation, and PM enrichment with Tween 40. Moreover, five different protein digestion protocols were applied on the same PM fraction. PM isolation and digestion protocols were evaluated by measuring the amount of transporter proteins by liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. RESULTS: Mouse liver homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation and PM enrichment with Tween 40 resulted in two times higher transporter protein quantity (Breast cancer resistance protein (Bcrp) 18.0 fmol/mug protein) in comparison with the PM samples isolated by extraction kit (Bcrp 9.8 fmol/mug protein). The evaluation of protein digestion protocols revealed that the most optimal protocol for PM protein digestion is with Lys-C and trypsin, in combination with trypsin enhancer and heat denaturation. Overall, quantities of Bcrp and Na+/K + ATPase proteins evaluated in mouse liver and kidney cortex by using our optimized PM isolation method, as well as, established digestion protocol were two to three times higher than previously reported and coefficient of variation (CV) for technical replicates was below 10%. CONCLUSION: We have established an improved transporter protein quantification methodology by optimizing PM isolation and protein digestion procedures. The optimized procedure resulted in a higher transporter protein yield and improved precision.

Place, publisher, year, edition, pages
Elsevier, 2018
National Category
Natural Sciences
Identifiers
urn:nbn:se:mau:diva-14606 (URN)10.1016/j.jpba.2018.10.013 (DOI)000456900700002 ()30339949 (PubMedID)2-s2.0-85054818718 (Scopus ID)27268 (Local ID)27268 (Archive number)27268 (OAI)
Available from: 2020-03-30 Created: 2020-03-30 Last updated: 2024-06-17Bibliographically approved
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