Name: cellular membrane affinity chromatography columns to identify specialized plant metabolites interacting with immobilized tropomyosin kinase receptor b
Uploaded: 2022-01-19T12:30:00
Description: Watch this Scientific Journal Video about Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B at JoVE

Z.C.A. was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) 2219- International Postdoctoral Research Fellowship Program. Research reported in this publication was supported by the National Center for Complimentary and Integrative Medicine of the National Institutes of Health under award number 1R41AT011716-01. This work was also partially supported by American Society of Pharmacognosy Research Starter Grant, Regis Technologies grant to L.C. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Cell culture of SH-SY5Y neuroblastoma cells (TrkB and TrkB-NULL (parental) cell lines)
NOTE: Cell lines (SH-SY5Y Cell Line (TrkB, BR6) and SH-SY5Y Parental Cell Line (TrkB NULL))49,50 were purchased from Kerafast. Cultured cells are used as a source of the transmembrane receptors to be immobilized for the preparation of CMAC columns. The following steps describe how to obtain cell membrane fragments and assemble funct...

Identification of active compounds present in complex mixtures of specialized metabolites is a very challenging task23. Traditionally, individual compounds are isolated, and their activity is tested in different assays. This approach is time-consuming and costly and often leads to isolation and identification of the most abundant and well-characterized compounds23.&#160;Currently used high-throughput screening assays rely heavily on screening combinatorial chemistry librari...

Botanical mixtures are rich in pharmacologically active compounds1, serving as a good source for the identification of new drug hits and leads2,3,4,5.&#160;The discovery of new medicines from natural products has been a fruitful approach and many currently approved drugs originated from compounds first identified in nature. The chemical diversity of natural compounds is hard to be matched by man-made libraries of chemically synthesized molecules. Many natural compounds interact with and modulate human protein targets and can be considered evolutionarily optimized drug-like molecules6. These natural compounds are particularly well suited for drug lead identification to use in neurological disorders6. Two of the currently FDA-approved drugs for the management of Alzheimer&#39;s disease (AD) are derived from natural alkaloids, namely: galantamine and rivastigmine (a derivative of physostigmine)6. L-DOPA, presently the most commonly prescribed drug for Parkinson&#39;s disease, was first identified from the broad bean (Vicia faba L.)7. Pergolide and lisuride, dopaminergic receptor agonists are the derivatives of natural ergot alkaloids from the parasitic fungus Claviceps purpurea8. Reserpine, an alkaloid isolated from Indian snakeroot (Rauvolfia serpentina (L.) Benth. ex Kurz)&#160;was one of the first antipsychotic drugs9. Recently, dysregulated immune response and systemic inflammation have been linked to the development of numerous neurological ailments, such as major depressive disorder or neurodegenerative diseases10. A plant-based diet together with other lifestyle interventions has been found to improve cognitive and functional abilities in the elderly11,12,13,14,15,16,17,18,19,20,21. Certain electrophilic molecules belonging to triterpenes and polyphenols have been found to modulate inflammatory responses in both in vitro and in vivo models12. For instance, natural compounds containing &#945;,&#946;-unsaturated carbonyl (e.g., curcumin, cinnamaldehyde), or isothiocyanate group (e.g., sulforaphane) interfere with Toll-like receptor-4 (TLR4) dimerization inhibiting the downstream synthesis of pro-inflammatory cytokines in a murine interleukin-3 dependent pro-B cell line12,22. Epidemiological evidence points strongly that dietary phytochemicals, present in complex food matrices, may also constitute a viable source of new drug leads6.
One of the major obstacles in the identification of biologically active molecules present in plant extracts, including plant-based food, is the complexity of the investigated samples. Traditionally, the individual compounds are isolated, purified, and subsequently tested for biological activity. This approach usually leads to the identification of the most abundant and well-characterized compounds. Phenotypic drug discovery approaches without a defined molecular target rely on the bio-guided-fractionation of complex mixtures23. In this approach, an extract is fractionated into less complex sub-fractions that are subsequently tested in phenotypic assays. The isolation and purification of active compounds are guided by biological activity verified in the assay. The knowledge of the identity of a specified drug target may significantly speed up the identification of pharmacologically active compounds present in complex mixtures. Those approaches are usually based on the immobilization of the molecular target, for example, an enzyme, on a solid surface, like magnetic beads23. The immobilized targets are subsequently used in the screening experiments resulting in the isolation of compounds interacting with the target. While this approach has been extensively used in the identification of compounds targeting cytosolic proteins, it has been less commonly applied in the identification of chemicals interacting with transmembrane proteins (TMPs)23. An additional challenge in the immobilization of TMPs stems from the fact that the activity of the protein depends on its interaction with cell membrane phospholipids and other molecules in the bilayer such as cholesterol23,24. It is important to preserve these subtle interactions between proteins and their native phospholipid bilayer environment when attempting to immobilize the transmembrane target.
In cellular membrane affinity chromatography (CMAC) cell membrane fragments, and not purified proteins, are immobilized on the artificial membrane (IAM) stationary phase particles23. IAM stationary phases are prepared by covalently bonding phosphatidylcholine analogs onto silica. Recently novel classes of IAM stationary phases have been developed in which free amine and silanol groups are end-capped (IAM.PC.DD2 particles). During CMAC columns preparation cell membrane fragments are immobilized onto the surface of IAM particles through adsorption.
CMAC columns have been used to date to immobilize different classes of TMPs including ion channels (e.g., nicotinic receptors), GPCRs (e.g., opioid receptors), protein transporters (e.g., p-glycoprotein), etc.24. The immobilized protein targets have been used in the characterization of pharmacodynamics (e.g., dissociation constant, Kd) or determining binding kinetics (kon and koff) of small molecule ligands interacting with the target as well as in the process of identification of potential new drug leads present in complex matrices24. Here we present the preparation of CMAC columns with the immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders.
Previous studies showed that the activation of the brain-derived neurotrophic factor (BDNF)/TrkB pathway is associated with the improvement of certain neurological ailments, such as AD or major depressive disorder25,26,27,28. It was reported that BDNF levels and its receptor TrkB expression decrease in AD, and similar reductions impair hippocampal function in animal models of AD29. Decreased levels of BDNF were reported in serum and brain of AD patients30,31,32. Tau overexpression or hyperphosphorylation were found to down-regulate BDNF expression in primary neurons and AD animal models33,34,35. Additionally, BDNF was reported to have protective effects on &#946;-amyloid induced neurotoxicity in vitro and in vivo36. Direct BDNF administration into the rat brain was shown to increase learning and memory in cognitively impaired animals37. BDNF/TrkB emerged as a valid target for ameliorating neurological and psychiatric disorders including AD28,38. Targeting the BDNF/TrkB signaling pathway for the development of therapies in AD will potentially enhance our understanding of the disease39. Unfortunately, BDNF itself cannot be used as a treatment because of its poor pharmacokinetic properties and adverse side effects40. Small molecule activators of TrkB/BDNF pathways have been explored as potential TrkB ligands41,42,43. Among tested small molecule agonists, 7,8-dihydroxyflavone (7,8-DHF), has been shown to activate the BDNF/TrkB pathway41,44,45,46. A derivative of 7,8-DHF (R13; 4-Oxo-2-phenyl-4H-chromene-7,8-diyl bis(methylcarbamate)) is currently under consideration as a possible drug for AD47. Recently, it was shown that several antidepressants work through directly binding to TrkB and promoting BDNF signaling, further stressing the importance of pursuing TrkB as a valid target to treat various neurological disorders48.
The protocol describes the process of assembling functional TrkB column and TrkB-NULL negative control column. The columns are characterized using a known natural product small-molecular ligand: 7,8-DHF. Additionally, we describe the process of screening complex matrices, using plant extract as an example, for the identification of compounds interacting with TrkB.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7-8 Dihydroxyflavone hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>D5446-10 mg</td>
			<td>&#8805;98% (HPLC)</td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate (ATP) disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A2383-1 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>VWR Chemicals BDH</td>
			<td>BDH9204-500 g</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF antibody</td>
			<td>Invitrogen</td>
			<td>PA5-15198-400 &#956;L</td>
			<td>Primary antibody; 2 mg/mL of concentration</td>
		</tr>
		<tr>
			<td>Benzamidine hydrochloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>B6506-25 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF) human</td>
			<td>Sigma-Aldrich</td>
			<td>B3795-10 &#956;g</td>
			<td>Recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>VWR Analytical</td>
			<td>BDH9224-1 kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholic acid sodium salt</td>
			<td>Alfa Aesar</td>
			<td>J62050-100 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>VWR</td>
			<td>71000-516</td>
			<td>40 mL, 285 mm (overall lenght), 32 x 140 mm (O.D. x H)</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>493511</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>VWR Analytical</td>
			<td>BDH-9232-500 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442-500 mL</td>
			<td>sterile-filtered, suitable for cell culture</td>
		</tr>
		<tr>
			<td>G418 disulfate salt solution</td>
			<td>Sigma-Aldrich</td>
			<td>G8168-100 mL</td>
			<td>50 mg/mL in H2O, 0.1 &#956;m filtered, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR Life Science</td>
			<td>E520-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilized artificial membrane (IAM.PC.DD2)</td>
			<td>Regis Technologies, Inc.</td>
			<td>1-771050-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>VWR Analytical</td>
			<td>BDH9244-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322425</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Plan Fluor</td>
			<td>Nikon</td>
			<td></td>
			<td>Confocal laser scanning microscope</td>
		</tr>
		<tr>
			<td>Normal goat serum (10%)</td>
			<td>Life Technologies</td>
			<td>50197Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Thermo Scientific</td>
			<td>36978-5 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>VWR Life Science</td>
			<td>K812-500 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>VWR Chemicals BDH</td>
			<td>0395-1 kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>VWR Life Science Ambreso</td>
			<td>M221-1 mL</td>
			<td>Proteomics grade, containing 50 mM AEBSF, 30 &#181;M aprotonin, 1 mM bestatin, 1 mM E-64 and 1 mM leupeptin</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Sigma-Aldrich</td>
			<td>R8758-500 mL</td>
			<td>with L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Secondary antibody goat anti-rabbit IgG (H+L)</td>
			<td>Invitrogen Alexa Flour Plus 488</td>
			<td>A32731</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-SY5Y Neuroblastoma cell lines expressing Trk-B</td>
			<td>Kerafast</td>
			<td>ECP007</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-SY5Y Trk-NULL cell line</td>
			<td>Kerafast</td>
			<td>ECP005</td>
			<td></td>
		</tr>
		<tr>
			<td>Snake skin dialysis tubing</td>
			<td>Thermo Scientific</td>
			<td>88245</td>
			<td>10K MWCO, 35 mm dry I.D.</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BDH VWR Analytical</td>
			<td>BDH9286-2.5 kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricorn 5/20 column</td>
			<td>GE Healthcare</td>
			<td>24-4064-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>VWR Life Science</td>
			<td>0497-1 kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500 mL</td>
			<td>0.25%, sterile-filtered, suitable for cell culture, 2.5 g porcine trypsin and 0.2&#160;g EDTA</td>
		</tr>
	</tbody>
</table>

cellular membrane affinity chromatography columns to identify specialized plant metabolites interacting with immobilized tropomyosin kinase receptor b

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

Following the protocol, two CMAC chromatographic columns were assembled: one with the immobilized SH-SY5Y neuroblastoma cell membrane fragments with overexpressed TrkB and one with SH-SY5Y TrkB-NULL cell membrane fragments. The correctly assembled CMAC column is presented in Figure 1 and the steps involved in cell membrane fragment immobilization are presented in Figure 2.
Since the immobilization of TrkB receptors on IAM.PC.DD2 chrom...

Watch this Scientific Journal Video about Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B at JoVE

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...

This vertical presents important steps in assembling cell membrane affinity chromatography columns with functional transmembrane receptors. These columns can be used for the identification of small molecules present in complex extracts and interacting with immobilized transmembrane receptors. Cellular membrane affinity chromatography columns speed up the identification of biologically active compounds present in complex mixtures.The use of cellular membrane affinity chromatography columns can be of particular interest to those interested in the identification of pharmacologically active compounds present in complex natural matrices, such as plant, fungal, or bacterial extracts. To begin, prepare 200 millimolar benzamidine stock solution by dissolving 120 of benzamidine in five milliliters of ultrapure deionized water. Prepare a 10 millimolar PMSF stock solution by dissolving 0.017 grams of PMSF in 10 milliliters of ethanol in a fume hood.Prepare 100X proteases inhibitor cocktail by dissolving commercially available protease inhibitor mixture in one milliliter of ultrapure deionized water. Mix thoroughly and aliquot 200 to 300 microliters of the cocktail. Prepare 100 millimolar ATP stock solution by dissolving 55.114 milligrams of ATP disodium salt hydrate in one milliliter of the ionized ultrapure water.Mix thoroughly and aliquot 100 microliters of the mixture. Prepare buffer by dissolving 3.03 grams of Tris-HCL, 2.9 grams of sodium chloride, 0.22 grams of calcium chloride anhydrous, 0.2 grams of magnesium chloride hexahydrate, and 0.19 grams of potassium chloride in 500 milliliters of ultrapure deionized water. Prepare homogenization buffer by mixing 17.3 milliliters of the Tris buffer with 0.3 milliliters of benzamidine stock solution, 0.2 milliliters of PMSF stock solution, 0.2 milliliters of proteases inhibitor cocktail, 20 microliters of ATP stock solution, two milliliters of glycerol, and 0.029 grams of EDTA.Adjust pH to 7.4 using hydrochloric acid solution. Spin down the cells harvested at four degrees Celsius for five minutes at 400 x g and remove the supernatant and wash the remaining cell pellet with 10 milliliters of 1X pH 7.4 ice cold PBS. Discard the supernatant and add 20 milliliters of homogenization buffer.Place the conical tube on ice. Transfer the cell suspension into a 40 milliliter Dounce homogenizer tissue grinder, homogenize the suspension manually on ice using 40 up and down pestle strokes. Transfer homogenized cell suspension into a 50 milliliter conical tube, discard the pellet and transfer the supernatant to a new conical tube.Save the cell membrane pellet and discard the supernatant. Prepare solubilization buffer by mixing 8.7 milliliters of the buffer solution with 0.1 milliliters of PMSF stock solution, 0.15 milliliters of benzamidine stock solution, 0.1 milliliters of proteases inhibitor cocktail, 10 microliters of ATP stock solution, one milliliter of glycerol, and 0.2 grams of sodium cholate. Transfer the solubilization buffer into the conical tube with cell membrane fragments.And resuspend the pellet. Rotate the resulting mixture at 150 RPM at four degrees Celsius for 18 hours. After 18 hours, centrifuge the solubilization mixture at four degrees Celsius for 30 minutes at 47, 900 x g.Add 100 milligrams of IAM. PC.DD2 particles to the supernatant and rotate the resulting suspension mixture at 150 RPM at four degrees Celsius for one hour. Prepare dialysis buffer in four liters of ultrapure deionized water by adding 24 grams of Tris-HCl, 23.4 grams of sodium chloride, 0.06 grams of calcium chloride, 5.85 grams of EDTA, and 0.07 grams of PMSF.Dissolve PMSF first in a small volume of ethanol and then add slowly into the beaker with the buffer. Cut 10 centimeters of cellulose membrane dialysis tubing to prepare dialysis tube, and transfer the suspension containing IAM. PC.DD2 particles and cell membrane fragments into the dialysis tube.Close both ends of the dialysis tube with dialysis tubing clips. Place the dialysis tube in the dialysis buffer. After 24 hours, place the dialysis tubing in the freshly prepared dialysis buffer and continued dialysis for another 24 hours.Prepare 10 millimolar pH 7.4 ammonium acetate buffer that will be used to wash the IAM. PC.DD2 particles and in column characterization experiments by dissolving 0.778 grams of ammonium acetate in one liter of ultrapure deionized water. After 48 hours of dialysis, remove the dialysis tube from the dialysis buffer and transfer the content of the dialysis tube into a 15 milliliter conical tube.Discard the supernatant and wash the remaining pellet three times with 10 milliliters of ammonium acetate buffer. After the third wash, resuspend the remaining pellet in one milliliter of ammonium acetate buffer. Mix it thoroughly and use the resulting slurry to pack the 5 x 20 glass column to yield the CMAC chromatography column.To pack the column, place a bottom filter previously soaked with ammonium acetate buffer into the filter holder. Fit the filter holder in the glass column and screw the column cap to secure the holder's position. Place the column vertically in a finger clamp and secure it in the lab stand.Place a beaker below the column, transfer a small volume of the slurry into the glass column slowly with a single channel pipetter holding the pipette tip against the glass column wall. To speed up the packing process, remove the buffer from above the stationary bed with a micropipette between each step. Place a top filter and screw the adapter unit so that there is no remaining buffer above the stationary phase.Secure the position of the adapter unit with the adapter lock. Connect the column to a high performance liquid chromatography pump, and to wash the column overnight with ammonium acetate buffer, set the flow rate to 0.2 milliliters per minute. For longer storage, run the column with 0.05%sodium azide solution in ammonium acetate buffer, wearing a lab coat, safety glasses, and gloves when working with sodium azide and store at four degrees Celsius.IAM.PC.DD2 particles with immobilized neuroblastoma TrkB cell membrane fragments and in the presence of BDNF resulted in fluorescent particles. No fluorescence was observed in TrkB null cell membrane encapsulated IAM particles or when no BDNF was used as in TrkB cell membrane encapsulated IAM particles. A functional CMAC columns typical frontal affinity chromatogram of increasing 7, 8-DHF concentrations of one millimolar, 750 nanomolar, 500 nanomolar, and 300 nanomolar to the immobilized TrkB receptors are shown, which showed a concentration dependent change in retention time.A nonfunctional CMAC column showed the lack of concentration dependent changes in retention of the marker ligand. A CMAC negative control column was prepared by immobilizing cell membrane fragments, not expressing the targeted protein to rule out non-specific interactions. The addition of 0.2%aqueous Gotu kola extract resulted in a significant reduction of 7, 8-DHF retention, indicating the presence of competing ligands for the agonist binding site.The lack of reduction of 7, 8-DHF retention on the CMAC negative TrkB null column further confirmed the lack of functional TrkB receptors on this column. The missing peak chromatography approach identified a compound strongly retained on TrkB column, while eluting early on TrkB null column, indicating specific interaction. Its crucial to know your source of receptors for fast cellular membrane affinity chromatography column.Make sure to check the literature to properly design homogenization and solubilization buffers. Cellular membrane affinity chromatography column prepared in this protocol was used to identify new potential drug hits and to characterize the nature of the interaction between immobilized receptor and its natural ligands.

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Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box - equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.

The goal of this protocol is to detect phenolic metabolites in plasma using a semi-targeted chromatography-mass spectrometry method.

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss ...