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 ANL
<ol>
	<li>Drinking water:
	<ol>
		<li>Dissolve ANL and maltose in the regular drinking water of the mice. The recommended maltose concentration is 0.7% (wt/vol). The maximum amount of ANL added to the drinking water is 1% (wt/vol). Once the mixture is ready, sterilize it by filtration and store it at 4 &#176;C.</li>
		<li>Provide the mixture prepared in step 1.1 to the mice. Change bottles frequently (e.g., every 3 days) to avoid contaminations, and check them every day to look for possible contamination or clogging. Monitor the amount of drunk water daily by weighing it, to know mice&#39;s ANL intake. 
		NOTE: The maximum labeling period evaluated here is 3 weeks using an ANL concentration of 1% in the drinking water, which resulted in well-detectable labeling in the brain. Good labeling can also be achieved in 2 weeks. Neither shorter labeling times, nor longer time points, nor other ANL amounts have been studied by us using this administration method, which does not imply that the foregoing would not work. ANL incorporation rates can vary depending on the studied tissue or cell-type. The time of labeling can vary depending on the scientific question; for example, if proteomes were to be labeled, then labeling time should be calculated in function of the average half-lives of the proteins in the studied tissue. If the interest is only to identify newly synthesized proteins, the times can be shortened. The labeling periods and ANL dosages have to be empirically tested for each experimental condition and cell-type of interest.</li>
	</ol>
	</li>
	<li>Intraperitoneal administration:
	<ol>
		<li>Dissolve ANL in physiological NaCl solution to 400 mM, phosphate saline buffer (PBS), or water.</li>
		<li>Make sure that the osmolarity of the solution is within the physiological range. Here, 10 mL/kg body weight of ANL solution is administered to the mice. 
		&#8203;NOTE: Good labeling in the excitatory neurons of the brain is successfully obtained by one daily intraperitoneal injection (IP) with 400 mM ANL for 1 week. Neither lower ANL concentrations nor other labeling periods by IP injections have been tested in this study, which does not imply they would not work. ANL is supplied as hydrochloride by some companies. In this case, the pH of the solutions must be adjusted to the adequate pH (depending on the administration route). ANL can also be synthesized following the method published by Link et al.11 with some modifications5. ANL labeling can be performed using other ANL concentrations, timespans, and administration routes. For tissues/cell-types with low metabolic rates, a low content methionine diet can be used, always starting 1 week before ANL administration. When 400 mM ANL is used, it can precipitate while stored at 4 &#176;C; heating up to 37 &#176;C brings ANL back into the solution. Let it reach room temperature before injection. In most world regions, ANL administration to mice is an animal experiment and needs to be approved by the pertinent authorities.</li>
	</ol>
	</li>
</ol>
2. Tissue harvesting, lysis, and protein extraction
<ol>
	<li>Tissue dissection: Dissect the area of tissue containing the cell-type of interest and note the weight of the collected piece of tissue. 
	NOTE: Samples can be stored at -80 &#176;C for several months (stop point). In the present protocol, mice cortex and hippocampus were dissected.</li>
	<li>Tissue lysis: Homogenize the tissue at room temperature by adding a volume 12-15 times the wet weight of the tissue of the lysis buffer (PBS pH 7.4, 1% wt/vol SDS, 1% (vol/vol) Triton X-100, Benzonase (1:1000 vol/vol), a protease inhibitor (PI) (EDTA free 1:4000)). For example, for a 20 mg tissue piece, add 240-300 &#956;L of lysis buffer. Triturate the tissue until it is homogenized. 
	NOTE: Samples can be stored at -80 &#176;C for several months (stop point). For direct homogenization in 1.5 mL tubes, the use of a handheld, battery-operated homogenizer is recommended, especially when small pieces of tissue are processed. For bigger pieces, a Dounce homogenizer can be used. To every buffer where protease inhibitors (PI) are included, they should be added immediately before use and not earlier.</li>
	<li>Protein denaturation: Heat the homogenate to 75 &#176;C for 15 min and centrifuge at 17,000 x g for 15 min at 10 &#176;C. Transfer the supernatant to a new tube.</li>
	<li>Protein content measurement: Measure the protein concentration of each sample, and adjust all the samples to be compared to the same protein concentration; adjust the concentration by adding lysis buffer. An optimal concentration range is between 2-4 &#956;g/&#956;L. 
	NOTE: Samples can be stored at -80 &#176;C for several months (stop point).</li>
	<li>Alkylation
	<ol>
		<li>Dilute the homogenized samples by 2-3 times in PBS (pH 7.8) + PI (EDTA free 1:4000).</li>
		<li>Add iodoacetamide (IAA) to a final concentration of 20 mM (stock solution 500 mM).</li>
		<li>Leave the samples for 1-2 h at 20 &#176;C in the dark. Do steps 2.5.2-2.5.3 twice. 
		NOTE: For some tissues, if the non-specific click in the negative control remains high, it might be necessary to perform a reduction step prior to alkylation12. Prepare the IAA stock freshly just before adding it to the samples. Samples can be stored at -80 &#176;C for several months (stop point).</li>
	</ol>
	</li>
	<li>Buffer exchange: Equilibrate buffer exchange columns using the click chemistry exchange buffer (PBS pH 7.8, 0.04% (wt/vol) SDS, 0.08% (vol/vol) Triton X-100 and PI (1:4000)). Follow the manufacturer&#39;s instructions. Exchange all alkylated samples. 
	NOTE: Eluted samples can be stored at -80 &#176;C for several months (stop point). In this step, the IAA is eliminated, and the buffer is exchanged by the click chemistry buffer. Protein can be measured again after the buffer exchange step to make sure that the proteins were not lost during the buffer exchange process. Depending on the type of columns used for buffer exchange, protein can be massively lost. Use the recommended columns to avoid protein loss. For sample storage, the preparation of aliquots is recommended.</li>
	<li>Click reaction to evaluate ANL incorporation in the experiment
	<ol>
		<li>Take 40 &#956;L of the eluted samples in step 2.6 and add PBS (pH 7.8) to a final volume of 120 &#181;L.</li>
		<li>To set up the click chemistry reaction, add the following reagents in the given order and vortex for 20 s following each addition: 1.5 &#181;L of triazole ligand (stock 40 mM), 1.5 &#181;L of biotin alkyne (stock 5 mM) and 1 &#181;L of Cu(I) bromide (stock 10 mg/mL, dissolved in DMSO by careful pipetting up and down, do not vortex). Add the reagents as fast as possible.</li>
		<li>Incubate the samples overnight in the dark at 4 &#176;C with continuous rotation.</li>
		<li>Centrifuge the samples at 4 &#176;C for 5 min at 17,000 x g. A slightly turquoise pellet will be visible. Transfer the supernatant to a new tube. 
		NOTE: Prepare Cu (I) bromide stock immediately before use to avoid copper oxidation. Keeping the ratio of volumes between the lysed sample and PBS constant between samples is essential to ensure the same final detergent concentration in each tube. To speed up the assembly of the click chemistry reaction, a table vortexer with racks for tubes is recommended. Store the clicked samples at -80 &#176;C for several months, or at -20 &#176;C for a couple of weeks until further processing (stop point).</li>
	</ol>
	</li>
	<li>Western blot analysis to evaluate ANL incorporation in the experiment
	<ol>
		<li>Run an SDS-PAGE with 20-40 &#956;L of the supernatant from the click chemistry reaction (step 2.7). Use 12% acrylamide gels, run in 1% SDS, 250 mM Tris-HCl, 2 mM glycine. Run until the front is out of the gel.</li>
		<li>Transfer the proteins to a nitrocellulose or PVDF membrane13.</li>
		<li>Incubate the membrane overnight at 4 &#176;C with GFP antibody (1:500, GFP protein is co-translated with MetRS*5), and biotin antibody (1:1000) for clicked alkyne detection, which is conjugated to biotin.</li>
		<li>Wash the primary antibody 2 times for 10 min and add the secondary antibody for 1.5 h.</li>
		<li>Wash the secondary antibody 2 times for 10 min and scan (if using fluorophore-conjugated antibodies) or expose to films (if using horseradish peroxidase-conjugated antibodies).</li>
		<li>Quantify the biotin signal using ImageJ or similar software, evaluate the general labeling of each sample of the experiment, and detect possible outliers. Evaluate the signal-to-noise ratio (ANL sample labeling to background from the negative controls) of the experiment and detect possible animals which failed to take up/incorporate ANL. 
		NOTE: For samples with high ANL incorporation, protein purification is theoretically possible using non-cleavable alkynes like the one used in this section for the evaluation of ANL incorporation. Using brain samples, the background obtained in the negative controls by MS after protein purification using non-cleavable alkynes is too high14. Therefore, only the easier-to-handle, non-cleavable alkynes were used for a first general sample and experiment evaluation. Regardless of the type of alkyne intended for protein purification, it is advisable to perform the steps for alkyne dosage optimization described in the following section (step 2.9).</li>
	</ol>
	</li>
	<li>Click reaction for cleavable alkyne dosage optimization
	<ol>
		<li>Prepare four tubes with 40 &#956;L of one representative sample (obtained in step 2.6 and evaluated in step 2.8) and add PBS (pH 7.8) to a final volume of 120 &#956;L.</li>
		<li>Set up the click chemistry reaction as explained in step 2.7 but this time, add four different amounts of the DST-alkyne. For said alkyne, testing 5 &#956;M, 15 &#956;M, 30 &#956;M, and 60 &#956;M is recommended.</li>
		<li>Repeat step 2.8 to decide which is the most suitable alkyne dosage for the specific experimental question under study, based on the highest labeling efficiency with the lowest background signal. 
		NOTE: Before subjecting the remaining samples to a click reaction, the optimal amount of any alkyne needs to be determined. There are several options of commercially available alkynes that can be used. They differ in the way of cleavage (e.g., light or reducing agents). Copper-free click chemistry using strain-promoted reagents (e.g., DBCO reagents) is also possible. Tiny changes in the amount of either alkynes or DBCO reagents often increase the signal in the negative control (background) significantly. Here, the protein purification using an alkyne with a disulfide bridge cleavable by reducing agents (Disulfide biotin alkyne or DST-alkyne15) is described. When using the DST-alkyne, for running the SDS-PAGE (step 8.1), avoid loading buffers with strong reducing agents such as DTT or &#946;-Mercaptoethanol to prevent alkyne cleavage. In this study, heating at 72 &#176;C, for 5 min, using 5 mM of N-ethylmaleimide (NEM) in the loading buffer does not affect DST-alkyne stability. Step 2.9.2 can be repeated, testing more fine-grained dosages in the range of the best one observed.</li>
	</ol>
	</li>
	<li>Preparative click reaction for protein purification
	<ol>
		<li>Click all the samples obtained in step 2.6 with the optimal alkyne dosage determined in step 2.9 and analyze a fraction by SDS-PAGE and Western blot (step 2.8). 
		&#8203;NOTE: Make sure that the pipettes are properly calibrated to ensure sample upscaling accuracy. Clicked samples can be stored at -80 &#176;C for several months (stop point).</li>
	</ol>
	</li>
</ol>
3. Protein purification
<ol>
	<li>Buffer exchange
	<ol>
		<li>Subject all clicked samples to a buffer exchange procedure as described in step 2.6, but use Neutravidin binding buffer as equilibration buffer (PBS pH 7.4, 0.15% (wt/vol) SDS, 1% (vol/vol) Triton X-100 and PI (1:2000)). 
		NOTE: Eluted samples can be stored at -80 &#176;C for several months (stop point). In this step, the non-clicked free alkyne is eliminated, and the buffer is exchanged by the Neutravidin binding buffer.</li>
	</ol>
	</li>
	<li>Binding to Neutravidin beads
	<ol>
		<li>Wash the Neutravidin high-capacity beads with the Neutravidin binding buffer (see step 3.1); mix the beads with the buffer and centrifuge the mixture for 5 min at 3000 x g, and discard the supernatant. Repeat three times.</li>
		<li>Prepare a 1:1 slurry of Neutravidin beads by adding the same volume of dry beads and Neutravidin binding buffer.</li>
		<li>Set aside 20-40 &#956;L of each sample as pre-purified lysate and store it at -20 &#176;C.</li>
		<li>Measure protein concentration (see step 2.4), and mix 100 &#956;g of protein with 4 &#956;L of the bead slurry obtained in step 3.2.2.</li>
		<li>Incubate the mixture overnight at 4 &#176;C with continuous rotation, enabling labeled proteins to bind to the beads. 
		NOTE: As for the click chemistry reaction, the basic affinity purification reaction described in step 3.2.4 can be upscaled. For example, for purification of proteins from the excitatory neurons of the hippocampus, 1 mg of protein lysate is mixed with 40 &#956;L of Neutravidin beads (1:1 slurry). The amount of Neutravidin beads can be scaled up or down according to the amount of labeled proteins per mg of total protein, which will depend on the chosen cell-type and labeling period and needs to be determined empirically.</li>
	</ol>
	</li>
	<li>Neutravidin beads washes and protein elution
	<ol>
		<li>Collect the supernatants by centrifugation (see step 3.2.1). Set a 20-40 &#956;l aliquot of each supernatant aside for later analysis and freeze the rest at -80 &#176;C.</li>
		<li>Wash the beads with chilled Neutravidin washing buffer 1 (PBS pH 7.4, 0.2% (wt/vol) SDS, 1% (vol/vol) Triton X-100 and PI (1:2000)) by adding the buffer, sedimenting the beads, and discarding the supernatant. Repeat this step three times.</li>
		<li>Then, add the same buffer and incubate the beads for 10 min under continuous rotation at 4 &#176;C before discarding the supernatant. Repeat this step three times.</li>
		<li>Wash the beads as explained in steps 3.3.2-3.3.3 but using Neutravidin washing buffer 2 (1x PBS, pH 7.4, and PI 1:2000).</li>
		<li>Wash the beads as explained the step 3.3.2-3.3.3 but using Neutravidin washing buffer 3 (50 mM ammonium bicarbonate and PI 1:2000).</li>
		<li>Elute the clicked proteins by incubating the beads for 30 min at 20 &#176;C with a volume of Neutravidin elution buffer (5% (vol/vol) &#946;-mercaptoethanol and 0.03% (wt/vol) SDS) corresponding to one volume of the dry beads used.</li>
		<li>Maintain the beads in suspension during elution by continuous agitation (1000 rpm) in a thermoblock shaker. Elute twice and combine both eluates. 
		NOTE: The centrifugation set up for separation of the solid fraction of the slurry (beads) and the aqueous phase (supernatant), as explained in step 3.2.1, applies to steps 3.3.1-3.3.7. The SDS amount can be increased if protein elution is not complete. However, with amounts above 0.08%-0.1% of SDS, proteins non-specifically bound to the beads start to be eluted. &#946;-mercaptoethanol is volatile and toxic; the use of a hood for steps 3.3.6-3.3.7 is advisable. The samples collected in steps 3.2.3 and 3.3.1 should be run by SDS-PAGE and evaluated by Western blot (see step 2.8) for evaluating the efficiency of the affinity purification (step 3.3).</li>
	</ol>
	</li>
	<li>Evaluation of eluted proteins
	<ol>
		<li>Load 1/3 of the eluted samples to an SDS-PAGE (see step 2.8.1).</li>
		<li>Stain the gel to visualize proteins using a high sensitivity method, such as silver staining or a fluorescent method.</li>
		<li>If the samples have the expected quality, meaning that there is 3-4-fold more protein in the ANL labeled samples compared to the negative control, the eluted proteins can be identified by routine mass spectrometry methods like the one explained in reference5.</li>
	</ol>
	</li>
</ol>

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<li>David, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+translation+visualized+by+ribosome-bound+nascent+chain+puromycylation%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Nuclear translation visualized by ribosome-bound nascent chain puromycylation.</a> Journal of Cell Biology. 197 (1), 45-57 (2012).</li>
<li>Schmidt, E. K., Clavarino, G., Ceppi, M., Pierre, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SUnSET%2C+a+nonradioactive+method+to+monitor+protein+synthesis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">SUnSET, a nonradioactive method to monitor protein synthesis.</a> Nature Methods. 6 (4), 275-277 (2009).</li>
<li>Roux, K. J., Kim, D. I., Burke, B., May, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BioID%3A+A+screen+for+protein-protein+interactions%5BTitle%5D)+AND+%22Current+Protocols+in+Protein+Science%22%5BJournal%5D)">BioID: A screen for protein-protein interactions.</a> Current Protocols in Protein Science. 91, 11-15 (2018).</li>
</ol>

The authors declare no competing financial interests.

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><tbody><tr><td>12% Acrylamide gels</td><td>GenScript</td><td>SurePAGE, Bis-Tris, 10 x 8, 12%</td><td></td></tr><tr><td>&amp;beta;-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&amp;nbsp; 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 ...

cell-type-specific-protein-purification-identification-from-complex

Research

JoVE Journal

Neuroscience

2.8K Views.  Synaptic Plasticity Department. Dit protocol maakt het mogelijk om eiwitten op een celtype specifieke manier te labelen. Dit is de eerste techniek die een dergelijke mogelijkheid biedt en het kan in vitro of in vivo worden gedaan. Het belangrijkste voordeel van de techniek is dat de gelabelde celtypespecifieke eiwitten ongeïdentificeerd kunnen worden gezuiverd of in situ kunnen worden gevisualiseerd door immunofluorescentie.Om te beginnen met het voorbereiden van het weefsel lysaat door het toevoegen van lysis buffe...

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

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Biochemistry

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, characterizing differences in the translatome (the whole of translated mRNAs) provides an additional layer of information particularly useful when trying to understand the function of RNA regulating or binding proteins. A number of methods for accomplishing this have been developed, including ribosome profiling and polysome analysis. However, both methods carry significant technical challenges and cannot be applied to specific cell populations within a tissue unless combined with additional sorting methods. In contrast, the RiboTag method is a genetic-based, efficient, and technically straightforward alternative that allows the identification of ribosome associated RNAs from specific cell populations without added sorting steps, provided an applicable cell-specific Cre driver is available. This method consists of breeding to generate the genetic models, sample collection, immunoprecipitation, and downstream RNA analyses. Here, we outline this process in adult male mouse germ cells mutant for an RNA binding protein required for male fertility. Special attention is paid to considerations for breeding with a focus on efficient colony management and the generation of correct genetic backgrounds and immunoprecipitation in order to reduce background and optimize output. Discussion of troubleshooting options, additional confirmatory experiments, and potential downstream applications is also included. The presented genetic tools and molecular protocols represent a powerful way to describe the ribosome-associated RNAs of specific cell populations in complex tissues or in systems with aberrant mRNA storage and translation with the goal of informing on the molecular drivers of mutant phenotypes.

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, ...

Developmental Biology

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Biology

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the importance of intercellular signaling processes in the regulation of cell-nonautonomous proteostasis. Recent studies are now beginning to uncover signaling components and pathways that coordinate protein quality control from one tissue to another. It is therefore important to identify mechanisms and components of the cell-nonautonomous proteostasis network (PN) and its relevance for aging, stress responses and protein misfolding diseases. In the laboratory, we use genetic knockdown by tissue-specific RNAi in combination with stress reporters and tissue-specific proteostasis sensors to study this. We describe methodologies to examine and to identify components of the cell-nonautonomous PN that can act in tissues perceiving a stress condition and in responding cells to activate a protective response. We first describe how to generate hairpin RNAi constructs for constitutive genetic knockdown in specific tissues and how to perform tissue-specific genetic knockdown by feeding RNAi at different life stages. Stress reporters&#160;and behavioral assays function as&#160;valuable readouts&#160;that enable&#160;the fast screening of genes and conditions modifying systemic stress signaling processes. Finally, proteostasis sensors expressed in different tissues are utilized to determine changes in the tissue-specific capacity of the PN at different stages of development and aging. Thus, these tools should help clarify and allow monitoring the capacity of PN in specific tissues, while helping to identify components that function in different tissues to mediate cell-nonautonomous PN in an organism.

Maintenance of organismal proteostasis requires the coordination of protein quality control responses such as chaperone expression from one tissue to another. Here, we provide tools used in C. elegans that allow monitoring of proteostasis capacity in specific tissues and determine intercellular signaling responses.

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the ...

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids potential pitfalls caused by the heterogeneity of tissues and unleashes the powerful transcriptome analysis. The protocol described here demonstrates how to use the Translating Ribosome Affinity Purification (TRAP) method to isolate ribosome-bound RNAs from a small amount of EGFP-expressing cells in a complex tissue without cell sorting. This protocol is suitable for isolating cell-type-specific RNAs using the recently available NuTRAP mouse model and could also be used to isolate RNAs from any EGFP-expressing cells.

This protocol aims to isolate cell-type-specific translating ribosomal mRNAs using the NuTRAP mouse model.

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids ...

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

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