lnu.sePublications
Change search
Link to record
Permanent link

Direct link
BETA
Ohlson, Sten
Publications (10 of 100) Show all publications
Ohlson, S., Kaur, J., Raida, M., Niss, U., Bengala, T., Drum, C. L., . . . Torres, A. R. (2017). Direct analysis - no sample preparation - of bioavailable cortisol in human plasma by weak affinity chromatography (WAC). Journal of chromatography. B, 1061, 438-444
Open this publication in new window or tab >>Direct analysis - no sample preparation - of bioavailable cortisol in human plasma by weak affinity chromatography (WAC)
Show others...
2017 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 1061, p. 438-444Article in journal (Refereed) Published
Abstract [en]

Pre-analytical treatment of blood plasma is a time consuming and often rate limiting step in the workflow of LC/MS analysis. We present in this pilot study a new approach for quantitative LC/MS based on weak affinity chromatography (WAC) of crude plasma. The steroid hormone cortisol was selected as a clinically relevant biomarker, as it currently requires extensive pre-analytical preparation. A WAC unit with saturating, immobilized albumin as a prototypic weak binder was used in combination with an ion-funnel MS/MS detector to perform zonal affinity chromatography of cortisol directly from a plasma sample, followed by quantitative multiple reaction monitoring (MRM). This procedure also allowed us to determine the amount of bioavailable cortisol in the clinical plasma sample which is of significant therapeutic interest. This WAC-MS approach showed an excellent correlation (R-2 = 0.86 (P < 0.0001 (highly significant); n = 60) with a state-of-the-art, clinical competitive immunoassay procedure for plasma cortisol analysis. With integration of WAC into LC/MS workflow, it may be possible to both accelerate and improve assay performance by eliminating the sample extraction step. Preliminary data with other steroid hormones indicate that WAC-MS can be applied to various biomolecules using a plasma transport protein such as albumin.

Place, publisher, year, edition, pages
Elsevier, 2017
Keywords
Weak affinity chromatography, WAC-MS, Sample preparation, Cortisol analysis, Bioavailable cortisol
National Category
Analytical Chemistry
Research subject
Chemistry, Analytical Chemistry
Identifiers
urn:nbn:se:lnu:diva-68334 (URN)10.1016/j.jchromb.2017.07.035 (DOI)000411542000058 ()28820982 (PubMedID)
Available from: 2017-10-12 Created: 2017-10-12 Last updated: 2017-10-12Bibliographically approved
Duong-Thi, M.-D., Bergström, M., Edwards, K., Eriksson, J., Ohlson, S., To Yiu Ying, J., . . . Agmo Hernández, V. (2016). Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins. The Analyst, 141(3), 981-988
Open this publication in new window or tab >>Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins
Show others...
2016 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 141, no 3, p. 981-988Article in journal (Refereed) Published
Abstract [en]

Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures.

National Category
Analytical Chemistry
Research subject
Chemistry, Analytical Chemistry
Identifiers
urn:nbn:se:lnu:diva-50634 (URN)10.1039/c5an02105g (DOI)000368942600028 ()26673836 (PubMedID)2-s2.0-84956759926 (Scopus ID)
Available from: 2016-03-11 Created: 2016-03-11 Last updated: 2017-11-30Bibliographically approved
Duong-Thi, M.-D., Bergström, G., Mandenius, C.-F., Bergström, M., Fex, T. & Ohlson, S. (2014). Comparison of weak affinity chromatography and surface plasmon resonance in determining affinity of small molecules. Analytical Biochemistry, 461, 57-59
Open this publication in new window or tab >>Comparison of weak affinity chromatography and surface plasmon resonance in determining affinity of small molecules
Show others...
2014 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 461, p. 57-59Article in journal (Refereed) Published
Abstract [en]

In this study, we compared affinity data from surface plasmon resonance (SPR) and weak affinity chromatography (WAC), two established techniques for determination of weak affinity (mM-mu M) small molecule-protein interactions. In the current comparison, thrombin was used as target protein. In WAC the affinity constant (K-D) was determined from retention times, and in SPR it was determined by Langmuir isotherm fitting of steady-state responses. Results indicate a strong correlation between the two methods (R-2 = 0.995, P < 0.0001). (C) 2014 Elsevier Inc. All rights reserved.

Keywords
Affinity determination, Fragment-based drug discovery, Surface plasmon resonance, Thrombin, Weak affinity chromatography
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-36862 (URN)10.1016/j.ab.2014.05.023 (DOI)000340077600009 ()2-s2.0-84903973479 (Scopus ID)
Available from: 2014-09-10 Created: 2014-09-10 Last updated: 2017-12-05Bibliographically approved
Meiby, E., Simmonite, H., le Strat, L., Davis, B., Matassova, N., Moore, J. D., . . . Ohlson, S. (2013). Fragment Screening by Weak Affinity Chromatography: Comparison with Established Techniques for Screening against HSP90. Analytical Chemistry, 85(14), 6756-6766
Open this publication in new window or tab >>Fragment Screening by Weak Affinity Chromatography: Comparison with Established Techniques for Screening against HSP90
Show others...
2013 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 85, no 14, p. 6756-6766Article in journal (Refereed) Published
Abstract [en]

The increasing use of fragment-based lead discovery (FBLD) in industry as well as in academia creates a high demand for sensitive and reliable methods to detect the binding of fragments to act as starting points in drug discovery programs. Nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and X-ray crystallography are well-established methods for fragment finding, and thermal shift and fluorescence polarization (FP) assays are used to a lesser extent. Weak affinity chromatography (WAC) was recently introduced as a new technology for fragment screening. The study presented here compares screening of 111 fragments against the ATPase domain of HSP90 by all of these methods, with isothermal titration calorimetry (ITC) used to confirm the most potent hits. The study demonstrates that WAC is comparable to the established methods of ligand-based NMR and SPR as a hit-id method, with hit correlations of 88% and 83%, respectively. The stability of HSP90 WAC columns was also evaluated and found to give 90% reproducibility even after 207 days of storage. A good correlation was obtained between the various technologies, validating WAC as an effective technology for fragment screening.

National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-29213 (URN)10.1021/ac400715t (DOI)000322059600031 ()2-s2.0-84880567242 (Scopus ID)
Available from: 2013-10-04 Created: 2013-10-03 Last updated: 2017-12-06Bibliographically approved
Duong-Thi, M.-D., Bergström, M., Fex, T., Isaksson, R. & Ohlson, S. (2013). High-Throughput Fragment Screening by Affinity LC-MS. Journal of Biomolecular Screening, 18(2), 160-171
Open this publication in new window or tab >>High-Throughput Fragment Screening by Affinity LC-MS
Show others...
2013 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 18, no 2, p. 160-171Article in journal (Refereed) Published
Abstract [en]

Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in < 4 h (corresponding to > 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K-D) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

Keywords
drug discovery, fragment screening, mass spectrometry, thrombin, weak affinity chromatography
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry; Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-24535 (URN)10.1177/1087057112459271 (DOI)000313661900002 ()2-s2.0-84872546422 (Scopus ID)
Available from: 2013-02-25 Created: 2013-02-25 Last updated: 2017-12-06Bibliographically approved
Meiby, E., M Zetterberg, M., Victor, H., Ohlson, S. & Edwards, K. (2013). Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis.. Analytical and Bioanalytical Chemistry, 405(14), 4859-4869
Open this publication in new window or tab >>Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis.
Show others...
2013 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 405, no 14, p. 4859-4869Article in journal (Refereed) Published
Abstract [en]

Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography.

Keywords
Lipodisks COX-1 HPLC-MS Model membrane Drug partition studies Membrane protein WAC Weak affinity chromatography
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology Biophysics Analytical Chemistry
Research subject
Natural Science, Biochemistry; Natural Science, Biotechnology
Identifiers
urn:nbn:se:lnu:diva-25968 (URN)10.1007/s00216-013-6892-3 (DOI)000318312400018 ()2-s2.0-84881365134 (Scopus ID)
Available from: 2013-05-30 Created: 2013-05-30 Last updated: 2017-12-06Bibliographically approved
Ramos-Soriano, J., Niss, U., Angulo, J., Angulo, M., Moreno-Vargas, A. J., Carmona, A. T., . . . Robina, I. (2013). Synthesis, Biological Evaluation, WAC and NMR Studies of S-Galactosides and Non-Carbohydrate Ligands of Cholera Toxin Based on Polyhydroxyalkylfuroate Moieties. Chemistry - A European Journal, 19(52), 17989-18003
Open this publication in new window or tab >>Synthesis, Biological Evaluation, WAC and NMR Studies of S-Galactosides and Non-Carbohydrate Ligands of Cholera Toxin Based on Polyhydroxyalkylfuroate Moieties
Show others...
2013 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 19, no 52, p. 17989-18003Article in journal (Refereed) Published
Abstract [en]

The synthesis of several non-carbohydrate ligands of cholera toxin based on polyhydroxyalkylfuroate moieties is reported. Some of them have been linked to D-galactose through a stable and well-tolerated S-glycosidic bond. They represent a novel type of non-hydrolyzable bidentate ligand featuring galactose and polyhydroxyalkylfuroic esters as pharmacophoric residues, thus mimicking the GM1 ganglioside. The affinity of the new compounds towards cholera toxin was measured by weak affinity chromatography (WAC). The interaction of the best candidates with this toxin was also studied by saturation transfer difference NMR experiments, which allowed identification of the binding epitopes of the ligands interacting with the protein. Interestingly, the highest affinity was shown by non-carbohydrate mimics based on a polyhydroxyalkylfuroic ester structure.

Keywords
weak affinity chromatography (WAC), biomimetic synthesis, carbohydrates, cholera toxin, NMR spectroscopy
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biotechnology
Identifiers
urn:nbn:se:lnu:diva-32107 (URN)10.1002/chem.201302786 (DOI)000328531000041 ()2-s2.0-84891011906 (Scopus ID)
Available from: 2014-02-05 Created: 2014-02-05 Last updated: 2017-12-06Bibliographically approved
Duong-Thi, M.-D., Bergström, M., Fex, T., Svensson, S., Ohlson, S. & Isaksson, R. (2013). Weak Affinity Chromatography for Evaluation of Stereoisomers in Early Drug Discovery. Journal of Biomolecular Screening, 18(6), 748-755
Open this publication in new window or tab >>Weak Affinity Chromatography for Evaluation of Stereoisomers in Early Drug Discovery
Show others...
2013 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 18, no 6, p. 748-755Article in journal (Refereed) Published
Abstract [en]

In early drug discovery (e.g. in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations. In this work, we concurrently screened mixtures of stereoisomers and estimated their affinities to a protein target (thrombin) using weak affinity chromatography-mass spectrometry (WAC-MS). Affinity determinations by WAC showed that minor changes in stereoisomeric configuration could have major impact on affinity. The ability of WAC-MS to provide instant information about stereoselectivity and binding affinities directly from analyte mixtures is a great advantage in fragment library screening and drug lead development.

Place, publisher, year, edition, pages
Sage Publications, 2013
Keywords
fragment-based drug discovery, mixture screening, stereoisomers, thrombin, weak affinity chromatography
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Natural Science, Biomedical Sciences; Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-24641 (URN)10.1177/1087057113480391 (DOI)000320888100013 ()2-s2.0-84879476954 (Scopus ID)
Available from: 2013-03-01 Created: 2013-03-01 Last updated: 2017-12-06Bibliographically approved
Landström, J., Bergström, M., Hamark, C., Ohlson, S. & Widmalm, G. (2012). Combining weak affinity chromatography, NMR spectroscopy and molecular simulations in carbohydrate-lysozyme interaction studies.. Organic and biomolecular chemistry, 10(15), 3019-3032
Open this publication in new window or tab >>Combining weak affinity chromatography, NMR spectroscopy and molecular simulations in carbohydrate-lysozyme interaction studies.
Show others...
2012 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 10, no 15, p. 3019-3032Article in journal (Refereed) Published
Abstract [en]

By examining the interactions between the protein hen egg-white lysozyme (HEWL) and commercially available and chemically synthesized carbohydrate ligands using a combination of weak affinity chromatography (WAC), NMR spectroscopy and molecular simulations, we report on new affinity data as well as a detailed binding model for the HEWL protein. The equilibrium dissociation constants of the ligands were obtained by WAC but also by NMR spectroscopy, which agreed well. The structures of two HEWL-disaccharide complexes in solution were deduced by NMR spectroscopy using (1)H saturation transfer difference (STD) effects and transferred (1)H,(1)H-NOESY experiments, relaxation-matrix calculations, molecular docking and molecular dynamics simulations. In solution the two disaccharides β-d-Galp-(1→4)-β-D-GlcpNAc-OMe and β-D-GlcpNAc-(1→4)-β-D-GlcpNAc-OMe bind to the B and C sites of HEWL in a syn-conformation at the glycosidic linkage between the two sugar residues. Intermolecular hydrogen bonding and CH/π-interactions form the basis of the protein-ligand complexes in a way characteristic of carbohydrate-protein interactions. Molecular dynamics simulations with explicit water molecules of both the apo-form of the protein and a ligand-protein complex showed structural change compared to a crystal structure of the protein. The flexibility of HEWL as indicated by a residue-based root-mean-square deviation analysis indicated similarities overall, with some residue specific differences, inter alia, for Arg61 that is situated prior to a flexible loop. The Arg61 flexibility was notably larger in the ligand-complexed form of HEWL. N,N'-Diacetylchitobiose has previously been observed to bind to HEWL at the B and C sites in water solution based on (1)H NMR chemical shift changes in the protein whereas the disaccharide binds at either the B and C sites or the C and D sites in different crystal complexes. The present study thus highlights that protein-ligand complexes may vary notably between the solution and solid states, underscoring the importance of targeting the pertinent binding site(s) for inhibition of protein activity and the advantages of combining different techniques in a screening process.

National Category
Chemical Sciences
Research subject
Natural Science
Identifiers
urn:nbn:se:lnu:diva-18565 (URN)10.1039/c2ob07066a (DOI)22395160 (PubMedID)2-s2.0-84858969506 (Scopus ID)
Available from: 2012-05-05 Created: 2012-05-05 Last updated: 2017-12-07Bibliographically approved
Bergström, M., Åström, E., Påhlsson, P. & Ohlson, S. (2012). Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography. Journal of chromatography. B, 885-886, 66-72
Open this publication in new window or tab >>Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography
2012 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 885-886, p. 66-72Article in journal (Refereed) Published
Abstract [en]

Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in E.coli as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from Aleuria aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms towards a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked α1-3 to GlcNAc interacted with higher affinity compared to fucose linked α1-6 or α1-4 and the obtained dissociation constants (Kd) were in the range of 10 μM for all AAL forms. Tetra- and pentasaccharides with fucose in α1-2, α1-3 or α1-4 had Kd values ranging from 0.1–7 mM while a large α1-6 fucosylated oligosaccharide had a Kd of about 20 μM. The recombinant multivalent AAL forms and native AAL exhibited similar affinities towards all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.

Place, publisher, year, edition, pages
Elsevier, 2012
Keywords
Affinity; Aleuria aurantia lectin; Glycan interaction; Recombinant protein; Weak Affinity Chromatography
National Category
Chemical Sciences
Research subject
Chemistry, Biotechnology
Identifiers
urn:nbn:se:lnu:diva-16303 (URN)10.1016/j.jchromb.2011.12.015 (DOI)2-s2.0-84856642608 (Scopus ID)
Available from: 2012-05-04 Created: 2011-12-22 Last updated: 2017-12-08Bibliographically approved
Organisations

Search in DiVA

Show all publications