Publications by authors named "Jos Buijs"

33 Publications

Novel multivalent design of a monoclonal antibody improves binding strength to soluble aggregates of amyloid beta.

Transl Neurodegener 2021 Sep 28;10(1):38. Epub 2021 Sep 28.

Protein Drug Design, Faculty of Pharmacy, Uppsala University, 75124, Uppsala, Sweden.

Background: Amyloid-β (Aβ) immunotherapy is a promising therapeutic strategy in the fight against Alzheimer's disease (AD). A number of monoclonal antibodies have entered clinical trials for AD. Some of them have failed due to the lack of efficacy or side-effects, two antibodies are currently in phase 3, and one has been approved by FDA. The soluble intermediate aggregated species of Aβ, termed oligomers and protofibrils, are believed to be key pathogenic forms, responsible for synaptic and neuronal degeneration in AD. Therefore, antibodies that can strongly and selectively bind to these soluble intermediate aggregates are of great diagnostic and therapeutic interest.

Methods: We designed and recombinantly produced a hexavalent antibody based on mAb158, an Aβ protofibril-selective antibody. The humanized version of mAb158, lecanemab (BAN2401), is currently in phase 3 clinical trials for the treatment of AD. The new designs involved recombinantly fusing single-chain fragment variables to the N-terminal ends of mAb158 antibody. Real-time interaction analysis with LigandTracer and surface plasmon resonance were used to evaluate the kinetic binding properties of the generated antibodies to Aβ protofibrils. Different ELISA setups were applied to demonstrate the binding strength of the hexavalent antibody to Aβ aggregates of different sizes. Finally, the ability of the antibodies to protect cells from Aβ-induced effects was evaluated by MTT assay.

Results: Using real-time interaction analysis with LigandTracer, the hexavalent design promoted a 40-times enhanced binding with avidity to protofibrils, and most of the added binding strength was attributed to the reduced rate of dissociation. Furthermore, ELISA experiments demonstrated that the hexavalent design also had strong binding to small oligomers, while retaining weak and intermediate binding to monomers and insoluble fibrils. The hexavalent antibody also reduced cell death induced by a mixture of soluble Aβ aggregates.

Conclusion: We provide a new antibody design with increased valency to promote binding avidity to an enhanced range of sizes of Aβ aggregates. This approach should be general and work for any aggregated protein or repetitive target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40035-021-00258-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8477473PMC
September 2021

Complement-Dependent Activity of CD20-Specific IgG Correlates With Bivalent Antigen Binding and C1q Binding Strength.

Front Immunol 2020 11;11:609941. Epub 2021 Jan 11.

Department of Genetics, Friedrich-Alexander University, Erlangen, Germany.

Monoclonal antibodies directed against the CD20 surface antigen on B cells are widely used in the therapy of B cell malignancies. Upon administration, the antibodies bind to CD20 expressing B cells and induce their depletion cell- and complement-dependent cytotoxicity or by induction of direct cell killing. The three antibodies currently most often used in the clinic are Rituximab (RTX), Ofatumumab (OFA) and Obinutuzumab (OBI). Even though these antibodies are all of the human IgG1 subclass, they have previously been described to vary considerably in the effector functions involved in therapeutic B cell depletion, especially in regards to complement activation. Whereas OFA is known to strongly induce complement-dependent cytotoxicity, OBI is described to be far less efficient. In contrast, the role of complement in RTX-induced B cell depletion is still under debate. Some of this dissent might come from the use of different systems for characterization of antibody effector functions. We therefore set out to systematically compare antibody as well as C1q binding and complement-activation by RTX, OFA and OBI on human B cell lines that differ in expression levels of CD20 and complement-regulatory proteins as well as human primary B cells. Applying real-time interaction analysis, we show that the overall strength of C1q binding to live target cells coated with antibodies positively correlated with the degree of bivalent binding for the antibodies to CD20. Kinetic analysis revealed that C1q exhibits two binding modes with distinct affinities and binding stabilities, with exact numbers varying both between antibodies and cell lines. Furthermore, complement-dependent cell killing by RTX and OBI was highly cell-line dependent, whereas the superior complement-dependent cytotoxicity by OFA was independent of the target B cells. All three antibodies were able to initiate deposition of C3b on the B cell surface, although to varying extent. This suggests that complement activation occurs but might not necessarily lead to induction of complement-dependent cytotoxicity. This activation could, however, initiate complement-dependent phagocytosis as an alternative mechanism of therapeutic B cell depletion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.609941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829346PMC
June 2021

Bivalent binding on cells varies between anti-CD20 antibodies and is dose-dependent.

MAbs 2020 Jan-Dec;12(1):1792673

Department of Immunology, Genetics and Pathology, Uppsala University , Uppsala, Sweden.

Based on their mechanism of action, two types of anti-CD20 antibodies are distinguished: Type I, which efficiently mediate complement-dependent cytotoxicity, and Type II, which instead are more efficient in inducing direct cell death. Several molecular characteristics of these antibodies have been suggested to underlie these different biological functions, one of these being the manner of binding to CD20 expressed on malignant B cells. However, the exact binding model on cells is unclear. In this study, the binding mechanism of the Type I therapeutic antibodies rituximab (RTX) and ofatumumab (OFA) and the Type II antibody obinutuzumab (OBI) were established by real-time interaction analysis on live cells. It was found that the degree of bivalent stabilization differed for the antibodies: OFA was stabilized the most, followed by RTX and then OBI, which had the least amount of bivalent stabilization. Bivalency inversely correlated with binding dynamics for the antibodies, with OBI displaying the most dynamic binding pattern, followed by RTX and OFA. For RTX and OBI, bivalency and binding dynamics were concentration dependent; at higher concentrations the interactions were more dynamic, whereas the percentage of antibodies that bound bivalent was less, resulting in concentration-dependent apparent affinities. This was barely noticeable for OFA, as almost all molecules bound bivalently at the tested concentrations. We conclude that the degree of bivalent binding positively correlates with the complement recruiting capacity of the investigated CD20 antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/19420862.2020.1792673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531561PMC
July 2021

A real-time cell-binding assay reveals dynamic features of STxB-Gb3 cointernalization and STxB-mediated cargo delivery into cancer cells.

FEBS Lett 2020 08 23;594(15):2406-2420. Epub 2020 Jun 23.

Ridgeview Instruments AB, Uppsala, Sweden.

The interaction between the Shiga toxin B-subunit (STxB) and its globotriaosylceramide receptor (Gb3) has a high potential for being exploited for targeted cancer therapy. The primary goal of this study was to evaluate the capacity of STxB to carry small molecules and proteins as cargo into cells. For this purpose, an assay was designed to provide real-time information about the StxB-Gb3 interaction as well as the dynamics and mechanism of the internalization process. The assay revealed the ability to distinguish the process of binding to the cell surface from internalization and presented the importance of receptor and STxB clustering for internalization. The overall setup demonstrated that the binding mechanism is complex, and the concept of affinity is difficult to apply. Hence, time-resolved methods, providing detailed information about the interaction of STxB with cells, are critical for the optimization of intracellular delivery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.13847DOI Listing
August 2020

Carbon Flux as a Measure of Prostate Cancer Aggressiveness: [C]-Acetate PET/CT.

Int J Med Sci 2020 14;17(2):214-223. Epub 2020 Jan 14.

Division of Nuclear Medicine and PET, Department of Surgical Sciences, Uppsala University, Uppsala, Sweden.

: Dynamic [C]-acetate positron emission tomography (PET) can be used to study tissue perfusion and carbon flux simultaneously. In this study, the feasibility of the quantification of prostate cancer aggressiveness using parametric methods assessing [C]-acetate kinetics was investigated in prostate cancer subjects. The underlying uptake mechanism correlated with [C]-acetate influx and efflux measured in real-time in vitro in cell culture. : Twenty-one patients with newly diagnosed low-to-moderate risk prostate cancer underwent magnetic resonance imaging (MRI) and dynamic [C]-acetate PET/CT examinations of the pelvis. Parametric images of K (extraction × perfusion), k (oxidative metabolism) and V (=K/k, anabolic metabolism defined as carbon retention) were constructed using a one-tissue compartment model with an arterial input function derived from pelvic arteries. Regions of interest (ROIs) of the largest cancer lesion in each patient and normal prostate tissue were drawn using information from MRI (T2 and DWI images), biopsy results, and post-surgical histopathology of whole prostate sections (n=7). In vitro kinetics of [C]-acetate were studied on DU145 and PC3 cell lines using LigandTracer White equipment for the measurement of the radioactivity uptake in real-time at 37°C. : Mean prostate specific antigen (PSA) was 8.33±3.92 ng/mL and median Gleason Sum 6 (range 5-7). K, V and standardized uptake values (SUVs) were significantly higher in cancerous prostate tissues compared to normal ones for all patients (p<0.001), while k was not (p=0.26). PSA values correlated to early SUVs (r=0.50, p=0.02) and K (r=0.48, p=0.03). Early and late SUVs correlated to V (r>0.76, p<0.001) and K (r>0.64, p<0.005). In vitro studies demonstrated higher extraction and retention (p<0.01) of [C]-acetate in the more aggressive PC3 cells. : Parametric images could be used to visualize the [C]-acetate kinetics of the prostate cancer exhibiting elevated extraction associated with the cancer aggressiveness. The influx rate of [C]-acetate studied in cell culture also showed dependence on the cancer aggressiveness associated with elevated lipogenesis. Dynamic [C]-acetate/PET demonstrated potential for prostate cancer aggressiveness estimation using parametric-based K and V values.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7150/ijms.39542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6990881PMC
November 2020

Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture.

Biotechnol Prog 2019 05 30;35(3):e2775. Epub 2019 Jan 30.

AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.

High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25-42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 × 10 cells/mL as in clarified supernatant. Two pilot-scale purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L, where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient single step, which directly connected the culture to the downstream process without cell clarification. Purification of mAb directly from non-clarified cell broth without cell separation can provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2775, 2019.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/btpr.2775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617771PMC
May 2019

Comparative Evaluation of Two DARPin Variants: Effect of Affinity, Size, and Label on Tumor Targeting Properties.

Mol Pharm 2019 03 8;16(3):995-1008. Epub 2019 Feb 8.

Nuclear Medicine Department, Cancer Research Institute , Tomsk National Research Medical Center Russian Academy of Sciences , Tomsk , Russia.

Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins that can be selected for binding to desirable molecular targets. High affinity and small size of DARPins render them promising probes for radionuclide molecular imaging. However, detailed knowledge on many factors influencing their imaging properties is still lacking. We have evaluated two human epidermal growth factor 2 (HER2)-specific DARPins with different size and binding properties. DARPins 9_29-H and G3-H were radiolabeled with iodine-125 and tricarbonyl technetium-99m and evaluated in vitro. A side-by-side comparison of biodistribution and tumor targeting was performed. HER2-specific tumor accumulation of G3-H was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H in comparison to 9_29-H. Technetium-99m labeled G3-H demonstrated a better biodistribution profile than 9_29-H, with several-fold lower uptake in liver. Radioiodinated G3-H showed the best tumor-to-organ ratios. The combined effect of affinity, molecular weight, scaffold composition, and nonresidualizing properties of iodine label provided radioiodinated G3-H with high clinical potential for imaging of HER2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.8b00922DOI Listing
March 2019

Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours.

Eur J Pharm Biopharm 2019 Jan 5;134:37-48. Epub 2018 Nov 5.

Department of Protein Technology, KTH - Royal Institute of Technology, SE-10691 Stockholm, Sweden.

ADAPTs are small engineered non-immunoglobulin scaffold proteins, which have demonstrated very promising features as vectors for radionuclide tumour targeting. Radionuclide imaging of human epidermal growth factor 2 (HER2) expression in vivo might be used for stratification of patients for HER2-targeting therapies. ADAPT6, which specifically binds to HER2, has earlier been shown to have very promising features for in vivo targeting of HER2 expressing tumours. In this study we tested the hypothesis that dimerization of ADAPT6 would increase the apparent affinity to HER2 and accordingly improve tumour targeting. To find an optimal molecular design of dimers, a series of ADAPT dimers with different linkers, -SSSG- (DiADAPT6L1), -(SSSG)- (DiADAPT6L2), and -(SSSG)- (DiADAPT6L3) was evaluated. Dimers in combination with optimal linker lengths demonstrated increased apparent affinity to HER2. The best variants, DiADAPT6L2 and DiADAPT6L3 were site-specifically labelled with In and I, and compared with a monomeric ADAPT6 in mice bearing HER2-expressing tumours. Despite higher affinity, both dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer. We conclude that improved affinity of a dimeric form of ADAPT does not compensate the disadvantage of increased size. Therefore, increase of affinity should be obtained by affinity maturation and not by dimerization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejpb.2018.11.004DOI Listing
January 2019

Enhanced protection of the renal vascular endothelium improves early outcome in kidney transplantation: Preclinical investigations in pig and mouse.

Sci Rep 2018 03 26;8(1):5220. Epub 2018 Mar 26.

Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Ischemia reperfusion injury is one of the major complications responsible for delayed graft function in kidney transplantation. Applications to reduce reperfusion injury are essential due to the widespread use of kidneys from deceased organ donors where the risk for delayed graft function is especially prominent. We have recently shown that coating of inflamed or damaged endothelial cells with a unique heparin conjugate reduces thrombosis and leukocyte recruitment. In this study we evaluated the binding capacity of the heparin conjugate to cultured human endothelial cells, to kidneys from brain-dead porcine donors, and to murine kidneys during static cold storage. The heparin conjugate was able to stably bind cultured endothelial cells with high avidity, and to the renal vasculature of explanted kidneys from pigs and mice. Treatment of murine kidneys prior to transplantation reduced platelet deposition and leukocyte infiltration 24 hours post-transplantation, and significantly improved graft function. The present study thus shows the benefits of enhanced protection of the renal vasculature during cold storage, whereby increasing the antithrombotic and anti-adhesive properties of the vascular endothelium yields improved renal function early after transplantation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-21463-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979943PMC
March 2018

New insights in Type I and II CD20 antibody mechanisms-of-action with a panel of novel CD20 antibodies.

Br J Haematol 2018 03 22;180(6):808-820. Epub 2018 Feb 22.

Laboratory of Translational Immunology, UMC Utrecht, Utrecht, The Netherlands.

Based on their mechanisms-of-action, CD20 monoclonal antibodies (mAbs) are grouped into Type I [complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)] and Type II [programmed cell death (PCD) and ADCC] mAbs. We generated 17 new hybridomas producing CD20 mAbs of different isotypes and determined unique heavy and light chain sequence pairs for 13 of them. We studied their epitope binding, binding kinetics and structural properties and investigated their predictive value for effector functions, i.e. PCD, CDC and ADCC. Peptide mapping and CD20 mutant screens revealed that 10 out of these 11 new mAbs have an overlapping epitope with the prototypic Type I mAb rituximab, albeit that distinct amino acids of the CD20 molecule contributed differently. Binding kinetics did not correlate with the striking differences in CDC activity among the mIgG2c mAbs. Interestingly, chimerization of mAb m1 resulted in a mAb displaying both Type I and II characteristics. PCD induction was lost upon introduction of a mutation in the framework of the heavy chain affecting the elbow angle, supporting that structural changes within this region can affect functional activities of CD20 mAbs. Together, these new CD20 mAbs provide further insights in the properties dictating the functional efficacy of CD20 mAbs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bjh.15132DOI Listing
March 2018

Evaluation of HER2-specific peptide ligand for its employment as radiolabeled imaging probe.

Sci Rep 2018 02 14;8(1):2998. Epub 2018 Feb 14.

Institute of Biostructures and Bioimaging, National Research Council, Naples, Italy.

HER2 transmembrane receptor is an important target in immunotherapy treatment of breast and gastroesophageal cancer. Molecular imaging of HER2 expression may provide essential prognostic and predictive information concerning disseminated cancer and aid in selection of an optimal therapy. Radiolabeled low molecular weight peptide ligands are particularly attractive as probes for molecular imaging, since they reach and bind to the target and clear from non-target organs and blood stream faster than bulky antibodies. In this study, we evaluated a potential HER2-imaging probe, an A9 nonapeptide, derived from the trastuzumab-Fab portion. Its cellular uptake was investigated by mass spectrometry analysis of the cytoplasmic cellular extracts. Moreover, based on in-silico modeling, DTPA chelator was conjugated to N-terminus of A9. In-labeled A9 demonstrated nanomolar affinity to HER2-expressing BT474 cells and favorable biodistribution profile in NMRI mice. This study suggests that the peptide A9 represents a good lead candidate for development of molecular probe, to be used for imaging purposes and for the delivery of cytotoxic agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-21283-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812989PMC
February 2018

Novel Real-Time Proximity Assay for Characterizing Multiple Receptor Interactions on Living Cells.

Anal Chem 2017 12 5;89(24):13212-13218. Epub 2017 Dec 5.

Department of Immunology, Genetics and Pathology, Uppsala University , 751 05 Uppsala, Sweden.

Cellular receptor activity is often controlled through complex mechanisms involving interactions with multiple molecules, which can be soluble ligands and/or other cell surface molecules. In this study, we combine a fluorescence-based technology for real-time interaction analysis with fluorescence quenching to create a novel time-resolved proximity assay to study protein-receptor interactions on living cells. This assay extracts the binding kinetics and affinity for two proteins if they bind in proximity on the cell surface. One application of real-time proximity interaction analysis is to study relative levels of receptor dimerization. The method was primarily evaluated using the HER2 binding antibodies Trastuzumab and Pertuzumab and two EGFR binding antibodies including Cetuximab. Using Cetuximab and Trastuzumab, proximity of EGFR and HER2 was investigated before and after treatment of cells with the tyrosine-kinase inhibitor Gefitinib. Treated cells displayed 50% increased proximity signal, whereas the binding characteristics of the two antibodies were not significantly affected, implying an increase in the EGFR-HER2 dimer level. These results demonstrate that real-time proximity interaction analysis enables determination of the interaction rate constants and affinity of two ligands while simultaneously quantifying their relative colocalization on living cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.7b02983DOI Listing
December 2017

Comparative evaluation of tumor targeting using the anti-HER2 ADAPT scaffold protein labeled at the C-terminus with indium-111 or technetium-99m.

Sci Rep 2017 11 7;7(1):14780. Epub 2017 Nov 7.

School of Biotechnology, Division of Protein Technology, KTH Royal Institute of Technology, Stockholm, Sweden.

ABD-Derived Affinity Proteins (ADAPTs) is a novel class of engineered scaffold proteins derived from an albumin-binding domain of protein G. The use of ADAPT6 derivatives as targeting moiety have provided excellent preclinical radionuclide imaging of human epidermal growth factor 2 (HER2) tumor xenografts. Previous studies have demonstrated that selection of nuclide and chelator for its conjugation has an appreciable effect on imaging properties of scaffold proteins. In this study we performed a comparative evaluation of the anti-HER2 ADAPT having an aspartate-glutamate-alanine-valine-aspartate-alanine-asparagine-serine (DEAVDANS) N-terminal sequence and labeled at C-terminus with Tc using a cysteine-containing peptide based chelator, glycine-serine-serine-cysteine (GSSC), and a similar variant labeled with In using a maleimido derivative of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Both Tc-DEAVDANS-ADAPT6-GSSC and In-DEAVDANS-ADAPT6-GSSC-DOTA accumulated specifically in HER2-expressing SKOV3 xenografts. The tumor uptake of both variants did not differ significantly and average values were in the range of 19-21%ID/g. However, there was an appreciable variation in uptake of conjugates in normal tissues that resulted in a notable difference in the tumor-to-organ ratios. The In-DOTA label provided 2-6 fold higher tumor-to-organ ratios than Tc-GSSC and is therefore the preferable label for ADAPTs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-15366-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5676751PMC
November 2017

Radionuclide Tumor Targeting Using ADAPT Scaffold Proteins: Aspects of Label Positioning and Residualizing Properties of the Label.

J Nucl Med 2018 01 1;59(1):93-99. Epub 2017 Sep 1.

Institute for Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; and.

Visualization of cancer-associated alterations of molecular phenotype using radionuclide imaging is a noninvasive approach to stratifying patients for targeted therapies. The engineered albumin-binding domain-derived affinity protein (ADAPT) is a promising tracer for radionuclide molecular imaging because of its small size (6.5 kDa), which satisfies the precondition for efficient tumor penetration and rapid clearance. Previous studies demonstrated that the human epidermal growth factor receptor type 2 (HER2)-targeting ADAPT6 labeled with radiometals at the N terminus is able to image HER2 expression in xenografts a few hours after injection. The aim of this study was to evaluate whether the use of a nonresidualizing label or placement of the labels at the C terminus would further improve the targeting properties of ADAPT6. Two constructs, Cys-ADAPT6 and Cys-ADAPT6, having the (HE)DANS sequence at the N terminus were produced and site-specifically labeled using In-DOTA or I-iodo-((4-hydroxyphenyl)ethyl) maleimide (HPEM). The conjugates were compared in vitro and in vivo. HER2-targeting properties and biodistribution were evaluated in BALB/C mice bearing ovarian carcinoma cell (SKOV-3) xenografts. Specific HER2 binding and high affinity were preserved after labeling. Both Cys-ADAPT6 and Cys-ADAPT6 were internalized slowly by HER2-expressing cancer cells. Depending on the label position, uptake at 4 h after injection varied from 10% to 22% of the injected dose per gram of tumor tissue. Regardless of terminus position, the I-HPEM label provided more than 140-fold lower renal uptake than the In-DOTA label at 4 after injection. The tumor-to-organ ratios were, in contrast, higher for both of the In-DOTA-labeled ADAPT variants in other organs. Tumor-to-blood ratios for In-labeled Cys-ADAPT6 and Cys-ADAPT6 did not differ significantly (250-280), but In-DOTA-Cys-ADAPT6 provided significantly higher tumor-to-lung, tumor-to-liver, tumor-to-spleen, and tumor-to-muscle ratios. Radioiodinated variants had similar tumor-to-organ ratios, but I-HPEM-Cys-ADAPT6 had significantly higher tumor uptake and a higher tumor-to-kidney ratio. Residualizing properties of the label strongly influence the targeting properties of ADAPT6. The position of the radiolabel influences targeting as well, although to a lesser extent. Placement of a label at the C terminus yields the best biodistribution features for both radiometal and radiohalogen labels. Low renal retention of the radioiodine label creates a precondition for radionuclide therapy using I-labeled HPEM-Cys-ADAPT6.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2967/jnumed.117.197202DOI Listing
January 2018

The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule.

Sci Rep 2017 07 20;7(1):5961. Epub 2017 Jul 20.

Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The In-labelled DOTA-conjugated Z Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-Z. DOTA-Z was labelled with Co (T = 271.8 d), Co (T = 17.5 h), and, for comparison, with the positron-emitting radionuclide Ga (T = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope Co was used in animal studies. Both Co-DOTA-Z and Ga-DOTA-Z demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-Z were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, Co-DOTA-Z demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for Ga-DOTA-Z. The results of this study suggest that the positron-emitting cobalt isotope Co would be an optimal label for DOTA-Z and further development should concentrate on this radionuclide as a label.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-05700-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519605PMC
July 2017

Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells.

Front Immunol 2017 24;8:455. Epub 2017 Apr 24.

Ridgeview Instruments AB, Vänge, Sweden.

Understanding molecular interactions on immune cells is crucial for drug development to treat cancer and autoimmune diseases. When characterizing molecular interactions, the use of a relevant living model system is important, as processes such as receptor oligomerization and clustering can influence binding patterns. We developed a protocol to enable time-resolved analysis of ligand binding to receptors on living suspension cells. Different suspension cell lines and weakly adhering cells were tethered to Petri dishes with the help of a biomolecular anchor molecule, and antibody binding was analyzed using LigandTracer. The protocol and assay described in this report were used to characterize interactions involving eight cell lines. Experiments were successfully conducted in three different laboratories, demonstrating the robustness of the protocol. For various antibodies, affinities and kinetic rate constants were obtained for binding to CD20 on both Daudi and Ramos B-cells, the T-cell co-receptor CD3 on Jurkat cells, and the Fcγ receptor CD32 on transfected HEK293 cells, respectively. Analyzing the binding of Rituximab to B-cells resulted in an affinity of 0.7-0.9 nM, which is similar to values reported previously for living B-cells. However, we observed a heterogeneous behavior for Rituximab interacting with B-cells, which to our knowledge has not been described previously. The understanding of complex interactions will be facilitated with the possibility to characterize binding processes in real-time on living immune cells. This provides the chance to broaden the understanding of how binding kinetics relate to biological function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.00455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5401896PMC
April 2017

Comparative Evaluation of Affibody Molecules for Radionuclide Imaging of in Vivo Expression of Carbonic Anhydrase IX.

Mol Pharm 2016 11 27;13(11):3676-3687. Epub 2016 Sep 27.

Department of Immunology, Genetics and Pathology, Uppsala University , SE-75285 Uppsala, Sweden.

Overexpression of the enzyme carbonic anhydrase IX (CAIX) is documented for chronically hypoxic malignant tumors as well as for normoxic renal cell carcinoma. Radionuclide molecular imaging of CAIX would be useful for detection of hypoxic areas in malignant tumors, for patients' stratification for CAIX-targeted therapies, and for discrimination of primary malignant and benign renal tumors. Earlier, we have reported feasibility of in vivo radionuclide based imaging of CAIX expressing tumors using Affibody molecules, small affinity proteins based on a nonimmunoglobulin scaffold. In this study, we compared imaging properties of several anti-CAIX Affibody molecules having identical scaffold parts and competing for the same epitope on CAIX, but having different binding paratopes. Four variants were labeled using residualizing Tc and nonresidualizing I labels. All radiolabeled variants demonstrated high-affinity detection of CAIX-expressing cell line SK-RC-52 in vitro and specific accumulation in SK-RC-52 xenografts in vivo. I-labeled conjugates demonstrated much lower radioactivity uptake in kidneys but higher radioactivity concentration in blood compared with Tc-labeled counterparts. Although all variants cleared rapidly from blood and nonspecific compartments, there was noticeable difference in their biodistribution. The best variant for imaging of expression of CAIX in disseminated cancer was Tc-(HE)-ZCAIX:2 providing tumor uptake of 16.3 ± 0.9% ID/g and tumor-to-blood ratio of 44 ± 7 at 4 h after injection. For primary renal cell carcinoma, the most promising imaging candidate was I-ZCAIX:4 providing tumor-kidney ratio of 2.1 ± 0.5. In conclusion, several clones of scaffold proteins should be evaluated to select the best variant for development of an imaging probe with optimal sensitivity for the intended application.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.6b00502DOI Listing
November 2016

ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers.

Cancer Res 2015 Oct 21;75(20):4364-71. Epub 2015 Aug 21.

Department of Protein Technology, KTH Royal Institute of Technology, Stockholm, Sweden.

Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, ¹¹¹In for SPECT imaging and ⁶⁸Ga for PET imaging. Pharmacologic studies in mice demonstrated that the fully engineered molecule (111)In/⁶⁸Ga-DOTA-(HE)3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-14-3497DOI Listing
October 2015

Automated functional characterization of radiolabeled antibodies: a time-resolved approach.

Nucl Med Commun 2014 Jul;35(7):767-76

aAlgeta ASA, Oslo, Norway bBiomedical Radiation Sciences, Uppsala University cRidgeview Instruments AB, Vänge dRidgeview Diagnostics AB, Uppsala, Sweden.

Background: The number of radiolabeled monoclonal antibodies (mAbs) used for medical imaging and cancer therapy is increasing. The required chemical modification for attaching a radioactive label and all associated treatment may lead to a damaged mAb subpopulation. This paper describes a novel method, concentration through kinetics (CTK), for rapid assessment of the concentration of immunoreactive mAb and the specific radioactivity, based on monitoring binding kinetics.

Methods: The interaction of radiolabeled mAb with either the antigen or a general mAb binder such as Protein A was monitored in real time using the instrument LigandTracer. As the curvature of the binding trace has a distinct shape based on the interaction kinetics and concentration of the functional mAb, the immunoreactive mAb concentration could be calculated through reverse kinetic fitting of the binding curves, using software developed for this project. The specific activity, describing the degree of radioactive labeling, was determined through the use of calibrated signal intensities.

Results: The performance of the CTK assay was evaluated on the basis of various mAb-based interaction systems and assay formats, and it was shown that the assay can provide accurate and repeatable results for immunoreactive concentration and specific activity, with both accuracy and relative SD values below 15%.

Conclusion: By applying reverse kinetics on real-time binding traces it is possible to estimate the functional concentration and specific activity of radiolabeled mAb. The CTK assay may in the future be included as a complement to current quality assessment methods of radiolabeled mAbs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MNM.0000000000000117DOI Listing
July 2014

Complexation and sequestration of BMP-2 from an ECM mimetic hyaluronan gel for improved bone formation.

PLoS One 2013 22;8(10):e78551. Epub 2013 Oct 22.

Division of Polymers Chemistry, Department of Chemistry-ångström, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Bone morphogenetic protein-2 (BMP-2) is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs) dermatan sulfate (DS) or heparin (HP), prior to loading it into a hyaluronic acid (HA) hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078551PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3805527PMC
May 2014

Effect-based proteomic detection of growth promoter abuse.

Anal Bioanal Chem 2013 Feb 14;405(4):1171-9. Epub 2012 Nov 14.

Institute of Agri-food and Land Use, School of Biological Sciences, Queen's University Belfast, Belfast, Northern Ireland, UK.

Unregulated growth promoter use in food-producing animals is an issue of concern both from food safety and animal welfare perspectives. However, the monitoring of such practices is analytically challenging due to the concerted actions of users to evade detection. Techniques based on the monitoring of biological responses to exogenous administrations have been proposed as more sensitive methods to identify treated animals. This study has, for the first time, profiled plasma proteome responses in bovine animals to treatment with nortestosterone decanoate and 17β-oestradiol benzoate, followed by dexamethasone administration. Two-dimensional fluorescence differential in-gel electrophoresis analysis revealed a series of hepatic and acute-phase proteins within plasma whose levels were up- or down-regulated within phases of the treatment regime. Surface plasmon resonance (SPR) immuno-assays were developed to quantify responses of identified protein markers during the experimental treatment study with a view to developing methods which can be used as screening tools for growth promoter abuse detection. SPR analysis demonstrated the potential for plasma proteins to be used as indicative measures of growth promoter administrations and concludes that the sensitivity and robustness of any detection approach based on plasma proteome analysis would benefit from examination of a range of proteins representative of diverse biological processes rather being reliant on specific individual markers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00216-012-6534-1DOI Listing
February 2013

Use of a novel micro-fluidic device to create arrays for multiplex analysis of large and small molecular weight compounds by surface plasmon resonance.

Biosens Bioelectron 2011 Feb 10;26(6):3029-36. Epub 2010 Dec 10.

Institute of Agri-Food and Land Use, School of Biological Sciences, Queen's University, Stranmillis Road, Belfast BT9 5AG, UK.

There is an increasing demand to develop biosensor monitoring devices capable of biomarker profiling for predicting animal adulteration and detecting multiple chemical contaminants or toxins in food produce. Surface plasmon resonance (SPR) biosensors are label free detection systems that monitor the binding of specific biomolecular recognition elements with binding partners. Essential to this technology are the production of biochips where a selected binding partner, antibody, biomarker protein or low molecular weight contaminant, is immobilised. A micro-fluidic immobilisation device allowing the covalent attachment of up to 16 binding partners in a linear array on a single surface has been developed for compatibility with a prototype multiplex SPR analyser. The immobilisation unit and multiplex SPR analyser were respectively evaluated in their ability to be fit-for-purpose for binding partner attachment and detection of high and low molecular weight molecules. The multiplexing capability of the dual technology was assessed using phycotoxin concentration analysis as a model system. The parent compounds of four toxin groups were immobilised within a single chip format and calibration curves were achieved. The chip design and SPR technology allowed the compartmentalisation of the binding interactions for each toxin group offering the added benefit of being able to distinguish between toxin families and perform concentration analysis. This model is particularly contemporary with the current drive to replace biological methods for phycotoxin screening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bios.2010.12.007DOI Listing
February 2011

Increased striatal mRNA and protein levels of the immunophilin FKBP-12 in experimental Parkinson's disease and identification of FKBP-12-binding proteins.

J Proteome Res 2007 Oct 18;6(10):3952-61. Epub 2007 Sep 18.

Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, P.O. Box 583 Biomedical Centre, SE-75123 Uppsala, Sweden.

FKBP-12, a 12 kDa FK506-binding protein (neuroimmunophilin), acts as a receptor for the immunosuppressant drug FK506. Neuroimmunophilins, including FKBP-12, are abundant in the brain and have been shown to be involved in reversing neuronal degeneration and preventing cell death. In this report, we have utilized several analytical techniques, such as in situ hybridization, Western blotting, two-dimensional gel electrophoresis, and liquid chromatography electrospray tandem mass spectrometry to study the transcriptional expression as well as protein levels of FKBP-12 in the unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. The FKBP-12 protein was also detected directly on brain tissue sections using mass spectrometry profiling. We found increased levels of FKBP-12 mRNA and protein in the dorsal and middle part of the 6-OHDA lesioned striatum. Thus, these studies clearly demonstrate that FKBP-12 is increased in the brain of a common animal model of Parkinson's disease (PD). Additionally, we have identified potential binding partners to FKBP-12 that may be implicated in the pathophysiology of Parkinson's disease, such as alpha-enolase, 14-3-3 zeta/delta, pyruvate kinase isozymes, and heat shock protein 70, using surface plasmon resonance sensor technology in combination with mass spectrometry. In conclusion, these data strongly suggests that FKBP-12 is altered in an experimental model of PD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr070189eDOI Listing
October 2007

Optimizing the surface plasmon resonance/mass spectrometry interface for functional proteomics applications: how to avoid and utilize nonspecific adsorption.

Proteomics 2006 Apr;6(8):2355-64

Department of Chemical Engineering, Mälardalen University, Eskilstuna, Sweden.

A great challenge in functional or interaction proteomics is to map protein networks and establish a functional relationship between expressed proteins and their effects on cellular processes. These cellular processes can be studied by characterizing binding partners to a "bait" protein against a complex background of other molecules present in cells, tissues, or biological fluids. This so-called ligand fishing process can be performed by combining surface plasmon resonance biosensors with MS. This combination generates a unique and automated method to quantify and characterize biomolecular interactions, and identify the interaction partners. A general problem in chip-based affinity separation systems is the large surface-to-volume ratio of the fluidic system. Extreme care, therefore, is required to avoid nonspecific adsorption, resulting in losses of the target protein and carry-over during the affinity purification process, which may lead to unwanted signals in the final MS analysis and a reduction in sensitivity. In this study, carry-over of protein and low-molecular weight substances has been investigated systematically and cleaning strategies are presented. Furthermore, it is demonstrated that by the introduction of colloidal particles as a capturing and transporting agent, the recovery yield of the affinity-purified ligand could be improved nearly twofold.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.200401353DOI Listing
April 2006

Structure, stability, and orientation of BSA adsorbed to silica.

J Colloid Interface Sci 2005 Sep;289(1):26-35

Department of Chemical Engineering, Mälardalen University, Eskilstuna, Sweden.

In this investigation, the structure, stability, and orientation of bovine serum albumin (BSA) adsorbed onto silica particles were studied using differential scanning calorimetry (DSC) and limited proteolysis in combination with mass spectrometry (MS). DSC gave information on the overall structural stability of BSA while limited proteolysis was used to probe the accessibility of enzymatic cleavage sites, thereby yielding information on the orientation and structure of BSA adsorbed to silica surfaces. Thermal investigation of BSA in various buffers, both free in solution and in the adsorbed state, showed that solutes that surround the protein played an important role with respect to the overall structural stability and the structural heterogeneity of BSA. Limited proteolysis with trypsin and chymotrypsin indicated that BSA in the adsorbed state is oriented with domain 2 facing the silica surface. Also, upon adsorption, no additional cleavage sites were exposed. The combination of the results presented in this study implied that BSA molecules adsorbed onto silica particles were significantly reduced in their structural stability, but not to an extent that internal residues within the native structure became fully exposed to the solution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcis.2005.03.064DOI Listing
September 2005

SPR-MS in functional proteomics.

Brief Funct Genomic Proteomic 2005 May;4(1):39-47

Biacore AB, Rapsgatan 7, 754 50 Uppsala, Sweden.

The mapping of protein networks and the establishment of the functional relationships between expressed proteins and their effects on cellular processes represents a great challenge for functional or interaction proteomics. The combination of surface plasmon resonance (SPR)-based technology with mass spectrometry (MS) has created a unique analytical tool for functional proteomics investigations. Proteins are affinity purified, quantified and characterised in terms of their interactions, while the mass spectrometer identifies and structurally characterises the biomolecules. Recent developments have led to a closer integration of these key technologies, providing a combined approach which enables identification of proteins selected on the basis of their functional binding criteria. In addition to a historical overview of this field, some recent detailed examples of combined SPR-MS approaches will be reviewed in a number of key application areas, including ligand fishing, peptide sequence and post-translational modification analysis by SPR-MS/MS and enzyme inhibitor screening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bfgp/4.1.39DOI Listing
May 2005

Thermodynamic analysis of lysozyme adsorbed to silica.

J Colloid Interface Sci 2004 Aug;276(2):261-8

Department of Chemical Engineering, Mälardalen University, Box 325, 63105 Eskilstuna, Sweden.

The structural stability of hen egg white lysozyme in solution and adsorbed to small colloidal silica particles at various surface concentrations was investigated using hydrogen-deuterium (H/D) exchange in combination with mass spectrometry (HDX-MS) and differential scanning calorimetry (DSC). The combination of HDX-MS and DSC allows a full thermodynamic analysis of the lysozyme structure as both the enthalpy and the Gibbs free energy can be derived from the various measurements. Moreover, both HDX-MS and DSC provide information on the relative structural heterogeneity of lysozyme in the adsorbed state compared to that in solution. Results demonstrated that at high surface coverage, the structural stability of lysozyme was only marginally affected by adsorption to silica particles whereas the unfolding enthalpy decreased by more than 10%, meaning that the entropy of lysozyme increased with a similar value upon adsorption. Furthermore, the structural heterogeneity increased considerably. At lower surface concentrations, the structural heterogeneity increased further whereas the enthalpy of unfolding decreased. Further analyses of the HDX-MS experiments clearly indicated that folding/unfolding of lysozyme occurs through a two-domain process. These two domains had a similar amount of structural elements and a difference in stabilization energy of 8 kJ/mol, regardless if lysozyme was in solution or adsorbed to silica.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcis.2004.03.056DOI Listing
August 2004

Integration of surface plasmon resonance with mass spectrometry: automated ligand fishing and sample preparation for MALDI MS using a Biacore 3000 biosensor.

J Biomol Tech 2004 Jun;15(2):112-9

Biacore AB, Rapsgatan 7, 75450 Uppsala, Sweden.

Integrating surface plasmon resonance analysis with mass spectrometry allows detection and characterization of molecular interactions to be complemented with identification of interaction partners. We have developed a procedure for Biacore 3000 that automatically performs all steps from ligand fishing and recovery to sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry including on-target digestion. In the model system used in this study a signal transduction protein, calmodulin, was selectively captured from brain extract by one of its interaction partners immobilized on a sensor chip. The bound material was eluted, deposited directly onto a MALDI target, and analyzed by mass spectrometry both as an intact protein and after on-target tryptic digestion. The procedure with direct deposition of recovered material on the MALDI target reduces sample losses and, in combination with automatic sample processing, increases the throughput of surface plasmon resonance mass spectrometry analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291676PMC
June 2004

Inter-molecular migration during collisional activation monitored by hydrogen/deuterium exchange FT-ICR tandem mass spectrometry.

J Am Soc Mass Spectrom 2004 May;15(5):639-46

Department of Engineering Sciences, Division of Ion Physics, Uppsala University, Uppsala, Sweden.

The difficulty with integrating solution-phase hydrogen/deuterium exchange (HDX) and tandem mass spectrometry is that the energy added to cause fragmentation might promote gas-phase migration of the added deuterium atoms. Here, we compare the solution-phase HDX profiles generated from a- b- and y-type fragment ion series originating from capillary-skimmer dissociation. The isotopic distributions of fragments from the different fragment ion types were used to determine the isotopic state of the amide hydrogen within a specific residue. Even though the same amide hydrogen was examined, the result was different for different fragment ion types. This observation indicates that different fragment series are not equally subjected to inter-molecular migration during collision-induced dissociation (CID). We also investigated the gas-phase reactivity of originally undeuterated CID fragments of penta-phenylalanine using gas-phase HDX in an external accumulation hexapole. The incorporation of deuterium into the different fragments was studied as a function of hexapole pressure. It was found that different b- and y-ions from the same peptide had different gas-phase reactivity. However, the a-ions did not display significant gas-phase reactivity. The observed behavior has significant impact on any method that involves comparing the isotopic distributions of different fragment ions. Great care has to be taken in the interpretation of the HDX data using CID to increase the spatial resolution. The isotopic state observed after solution-phase exchange might be more preserved for some CID-fragment types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jasms.2004.01.003DOI Listing
May 2004

Localized changes in the structural stability of myoglobin upon adsorption onto silica particles, as studied with hydrogen/deuterium exchange mass spectrometry.

J Colloid Interface Sci 2003 Jul;263(2):441-8

Division of Ion Physics, Angström Laboratory, Uppsala University, Box 534, 75121, Uppsala, Sweden.

A new method is presented for monitoring the conformational stability of various parts of a protein that is physically adsorbed onto nanometer-sized silica particles. The method employs hydrogen/deuterium (H/D) exchange of amide hydrogens, a process that is extremely sensitive to structural features of proteins. The resulting mass increase is analyzed with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Higher structural specificity is obtained by enzymatically cleaving the adsorbed proteins prior to mass spectrometric analysis. The mass increases of four peptic fragments of myoglobin are followed as a function of the H/D exchange time. The four peptic fragments cover 90% of the myoglobin structure. Two of the peptic fragments, located in the middle of the myoglobin sequence and close to the heme group, do not show any adsorption-induced changes in their structural stability, whereas the more stable C- and N-terminal fragments are destabilized. Interestingly, for the N-terminal fragment, comprising residues 1-29, two distinct and equally large conformational populations are observed. One of these populations has a stability similar to that in solution (-23 kJ/mol), whereas the other population is highly destabilized upon adsorption (-11 kJ/mol).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0021-9797(03)00401-6DOI Listing
July 2003
-->