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

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

1. Drinking water:
   1. 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 °C.
   2. 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'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.
2. Intraperitoneal administration:
   1. Dissolve ANL in physiological NaCl solution to 400 mM, phosphate saline buffer (PBS), or water.
   2. 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.
      ​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 °C; heating up to 37 °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.

2. Tissue harvesting, lysis, and protein extraction

1. 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 °C for several months (stop point). In the present protocol, mice cortex and hippocampus were dissected.
2. 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 μL of lysis buffer. Triturate the tissue until it is homogenized.
   NOTE: Samples can be stored at -80 °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.
3. Protein denaturation: Heat the homogenate to 75 °C for 15 min and centrifuge at 17,000 x g for 15 min at 10 °C. Transfer the supernatant to a new tube.
4. 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 μg/μL.
   NOTE: Samples can be stored at -80 °C for several months (stop point).
5. Alkylation
   1. Dilute the homogenized samples by 2-3 times in PBS (pH 7.8) + PI (EDTA free 1:4000).
   2. Add iodoacetamide (IAA) to a final concentration of 20 mM (stock solution 500 mM).
   3. Leave the samples for 1-2 h at 20 °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 °C for several months (stop point).
6. 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's instructions. Exchange all alkylated samples.
   NOTE: Eluted samples can be stored at -80 °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.
7. Click reaction to evaluate ANL incorporation in the experiment
   1. Take 40 μL of the eluted samples in step 2.6 and add PBS (pH 7.8) to a final volume of 120 µL.
   2. 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 µL of triazole ligand (stock 40 mM), 1.5 µL of biotin alkyne (stock 5 mM) and 1 µL 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.
   3. Incubate the samples overnight in the dark at 4 °C with continuous rotation.
   4. Centrifuge the samples at 4 °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 °C for several months, or at -20 °C for a couple of weeks until further processing (stop point).
8. Western blot analysis to evaluate ANL incorporation in the experiment
   1. Run an SDS-PAGE with 20-40 μL 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.
   2. Transfer the proteins to a nitrocellulose or PVDF membrane¹³.
   3. Incubate the membrane overnight at 4 °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.
   4. Wash the primary antibody 2 times for 10 min and add the secondary antibody for 1.5 h.
   5. 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).
   6. 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).
9. Click reaction for cleavable alkyne dosage optimization
   1. Prepare four tubes with 40 μL 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 μL.
   2. 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 μM, 15 μM, 30 μM, and 60 μM is recommended.
   3. 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 β-Mercaptoethanol to prevent alkyne cleavage. In this study, heating at 72 °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.
10. Preparative click reaction for protein purification
    1. 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).
       ​NOTE: Make sure that the pipettes are properly calibrated to ensure sample upscaling accuracy. Clicked samples can be stored at -80 °C for several months (stop point).

3. Protein purification

1. Buffer exchange
   1. 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 °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.
2. Binding to Neutravidin beads
   1. 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.
   2. Prepare a 1:1 slurry of Neutravidin beads by adding the same volume of dry beads and Neutravidin binding buffer.
   3. Set aside 20-40 μL of each sample as pre-purified lysate and store it at -20 °C.
   4. Measure protein concentration (see step 2.4), and mix 100 μg of protein with 4 μL of the bead slurry obtained in step 3.2.2.
   5. Incubate the mixture overnight at 4 °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 μL 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.
3. Neutravidin beads washes and protein elution
   1. Collect the supernatants by centrifugation (see step 3.2.1). Set a 20-40 μl aliquot of each supernatant aside for later analysis and freeze the rest at -80 °C.
   2. 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.
   3. Then, add the same buffer and incubate the beads for 10 min under continuous rotation at 4 °C before discarding the supernatant. Repeat this step three times.
   4. 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).
   5. 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).
   6. Elute the clicked proteins by incubating the beads for 30 min at 20 °C with a volume of Neutravidin elution buffer (5% (vol/vol) β-mercaptoethanol and 0.03% (wt/vol) SDS) corresponding to one volume of the dry beads used.
   7. 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. β-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).
4. Evaluation of eluted proteins
   1. Load 1/3 of the eluted samples to an SDS-PAGE (see step 2.8.1).
   2. Stain the gel to visualize proteins using a high sensitivity method, such as silver staining or a fluorescent method.
   3. 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.