| 1. |
- Jansson, Erik, 1984, et al.
(författare)
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Effect of cholesterol depletion on the pore dilation of TRPV1
- 2013
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Ingår i: Molecular Pain. - : SAGE Publications. - 1744-8069. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.
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| 2. |
- Jansson, Erik, 1984, et al.
(författare)
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Microfluidic Flow Cell for Sequential Digestion of Immobilized Proteoliposomes
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:13, s. 5582-5588
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50–150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 μg proteoliposomes/cm2, and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 μg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1–1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect 65% more unique membrane-associated protein (p
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| 3. |
- Stenum, Thomas Søndergaard, et al.
(författare)
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RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins
- 2023
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:9, s. 4572-4587
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Tidskriftsartikel (refereegranskat)abstract
- RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.
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| 4. |
- Trkulja, Carolina, 1984, et al.
(författare)
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Probing Structure and Function of Ion Channels Using Limited Proteolysis and Microfluidics
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 136:42, s. 14875-14882
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Tidskriftsartikel (refereegranskat)abstract
- Even though gain, loss, or modulation of ion channel function is implicated in many diseases, both rare and common, the development of new pharmaceuticals targeting this class has been disappointing, where it has been a major problem to obtain correlated structural and functional information. Here, we present a microfluidic method in which the ion channel TRPV1, contained in proteoliposomes or in excised patches, was exposed to limited trypsin proteolysis. Cleaved-off peptides were identified by MS, and electrophysiological properties were recorded by patch clamp. Thus, the structure-function relationship was evaluated by correlating changes in function with removal of structural elements. Using this approach, we pinpointed regions of TRPV1 that affect channel properties upon their removal, causing changes in current amplitude, single-channel conductance, and EC50 value toward its agonist, capsaicin. We have provided a fast "shotgun" method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions.
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| 5. |
- Aerts, Jordan
(författare)
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Capillary electrophoresis mass spectrometry applied to structural proteomics and small molecule analysis
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Capillary electrophoresis with mass spectrometric (CE–MS) detection offers a separation method without equal in terms of flexibility, utility, and cost efficiency. Here we demonstrate precisely this through the application of several laboratory-built CE–MS instruments for the separation of brain metabolites in non-primates, enantioselective separations of synthetic anesthetic metabolites in fractionated pony urine, application in structural proteomics workflows, and identification of exogenous alkaloid biotransformationproducts in human cerebrospinal fluid (CSF).We outline a method for quickly and affordably etching austenitic steel tubing, which is widely used in electrospray sources for CE–MS. The stainless steel tapered tip emitters provide robust electrospray with low sheath liquid flow rates and can be easily fabricated in-house, offering flexibility and cost-efficiency when commercial options areunavailable. We contribute a CE–MS method for enantiomer separation, specifically targeting 6-hydroxynorketamine (HNK). By introducing chiral selectors into the separation capillary, the method enables efficient enantiomer separation and offers a newtool to assist with research on HNK as a cure for depression.We explore the feasibility of cold CE–MS in hydrogen deuterium exchange workflows. The utilization of a lab-designed Peltier-cooled CE device achieves deuterium back exchange rates on par with commercial liquid chromatography-based platforms, offering new possibilities for studying protein structures and interactions.We also demonstrate the wide ranging versatility of CE–MS with contributions to the identification of specific tobacco related metabolites in CSF samples during the development of a high throughput mass spectrometry diagnostic tool for Parkinson’sDisease.This thesis showcases the versatility and value of CE–MS in various applications, a true blessing for analytical chemistry.
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| 6. |
- Aerts, Jordan, et al.
(författare)
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Electrochemically Etched Tapered-Tip Stainless-Steel Electrospray-Ionization Emitters for Capillary Electrophoresis-Mass Spectrometry
- 2023
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 22:4, s. 1377-1380
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Tidskriftsartikel (refereegranskat)abstract
- We have used household consumables to facilitate electrochemical etching of stainless-steel hypodermic tubing to produce tapered-tip emitters suitable for electrospray ionization for use in mass spectrometry. The process involves the use of 1% oxalic acid and a 5 W USB power adapter, commonly known as a phone charger. Further, our method avoids the otherwise commonly used strong acids that entail chemical hazards: concentrated HNO3 for etching stainless steel, or concentrated HF for etching fused silica. Hence, we here provide a convenient and self-inhibiting procedure with minimal chemical hazards to manufacture tapered-tip stainless-steel emitters. We show its performance in metabolomic analysis with CE-MS of a tissue homogenate where the metabolites acetylcarnitine, arginine, carnitine, creatine, homocarnosine, and valerylcarnitine were identified, all with basepeak separated electropherograms, within <6 min of separation. The mass spectrometry data are freely available through the MetaboLight public data repository via access number MTBLS7230.
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| 7. |
- Aerts, Jordan, et al.
(författare)
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Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry
- 2023
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Ingår i: Analytical Chemistry. - : Springer Nature. - 0003-2700 .- 1520-6882. ; 95:2, s. 1149-1158
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Tidskriftsartikel (refereegranskat)abstract
- Currently, fast liquid chromatographic separations at low temperatures are exclusively used for the separation of peptides generated in hydrogen deuterium exchange (HDX) workflows. However, it has been suggested that capillary electrophoresis may be a better option for use with HDX. We performed in solution HDX on peptides and bovine hemoglobin (Hb) followed by quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for benchmarking a laboratory-built HDX–MS platform. We found that capillaries with a neutral coating to eliminate electroosmotic flow and adsorptive processes provided fast separations with upper limit peak capacities surpassing 170. In contrast, uncoated capillaries achieved 30% higher deuterium retention for an angiotensin II peptide standard owing to faster separations but with only half the peak capacity of coated capillaries. Data obtained using two different separation conditions on peptic digests of Hb showed strong agreement of the relative deuterium uptake between methods. Processed data for denatured versus native Hb after deuterium labeling for the longest timepoint in this study (50,000 s) also showed agreement with subunit interaction sites determined by crystallographic methods. All proteomic data are available under DOI: 10.6019/PXD034245.
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| 8. |
- Ainla, Alar, 1982, et al.
(författare)
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A Microfluidic Pipette for Single-Cell Pharmacology
- 2010
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 82:11, s. 4529-4536
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Tidskriftsartikel (refereegranskat)abstract
- We report on a free-standing microfluidic pipette made in poly(dimethylsiloxane) having a circulating liquid tip that generates a self-con-fining volume in front of the outlet channels. The method is flexible and scalable as the geometry and the size of the recirculation zone is defined by pressure, channel number, and geometry. The pipette is capable of carrying out a variety of complex fluid processing operations, such as mixing, multiplexing, or gradient generation at selected cells in cell and tissue cultures. Using an uptake assay, we show that it is possible to generate dose response curves in situ from adherent Chinese hamster ovary cells expressing proton-activated human transient receptor potential vanilloid (hTRPV1) receptors. Using confined superfusion and cell stimulation, we could activate hTRPV1 receptors in single cells, measure the response by a patch-clamp pipette, and induce membrane bleb formation by exposing selected groups of cells to formaldehyde/dithiothreitol-containing solutions, respectively. In short, the microfluidic pipette allows for complex, contamination-free multiple-compound delivery for pharmacological screening of intact adherent cells.
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| 9. |
- Ainla, Alar, 1982, et al.
(författare)
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A multi-purpose microfluidic pipette for single-cell analysis
- 2010
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Ingår i: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010; Groningen; Netherlands; 3 October 2010 through 7 October 2010. - 9781618390622 ; 2, s. 932-934
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Konferensbidrag (refereegranskat)abstract
- We report a multi-purpose microfluidic pipette, with a recirculating liquid tip. This device, made in poly(dimethylsiloxane), enables contamination-free manipulation and chemical stimulation of selected single cells in cell collectives or tissue slices. The pipette is capable of carrying out a variety of complex fluid processing functionalities, such as mixing, multiplexing, or gradient generation. The concept is flexible and scalable as the geometry and the size of the recirculation zone is defined by pressure, channel number, and geometry. We have applied the pipette in a fluorescence uptake assay, electrophysiology studies and for chemical induction of membrane protrusion from biological cells.
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| 10. |
- Casillas Trujillo, Luis, et al.
(författare)
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Experimental and theoretical evidence of charge transfer in multi-component alloys : how chemical interactions reduce atomic size mismatch
- 2021
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Ingår i: Materials Chemistry Frontiers. - : Royal Society of Chemistry. - 2052-1537. ; 5:15, s. 5746-5759
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Tidskriftsartikel (refereegranskat)abstract
- Ab initio simulations of a multi-component alloy using density functional theory (DFT) were combined with experiments on thin films of the same material using X-ray photoelectron spectroscopy (XPS) to study the connection between the electronic and atomic structures of multi-component alloys. The DFT simulations were performed on an equimolar HfNbTiVZr multi-component alloy. Structure and charge transfer were evaluated using relaxed, non-relaxed, as well as elemental reference structures. The use of a fixed sphere size model allowed quantification of charge transfer, and separation into different contributions. The charge transfer was generally found to follow electronegativity trends and results in a reduced size mismatch between the elements, and thus causes a considerable reduction of the lattice distortions compared to a traditional assumption based on tabulated atomic radii. A calculation of the average deviation from the average radius (i.e. the so-called δ-parameter) based on the atomic Voronoi volumes gave a reduction of δ from ca. 6% (using the volumes in elemental reference phases) to ca. 2% (using the volumes in the relaxed multi-component alloy phase). The reliability of the theoretical results was confirmed by XPS measurements of a Hf22Nb19Ti18V19Zr21 thin film deposited by sputter deposition. The experimentally observed core level binding energy shifts (CLS), as well as peak broadening due to a range of chemical surroundings, for each element showed good agreement with the calculated DFT values. The single solid solution phase of the sample was confirmed by X-ray diffraction (XRD) and transmission electron microscopy (TEM) including energy dispersive spectroscopy (EDS) with nm-resolution. These observations show that the HfNbTiVZr solid solution phase is non-ideal, and that chemical bonding plays an important part in the structure formation, and presumably also in the properties. Our conclusions should be transferable to other multi-component alloy systems, as well as some other multi-component material systems, and open up interesting possibilities for the design of material properties via the electronic structure and controlled charge transfer between selected metallic elements in the materials.
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