Name: multimer-page: a method for capturing and resolving protein complexes in biological samples
Uploaded: 2017-05-05T14:00:00
Description: Watch this Scientific Journal Video about Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples at JoVE.com

Supported by the NIH/NIDA DA034783. We thank Bryan A. Killinger for technical assistance with the multimer-PAGE.

1. Tissue Preparation
<ol>
	<li>Prepare 10 mL of 4x BN-PAGE sample buffer (200 mM Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), 200 mM NaCl, 40% w/v glycerol, 0.004% Ponceau S, pH 7.2). 
	NOTE: This stock solution may be made in advance and stored at 4 &#176;C.</li>
	<li>Dilute 250 &#181;L of 4x sample buffer in 750 &#181;L dH2O containing 1x commercial protease inhibitor cocktail. Vortex and chill on ice.</li>
	<li>Homogenize 20 mg of target tissue in the 1 mL 1x ice-cold BN-PAGE samp...

<ol>
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Protein-protein interactions are important for every task living things carry out. Because of this, they are the subject of intense scrutiny and research. Multimer-PAGE is a novel method for capturing, separating, and analyzing a wide range of protein complexes. We have previously demonstrated its applicability to studying oligimerization of the disease-associated protein &#945;-synuclein11. However, it is extensible to many protein complexes, as demonstrated in Figure 2. When com...

Normal cellular function is dependent on protein-protein interactions1,2. As a result, human diseases are often marked by perturbations in the assembly and behavior of various protein complexes3. The ability to characterize such interactions is therefore critical. Current means of detecting these interactions require purification of target proteins, often followed by pull-down of their interacting partners. Conventional purification is accomplished by differential centrifugation, precipitation, and/or chromatography4. These methods are time-consuming, must be altered for each target protein, and often result in low yields. Modern purification methods involve the fusion of peptide tags to target proteins, followed by immunoprecipitation or extraction on columns loaded with beads bound to a capture molecule5,6. While this is extensible to many proteins, it requires sequence modification of the target, potentially resulting in constructs that differ in affinity for their usual binding partners. The delicate nature of some protein-protein interactions means this method may not be applicable to many scenarios. Additionally, the pull-down assays used to map protein-protein interactions may not capture an accurate cellular picture, due to restricted degrees of freedom and non-native levels of the bait protein.
Ideally, protein complexes could be detected in their native states, without the need for purification or pull-down. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was developed as a less-denaturing alternative to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and allows for the separation of some proteins and complexes from biological samples7. However, proteins in BN-PAGE separate based on a large number of variables, including size, charge, three-dimensional structure, and association with other molecules. The interactions of these factors often result in co-separation of proteins, aggregate formation, and poor protein band resolution. Two-dimensional native-polyacrylamide gel electrophoresis resolves some, but not all, of these problems8.
To circumvent the complications associated with native separation, some authors use amine-reactive cross-linking reagents, such as dithiobis(succinimidyl propionate) (DSP), to capture protein complexes in tissue lysates4. These treated lysates can then be denatured and separated by SDS-PAGE, while preserving the native size and makeup of protein complexes. However, since cross-linking reagents react based on proximity of one molecule to another, and proteins in tissue lysates have many degrees of freedom and can stochastically interact, nonspecific background cross-linking can be high, especially in concentrated samples. This can lead to difficult-to-interpret results.
Here, we demonstrate the use of a hybrid BN-PAGE/SDS-PAGE method, termed multimer-polyacrylamide gel electrophoresis (multimer-PAGE), to separate and detect protein assemblies in complex mixtures. Initially, cell lysate is suspended in polyacrylamide gel via BN-PAGE. The lysate-containing gel is then reacted with the cross-linking reagent DSP. Pseudo-immobilized and slightly separated on the gel, proteins are much less likely to react nonspecifically, meaning background cross-linker reactivity is reduced. After cross-linking, the gel bands are excised and separated via SDS-PAGE. The resultant gel can then be analyzed by any means typically associated with polyacrylamide gel electrophoresis. This method allows for the separation and detection of native protein complexes in unmodified tissue lysate, without the need for additional purification or pull-down.

<table border="0" cellpadding="0" cellspacing="0" width="817">
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			<td height="21" style="height:21px;width:336px;">Chemicals</td>
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			<td height="21" style="height:21px;width:336px;">&#949;-Aminocaproic acid</td>
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			<td style="width:119px;">A2504</td>
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			<td style="width:119px;">164855000</td>
			<td>Toxic.</td>
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			<td height="42" style="height:42px;width:336px;">Acrylamide/bisacrilamide 37.5:1 (40%T stock solution)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0148</td>
			<td>Toxic.</td>
		</tr>
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			<td height="21" style="height:21px;width:336px;">Ammonium persulfate</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">A3678</td>
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			<td height="21" style="height:21px;width:336px;">Anti rabbit IgG-HRP from goat</td>
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			<td style="width:119px;">sc-2004</td>
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			<td height="21" style="height:21px;width:336px;">Bicinchoninic acid assay kit</td>
			<td style="width:195px;">Thermofisher</td>
			<td style="width:119px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Bis-tris</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">B9754</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Bovine serum albumin</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">A9647</td>
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			<td height="21" style="height:21px;width:336px;">Butanol</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A399-1</td>
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			<td height="21" style="height:21px;width:336px;">Chemiluminescence substrate kit</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">24078</td>
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			<td height="21" style="height:21px;width:336px;">Coomassie blue G-250</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0770</td>
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			<td height="21" style="height:21px;width:336px;">Digitonin</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">D141</td>
			<td>Toxic.</td>
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			<td height="21" style="height:21px;width:336px;">Dimethyl sulfoxide</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">D128-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Dithiobis(succinimidylpropionate)</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">22585</td>
			<td></td>
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			<td height="21" style="height:21px;width:336px;">Dry nonfat milk</td>
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			<td style="width:119px;">M0841</td>
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			<td style="width:119px;">G9012</td>
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			<td height="21" style="height:21px;width:336px;">Glycine</td>
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			<td style="width:119px;">BP381-5</td>
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			<td height="21" style="height:21px;width:336px;">Halt Protease Inhibitor Cocktail</td>
			<td style="width:195px;">Thermofisher</td>
			<td>78430</td>
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			<td height="21" style="height:21px;width:336px;">Hydrochloric acid</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A144SI-212</td>
			<td>For titration. Caustic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Methanol</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A412-4</td>
			<td>For PVDF membrane activation. Toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Monoclonal anti &#945;-synuclein IgG from rabbit</td>
			<td style="width:195px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-7011-R</td>
			<td></td>
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			<td height="21" style="height:21px;width:336px;">N,N,N&#39;,N&#39;-tetramethylethylenediamine</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">N,N&#39;-methylenebisacrylamide</td>
			<td style="width:195px;">Acros Organics</td>
			<td style="width:119px;">16479</td>
			<td>Toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">NP40</td>
			<td style="width:195px;">Boston Bioproducts</td>
			<td style="width:119px;">P-872</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:336px;">Polysorbate 20 (tween-20)</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP337-500</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:336px;">Polyvinylidene fluoride transfer membranes</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">88518</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ponceau S</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">P3504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Potassium chloride</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">P217-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Potassium phosphate monobasic</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">P9791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Protease inhibitor cocktail</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">88265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">SDS solution (10% w/v)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium chloride</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP358-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium dodecyl sulfate</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">L37771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium phosphate monobasic</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP329-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tricine</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">T0377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris base</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris-HCl (0.5 M buffer, pH 6.8)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris-HCl (1.5 M buffer, pH 8.8)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0798</td>
			<td></td>
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			<td height="21" style="height:21px;width:336px;">Name</td>
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			<td>Comments</td>
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			<td height="21" style="height:21px;">Instruments</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">GE Imagequant LAS 4000</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">28-9558-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">ImageJ software</td>
			<td style="width:195px;">NIH</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Synergy H1 microplate reader</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Gel Former + Stand</td>
			<td style="width:195px;">Biorad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Microfuge 22R centrifuge</td>
			<td style="width:195px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">2 mL dounce homogenizer</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Vortex mixer</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ultrasonic tissue homogenizer</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">FB120220</td>
			<td></td>
		</tr>
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multimer-page: a method for capturing and resolving protein complexes in biological samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

In this demonstration experiment, multimer-PAGE was performed on whole rat brain lysate. The resultant separated proteins were blotted onto polyvinylidene diflouride (PVDF) membranes, and then probed with antibodies against proteins that are known to form complexes. Figure 1 shows a validation of the protocol by two means. First, we demonstrate that the cross-linked proteins are cleavable by addition of a reducing agent, meaning the observed higher molecular weight specie...

Watch this Scientific Journal Video about Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples at JoVE.com

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

The overall goal of this protocol is to enable researchers to capture, separate, and analyze native protein complexes extracted from tissue lysates using polyacrylamide gel electrophoresis. This method can aid researchers who study proteins because it is able to stabilize otherwise labile protein associations under near-native conditions, allowing for their identification and subsequent analysis. The main advantage of this technique is that it uses common biological methods so it is widely applicable and can be tailored to the goals of the individual researcher.To begin this procedure, prepare for blue native polyacrylamide gel electrophoresis as described in the text protocol. Connect the electrodes to the power supply and electrophorese the proteins in the gel at 150 volts until the dye band progresses approximately two centimeters into the resolving layer. At this point, stop and disconnect the power supply.After disassembling the electrophoresis apparatus, separate the glass panes of the gel cassette. Remove and discard the stacking layer. Carefully cut the gel just below the bottom edge of the dye front, taking care to make this cut as smooth and straight as possible.Then, discard the unused piece of gel. Trim away any unused portions along the edges of the gel strip. Carefully place the strip into 10 milliliters of phosophate buffered saline in a small container and gently mix by notation for 30 minutes to equilibrate.After equilibration, discard and replace the PBS with another 10 milliliters. Pipette 500 microliters of 25 millimolar DSP dissolved in dimethyl sulfoxide into the PBS and continue mixing as before for 30 minutes. After 30 minutes, pour off the DSP solution.At 10 milliliters of 0.375 molar HSCL, pH 8.8, containing 2%sodium dodecyl sulfate to quench the unreacted DSP. Continue the notation for 15 minutes. While the gel strip is quenching, prepare SDS-PAGE gel solutions according to standard methods.Do not add the polymerization reagents. After quenching, return the blue native gel strip to room temperature and cast the strip into a new gel cassette. To do this, carefully pick up the gel and place it onto a clean gel cassette spacer plate.Orient the strip so that it is flipped from its prior orientation and the bottom of the dye front is nearest to the top of the new cassette. Place the strip such that its top edge lies even with where the top edge of the cover plate will be. Make sure the dye front is parallel to the horizontal edges of the glass plate.Push one side of the excised strip against one of the spacer walls, leaving room on the other side for the gel to be poured and a protein standard or ladder to be loaded. If the bottom edge of the gel strip contains any jagged or uneven areas, carefully cut them away. Once the gel strip is positioned correctly, lay the cover plate over the spacer plate.Apply gentle pressure to push out any trapped air bubbles. Continue to assemble the gel-pouring apparatus according to the manufacturer's instructions. The gel strip sometimes shifts when the top plate is being placed.It helps to let the strip hang over the edge of the top plate a little so it can be adjusted with a spatula to correct any shifts. Add polymerization reagents to the resolving gel buffer and pour it into the prepared gel cassette using a serological pipette. Fill the gel cassette to approximately two centimeters below the excised blue native PAGE gel strip to leave room for the stacking layer.Then, add 100 microliters of butanol over the top of the poured gel and allow 30 minutes for polymerization of the resolving layer before pouring off the butanol. Next, add the polymerization reagents to the stacking gel solution. Using a serological pipette, pour the stacking layer to fill all remaining empty space in the gel cassette.Tilt the gel cassette as the stacking layer is poured so it fills the space below the gel strip and air bubbles are not trapped. As the stacking gel buffer fills the empty space below the gel strip, gradually return the gel cassette to level footing. Continue to fill the empty space next to the excised gel strip with the stacking gel buffer until it nearly overflows.Then, allow the stacking layer to polymerize for 30 minutes. After the stacking layer has polymerized, remove the gel cassette from the pouring apparatus, rinse it with distilled water, and assemble the electrophoresis apparatus according to the manufacturer's instructions. Fill the inner chamber completely with 1x SDS-PAGE running buffer.Then, fill the outer chamber to the level indicated by the manufacturer. Load the space next to the excised gel strip with a molecular weight ladder or the appropriate protein standard. Attach the electrodes to the power supply and electrophorese the samples at 120 volts.When the Coomassie dye runs off the gel, stop the run and disconnect the power supply. Analyze the gel using standard methods of electroblotting and antibody-based protein detection. Validation of multimer-PAGE is shown here via immunoblot.The captured kinesin complex is cleaved by diphyo-3-itol showing that the high molecular weight aggregates are formed by cross-linking smaller substituents. Depicted here, brain lysates treated with increasing concentrations of SDS or Triton increased detection of monomeric alpha-synuclein while decreasing the detection of the soluble oligomer tetromer. Here, representative results of multimer-PAGE used to capture soluble complexes in rat brain lysate are shown.Proteins were detected via immunoblot. The expected monomeric weight of each protein is shown below the lanes. Similarly, capture of membrane-bound complexes is shown here.Multimer-PAGE occasionally fails to capture a complex, as is shown on the SDHA respiratory complex two blot. A discussion of possible explanations for this can be found in the text protocol. Once mastered, this technique can be done in about eight hours if it is performed properly.After watching this video, you should have a good understanding of how to perform in-gel cross-linking of native protein complexes followed by gel re-casting and separation by secondary SDS-PAGE. Don't forget that working with acrylamide can be extremely hazardous and personal protective equipment should be worn while casting the gel. Following this procedure, the separated stabilized complexes can be eluted or cut from the gel and can be analyzed by many means, including immunodetection or mass spectrometry depending on the researcher's needs.

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Research

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Biochemistry

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

Sheathless Capillary Electrophoresis&amp;#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD,&#160;allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...