Name: cell-type specific protein purification and identification from complex tissues using a mutant methionine trna synthetase mouse line
Uploaded: 2022-04-13T14:00:00
Description: Watch this Scientific Journal Video about Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line at JoVE.com

B.A-C is funded by the Spanish Ministry of Science and Innovation (Ram&#243;n y Cajal-RYC2018-024435-I), by the Autonomous Community of Madrid (Atracci&#243;n de Talento-2019T1/BMD-14057), and MICINN (PID2020-113270RA-I00) grants. R. A-P is funded by Autonomous Community of Madrid (Atracci&#243;n de Talento-2019T1/BMD-14057). E.M.S. is funded by the Max Planck Society, an Advanced Investigator award from the European Research Council (grant 743216), DFG CRC 1080: Molecular and Cellular Mechanisms of Neural Homeostasis, and DFG CRC 902: Molecular Principles of RNA-based Regulation. We thank D.C Dieterich and P. Landgraf for their technical advise and the synthesis of the DST-Alkyne. We thank E. Northrup, S. Zeissler, S. Gil Mast, and the animal facility of the MPI for Brain Research for their excellent support. We thank Sandra Goebbels for sharing the Nex-Cre mouse line. We thank Antonio G. Carroggio for his help with English editing. B.A-C. designed, conducted, and analyzed experiments. B.N-A, D.O.C, R.A-P, C. E., and S. t. D. conducted and analyzed experiments. B.A-C and E.M.S. designed experiments, and supervised the project, B.A-C wrote the paper. All authors edited the paper.

All experiments with animals were performed with permission from the local government offices in Germany (RP Darmstadt; protocols: V54-19c20/15-F122/14, V54-19c20/15-F126/1012) or Spain (Committee of Animal Experiments at the UCM and Environmental Counselling of the Comunidad de Madrid, protocol number: PROEX 005.0/21) and are compliant with the Max Planck Society rules and Spanish regulations and follow the EU guidelines for animal welfare.
1. In vivo metabolic labeling with AN...

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The critical aspects of the protocol are; the inclusion of negative controls, having enough biological replicates, ANL administration route, amount, and duration, alkylation of the samples, alkyne concentration, and &#946;-mercaptoethanol elimination when using DST-alkyne.
It is key to include negative control samples proceeding from ANL-labeled animals without Cre driver and therefore no MetRS* expression. These samples must be subjected to every step described in the protocol in parallel to ...

Aberrant protein homeostasis is caused by an imbalance in protein synthesis and degradation. Several diseases are related to alterations in protein homeostasis. The hallmark of some diseases is the presence of aggregates in different subcellular locations and brain areas. Protein homeostasis is not only important in disease but also plays a crucial role in normal organ and cellular function1. For example, protein synthesis is necessary for many forms of neuronal plasticity2,3, as determined by the use of chemical inhibitors that block protein synthesis4. However, it is neither clear in which cell-types the proteome is altered to support learning and memory, nor is it understood which specific proteins in each cell-type increase or decrease in their synthesis or degradation. Thus, a comprehensive study of protein homeostasis requires the capability to differentiate proteomes coming from specific cell-types. Indeed, the identification of cell-type-specific proteomes to study cellular processes occurring in a multicellular environment has been an important hurdle in proteomics. For this reason, we developed a technique using MetRS* expression combined with bio-orthogonal methods that has proven to be an effective way to identify and purify cell-type specific proteomes, filling this gap5,6,7.
The expression of a mutant MetRS* (MetRS L274G) allows for the loading of the non-canonical methionine analog ANL into the corresponding tRNA8,9 and its subsequent incorporation&#160;into proteins. When MetRS* expression is regulated by a cell-type-specific promoter, the non-canonical amino acid will be incorporated into the proteins in a cell-selective manner. Once ANL is incorporated in the proteins, it can be selectively functionalized by click-chemistry and subsequently either visualized by imaging (FUNCAT) or by Western Immunoblot (BONCAT). Alternatively, proteins can be selectively purified and identified by mass spectrometry (MS). Using this technology, we created a mouse line expressing the MetRS* protein under the control of the Cre recombinase. Considering the increasing number of available Cre-mouse lines, the MetRS* system can be used in any field to study any cell-type from any tissue for which there is an existing Cre-line. Protein labeling with ANL is possible in vitro or in vivo, and does not alter mouse behavior or protein integrity6. Labeling timespan can be adapted to the scientific question of each researcher, labeling newly synthesized proteins (shorter labeling times) or entire proteomes (longer labeling times). The use of this technique is limited by the number of cells of the type that the researcher is willing to study; hence protein isolation from cell-types with low numbers or low metabolic rates is not possible by this method. The goal of the presented method is to identify cell-type-specific proteins/proteomes labeled in vivo. In this protocol, we describe how to label cell-type-specific proteomes with ANL in live mice and purify the labeled proteins. After purification, proteins can be identified by routine mass spectrometry protocols5,10. The reduction of sample complexity achieved in this method by the selective purification of proteins from specific cellular populations allows the experimenter to detect subtle changes in proteomes, for example, in response to environmental changes. Purification of the labeled proteins can be achieved in ~10 days, not including the MS analysis or the labeling period. Here, we describe two methods for ANL administration to MetRS* expressing mice, namely (1) adding the amino acid in the drinking water, and (2) introducing ANL by intraperitoneal injections. Regardless of the method chosen for ANL administration, the isolation and purification steps are the same (from step 2 on).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12% Acrylamide gels</td>
			<td>GenScript</td>
			<td>SurePAGE, Bis-Tris, 10 x 8, 12%</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>Toxic; use a lab coat, gloves and a fume hood.</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma</td>
			<td>9830</td>
			<td>Toxic; use a lab coat, gloves and a fume hood</td>
		</tr>
		<tr>
			<td>ANL</td>
			<td></td>
			<td></td>
			<td>Synthesized as described previously for AHA (see references 5 and 11)</td>
		</tr>
		<tr>
			<td>ANL-HCl</td>
			<td>IrishBotech</td>
			<td>HAA1625.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Sigma</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin alkyne</td>
			<td>Thermo,</td>
			<td>B10185</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken antibody anti-GFP</td>
			<td>Aves</td>
			<td>1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA-free protease inhibitor</td>
			<td>Roche</td>
			<td>4693132001</td>
			<td>Toxic; use a lab coat and gloves.</td>
		</tr>
		<tr>
			<td>Copper (I) bromide</td>
			<td>Sigma</td>
			<td>254185</td>
			<td>99.999% (wt/wt)</td>
		</tr>
		<tr>
			<td>Disulfide tag (DST)-alkyne</td>
			<td></td>
			<td></td>
			<td>Synthesized as reported in reference number 15, in which it is referred to as probe 20</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>Filters</td>
			<td>Merk</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodo acetamide (IAA)</td>
			<td>Sigma</td>
			<td>I1149</td>
			<td></td>
		</tr>
		<tr>
			<td>IR anti chicken 800</td>
			<td>LI-COR</td>
			<td>IRDye 800CW</td>
			<td>Donkey anti-Chicken Secondary Antibody</td>
		</tr>
		<tr>
			<td>IR anti rabit 680</td>
			<td>LI-COR</td>
			<td>IRDye 680RD</td>
			<td>Goat anti-Rabbit IgG Secondary Antibody</td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>Sigma</td>
			<td>M9171</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Mixer</td>
			<td>BioSpec Products</td>
			<td>1083</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Sigma</td>
			<td>4259</td>
			<td>Toxic; use a lab coat, gloves and a fume hood.</td>
		</tr>
		<tr>
			<td>NeutrAvidin beads</td>
			<td>Pierce</td>
			<td>29200</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Bio-rad</td>
			<td>1620112</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Thermo</td>
			<td>J62036.K2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X pH 7.8</td>
			<td></td>
			<td></td>
			<td>Preparation described in reference number 5</td>
		</tr>
		<tr>
			<td>PD SpinTrap G-25 columns</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>Buffer exchange</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo,</td>
			<td>23225</td>
			<td>Reagents in the Pierce BCA Protein Assay Kit are toxic to aquatic life.</td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-biotin antibody</td>
			<td>Cell Signaling</td>
			<td>5597</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS 10%</td>
			<td>Sigma</td>
			<td>71736</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE&#160; Running buffer MOPS</td>
			<td>GenScript</td>
			<td>M00138</td>
			<td></td>
		</tr>
		<tr>
			<td>SYPRO Ruby stain</td>
			<td>Sigma</td>
			<td>S4942</td>
			<td></td>
		</tr>
		<tr>
			<td>Table automatic Vortexer</td>
			<td>Eppendorf</td>
			<td>Mixmate</td>
			<td></td>
		</tr>
		<tr>
			<td>Triazole ligand</td>
			<td>Sigma</td>
			<td>678937</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W4502</td>
			<td>Molecular biology grade</td>
		</tr>
	</tbody>
</table>

cell-type specific protein purification and identification from complex tissues using a mutant methionine trna synthetase mouse line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

Following the described protocol (summarized in Figure 1), ANL was administered to mice either by daily intraperitoneal injections (400 mM ANL 10 mL/kg, Nex-Cre::MetRS*) for 7 days, or via drinking water (0.7% Maltose, 1% ANL, CamkII-Cre::MetRS*) for 21 days. After labeling, the corresponding brain areas were dissected, lysed, alkylated, and clicked. Click reactions were analyzed by SDS-PAGE and Western Immunoblot. Representative images of the experiments are shown ...

Watch this Scientific Journal Video about Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line at JoVE.com

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol ...

This protocol makes it possible to label proteins in a cell type specific manner. This is the first technique that offers such a possibility and it can be done in vitro or in vivo. The main advantage of the technique is that the labeled cell type specific proteins can be purified unidentified, or visualized in situ by immunofluorescent.To begin prepare the tissue lysate by adding lysis buffer at a volume of 12 to 15 times the wet weight of the tissue and triturate the tissue until it is homogenized. Heat this homogenate for 15 minutes to 75 degrees Celsius to denature the protein then centrifuge the sample and transfer the supernatant to a new tube. This sample can be stored at minus 80 degrees Celsius.Next, for alkylation dilute the homogenized sample two to three times in phosphate saline buffer containing an EDTA-free protease inhibitor. Then add freshly prepared iodoacetamide to a final concentration of 20 millimoles. Leave the sample for one to two hours at 20 degrees Celsius in the dark.Repeat the iodoacetamide addition and incubation steps twice. Prepare the column as per the manufacturer's instructions by first vortexing the columns. Then removing the lid cutting the bottom part and centrifuging them.Next, perform a buffer exchange by first equilibrating the buffer exchange columns using the click chemistry exchange buffer and then exchanging all alkylated samples. To evaluate the azidonorleucine incorporation take 40 microliters of the alluded sample and add phosphate saline buffer to a final volume of 120 microliters. Then set up the click chemistry reaction by vortexing the reaction for 20 seconds.After adding each reagent in the indicated order as fast as possible, incubate the samples in the dark at four degrees Celsius overnight with continuous rotation. The following day centrifuge the samples for five minutes at 17, 000 times G after centrifugation a slightly turquoise pellet will be visible. Transfer the supernatant to a new tube and store the sample at minus 80 degrees Celsius.For labeled protein purification. Subject all the samples to a buffer exchange procedure as demonstrated to clean leftovers of free alkyne using neutravidin binding buffer as the equilibration buffer. Then wash the neutravidin high capacity beads three times by mixing the beads with the binding buffer.Centrifuge the mixture, discard the supernatant and repeat this three times. Then prepare a one-to-one slurry by adding the same volume of neutravidin dry beads and neutravidin binding buffer. Set aside 20 to 40 microliters of each sample as pre-purified lysate and store it at minus 20 degrees Celsius.After measuring the protein concentration of the sample mix one milligram of protein with 40 microliters of the beads slurry. Incubate the mixture overnight at four degrees Celsius with continuous rotation enabling the labeled proteins to bind to the beads. On the following day collect the supernatant by centrifugation.Set a 20 to 40 microliter aliquot of the supernatant aside for later analysis and freeze the rest at minus 80 degrees Celsius. Next, wash the beads three times by adding chilled neutravidin washing buffer one. Sedimenting the beads and discarding the supernatant.Then add the same buffer and incubate the beads for 10 minutes under continuous rotation at four degrees Celsius before discarding the supernatant. Repeat the wash with neutravidin washing buffers two and three, and elute the clicked proteins by incubating the beads for 30 minutes at 20 degrees Celsius with a volume of neutravidin elution buffer, corresponding to one volume of the dry beads used. During the elution, maintain the beads in suspension at 1000 revolutions per minute in a thermo block shaker elude twice and combine both elutes.Click reactions were analyzed by SDS page and Western immuno blot. Representative images of the experiments are shown for azidonorleucine administration by intra peritoneal injection and for azidonorleucine administration via drinking water. A further experiment provides an example for the determination of the optimal disulphide biotin cleavable alkyne concentration.In this example, the fold change between the labeled sample and control is highest at 14 Micromolar alkyne concentration. An example of a mass spectrometry result with a clear enrichment in the azidonorleucine labeled sample compared to the control is shown here. This difference was already visible in the total protein stain.Besides changes in peptide intensity there are also unique proteins found in both samples. This is a technically complex protocol and its a step must be carefully followed using proper negative controls alkylation, proper(indistinct)and adequate cleaning of the non click archive. Our key steps in this protocol, purified proteins can be either identified by mass spectrometry if the researchers interest is to study proteins or load it into a gel for studying proteins of interest by western blot.The ability of this method to label proteins from a specific cellular origin has a lot of applications such as the tension of proteins or the study of protein synthesis in vitro or in vivo.

Research

JoVE Journal

Neuroscience

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, ...