Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

All animal studies were performed in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center.

1. Langendorff Perfusion of Mouse Heart

1. Langendorff perfusion of mouse heart with ischemia and reperfusion
   1. Administer intraperitoneal pentobarbital sodium 70 mg/kg to the adult mouse (8-week-old, male, C57BL6/j). Confirm deep anesthesia by lack of withdrawal to toe pinch.
   2. Anticoagulate with intraperitoneal heparin 500 U/kg.
   3. Open the chest via sternal incision. Open the diaphragm and cut along the costal border to the anterior axillary axis. Then, cut the anterior axillary axis up to the forelimb to open completely the thoracic cage. After, visualizing the heart, clamp the ascending aorta with forceps and rapidly excise the heart. The death is due to exsanguination. Place the heart in cold Krebs (NaCl 6.9 g/L, KCl 0.35 g/L, NaHCO₃ 2.1 g/L, KH₂PO₄ 0.16 g/L, MgSO₄ 0.141 g/L, glucose 2 g/L and CaCl₂ 0.373 g/L).
   4. Cannulate the aorta with a blunt-tip needle and perfuse the heart retrogradely with Krebs solution in constant pressure mode.
   5. After a stabilization period of 15 min, subject the heart to a global no-flow ischemia for 30 min followed by reperfusion for 30 min.
   6. Harvest hearts after 15 min baseline perfusion, after 30 min ischemia, or after 30 min reperfusion. Trim off the atria, and snap-freeze the tissue in liquid nitrogen.
   7. Store at -80 °C until use

2. Tissue Homogenization, Solubilization, and Sucrose Density Gradient Sedimentation

1. Gradient preparation
   1. Prepare 2 solutions (15% and 50%) of 100 mL sucrose, mixing 4 mL of NaCl 2.5 M;
      0.8 mL MgCl₂ 1.25 M; 1 mL Tris-HCl 1 M, pH 7.5; 15 g or 50 g of sucrose (for 15% and 50%, respectively); ultrapure water to reach 100 mL; and xylene cyanol (0.02 mg/mL) to color the 15% solution
   2. Filter each solution (0.22 µm filters)
   3. The day the gradient is to be used, prepare 40 mL of each sucrose solution and add cycloheximide to each solution to achieve final concentration of 100 µg/mL
      1. Use a gradient maker to prepare a mix between the 15% and 50% sucrose solutions
      2. Fill the left compartment with 5 mL of the 15% sucrose gradient
      3. Fill the right compartment with the 50% sucrose gradient
      4. Open the tap and switch on the pump to stir the two small compartments with magnetic stirrer
      5. Use a tube linked to the second tap to allow the mixed sucrose solution to flow in an ultra-centrifugation tube with thin walls. Final volume will be around 10 mL.
   4. Keep the gradients at 4 °C (overnight if needed, but not longer)
2. Homogenization of heart tissue
   1. Homogenize fresh or frozen heart with a polytron in filtered lysis buffer (modified for sucrose gradient fractionation) containing 100 mM KCl, 20 mM Tris pH 7.5, 5 mM MgCl₂, 0.4% NP-40. Include cycloheximide 100 µg/mL to maintain polysome structure, 0.1 U RNase inhibitor and protease inhibitor cocktail (1 tablet for 50 mL).
   2. Incubate the lysate on ice 15 min.
   3. Centrifuge at 18,000 x g for 15 min at 4 °C to remove insoluble material and collect the supernatant.
   4. Reserve 50 µL for protein determination, RNA isolation and RNA integrity analysis (see Figure 2).
   5. Layer the supernatant (500–100 µL) on top of the gradients and balance the tubes with lysis buffer.
3. Sedimentation and collection of fractions
   1. Perform ultracentrifugation for 120 min at 37,000 rpm at 4 °C in a swinging bucket rotor. According to the manufacturer, this corresponds to 228,000 x g at maximal radius.
   2. Collect each gradient as 17 fractions (around 600 µL each fraction) in microcentrifuge tubes with continuous monitoring of absorbance at 254 nm (OD₂₅₄).
   3. Program the fraction collector as follows:
      1. Discard the first 3 min of collection (corresponding to the liquid contained in the tubes before the gradient)
      2. From 4 to 19 min, collect the fractions at the speed of 1 mL/min in 17 fractions.
   4. Place the fractions on ice as soon as each is collected. Freeze samples at -80 °C, if they are not being processed immediately. A typical UV densitometric tracing of the gradient as it is collected is shown in Figure 1.

3. Isolation and Analysis of mRNAs from Polysome and Nontranslating Fractions

1. Extraction of mRNA
   1. Thaw the fractions on ice, if they were flash-frozen after collection
   2. Transfer 200 µL of each fraction to a fresh tube
      NOTE: At this step, two or more consecutive fractions can be pooled together. It should be done carefully after analyzing the OD₂₅₄ graph so that polysome and non-polysome fractions don't get mixed.
   3. Add 10 ng of luciferase RNA as internal control to each sample for normalization purpose.
      NOTE: Luciferase primers should be used as an internal control to normalize the results.
   4. Add 700 µL of RNA extraction reagent (monophasic solution of phenol and guanidine isothiocyanate; see Table of Materials) to each tube. Mix well and incubate the tubes for 5 min at RT (room temperature).
   5. Add 140 µL chloroform and vortex for 15 s. Incubate for 2-3 min at RT.
   6. Centrifuge the samples for 15 min at 12,000 x g at 4 °C.
   7. Carefully transfer the upper aqueous phase to a new tube.
   8. Since the RNA content of the fractions is not high, add 10 µg of glycogen as carrier to each tube.
   9. Add 350 µL isopropanol to each tube. Mix well and incubate at -20 °C for 1 h to increase the yield.
   10. Centrifuge for 15 min at 12,000 x g at 4 °C.
   11. Discard the supernatant and add 700 µL ethanol 75% to wash the RNA pellet.
   12. Centrifuge for 5 min at 7,500 x g at 4 °C.
   13. Remove ethanol and let the pellet air dry for 10–15 min.
       NOTE: Over-drying the RNA pellet will prevent its complete solubilization in water.
   14. Resuspend the RNA pellet in 50 μL of RNase-free ultrapure H₂O.
   15. Incubate the samples for 10 min at 55 °C.
   16. Use 2 μL of each sample to measure the quality and quantity of the extracted RNA and store the remainder at -80 °C.
2. Reverse transcription and qPCR
   1. Use 4 μL of reverse transcription cDNA synthesis kit (see Table of Materials) for a 20 μL reaction. Prepare the supermix according to the number of samples. Transfer the required volume to each tube and add 5 μL of the input RNA to each tube. This volume can be corrected to be relevant for the range of the Taq polymerase working conditions.
   2. Incubate the tubes in a thermal cycler with the following program recommended by the manufacturer: 5 min at 25 °C, 20 min reverse transcription for 20 min at 46 °C, and reverse transcriptase inactivation for 1 min at 95 °C.
   3. Run qPCR with the optimized program for each pair of primers.
      NOTE: To detect the presence of certain mRNAs in fractions, equal volume from each fraction (of the isolated RNA) should be used for reverse transcription.
3. Analysis of the results
   1. Use the Ct value of the interested gene and the luciferase to calculate the delta Ct values
   2. Express each fraction delta Ct value as the percentage of the total mRNA value contained in all the fractions to visualize the distribution of the mRNA across the different fractions (Table 1)

4. Isolation and Analysis of miRNAs from Polysome Fractions and Nontranslating Fractions

1. Extraction of mRNA and miRNA
   NOTE: This protocol follows manufacturer’s instructions, with a few changes.
   1. Thaw the glycogen and the spike-in control. Keep on ice.
   2. Add 700 µL of miRNA extraction reagent to 200 µL of pooled sample.
   3. Add 1 µg/µL of glycogen and homogenize it using the pipette. Incubate it for 5 min at room temperature.
   4. Add 3.5 µL of 1.6 x 10⁸ spike-in control and mix it.
   5. Add 200 µL of chloroform and vortex it for at least 15 sec.
   6. Incubate for 3 min at room temperature.
   7. Centrifuge at 12,000 x g for 15 min at 4 °C.
   8. Using a pipette, transfer the supernatant to a new 1.5 mL tube. Discard the middle and bottom phases.
   9. Add 1.5x volume of 100% ethanol to the supernatant and mix it.
   10. Transfer 700 µL of this mixture to the miRNA extraction column and centrifuge it at full speed for 15 s at RT and discard the flow-through; repeat this step with any remaining sample.
   11. Add 700 µL of buffer 1 to the column and centrifuge it at full speed for 15 s at RT; discard the flow-through.
   12. Add 500 µL of buffer 2 to the column and centrifuge it at full speed for 15 s at RT; discard the flow-through.
   13. Add 500 µL of 80% ethanol to the column and centrifuge it at full speed for 2 min at RT; discard the flow-through.
   14. Transfer the column to a new 2 mL uncapped tube, open the lid and centrifuge it at RT for 5 min at full speed; discard the flow-through.
   15. Add 16 µL of RNAse-free water to the column and centrifuge it at full speed for 1 min at RT. Keep the eluate on ice or store at -80 °C for future analysis.
       NOTE: Two microliters of each sample can be used for OD analysis.
2. Reverse Transcription
   1. Prepare on ice. For each 20 µL RT-PCR reaction, add 4 µL of the miRNA RT buffer, 2 µL of nucleic acids, 9 µL of total RNA, 2 µL of enzyme and 3 µL of RNAse-free water; mix it and spin it down briefly.
   2. Set the Thermal Cycler as follows:
      37 °C for 60 min;
      95 °C for 5 min;
      4 °C hold.
   3. Remove the tubes from the thermal cycler and add 200 µL of RNAse-free water. Store at -20 °C or proceed to the real-time PCR.
3. Real-time PCR
   NOTE: This protocol follows 96 well plate format. Prepare the reaction mix on ice.
   1. Prepare PCR reaction mix and add cDNA (from miRNAs above) according to the range accepted by the protocol. Multiple reactions can be prepared in batch.
   2. Add a total of 25 µL of PCR mix + cDNA in each well.
   3. Seal the plate using an optical plate seal.
   4. Centrifuge the plate for 1 min at 1,000 x g at room temperature.
   5. Program the real-time cycler as follows:
      Initial activation step at 95 °C for 15 min;
      3-step cycling: denaturation at 94 °C for 15 s; annealing at 55 °C for 30 s; extension at 70 °C for 30 s (perform fluorescence data collection); add 40 cycles;
      Melting-curve according to cycler default.
4. Comparative analysis
   1. Example of normalization (see Table 2):
      1. Find the SCM minimum value.
      2. Normalize all the SCM values to this minimum.
      3. Subtract this value from the Ct values of miRNAs of interest (MOI) and reference gene (REF).
      4. Analyze data using the 2^-ΔCt formula.

5. Proteomic Analysis

1. Extraction of proteins from polysome fractions
   1. Combine collected sucrose fractions into three final fractions. Mark the fractions as heavy (high efficiency polysomes in pooled fractions 4, 5, 6 and 7 with the highest concentration of sucrose; bottom of the gradient tube), light (low efficiency polysomes in pooled fraction 8, 9, 10 and 11; middle of the gradient tube) and non-translating (pooled fractions 12, 13, 14 and 15 with the lowest concentration of sucrose; top of the gradient tube). Perform protein assay on pooled fractions.
   2. Prepare 100% stock of trichloroacetic acid (TCA) and store it at 4 °C in dark. Mix 0.5 mL of sample with 60 µL of 100% TCA and incubate at -20 °C overnight in the dark.
      NOTE: Use 1.5 mL low protein retention tubes (see Table of Materials).
   3. After overnight incubation, thaw the sample on ice and centrifuge at 15,000 x g, 4 °C for 30 min. Discard supernatant.
      NOTE: 100 µg of total protein in sample gives visible protein pellet at the tube bottom.
   4. Pre-chill acetone in -20 °C freezer. Add 0.5 mL of cold acetone to the protein pellet and centrifuge at 15,000 x g, 4 °C for 15 min. Discard supernatant. Repeat this step twice.
   5. Air dry the pellet. Resuspend the pellet in 360 µL of 50 mM Tris-HCl buffer (pH 8).
      NOTE: If needed, use a pulse sonicator to resuspend the pellet.
   6. Add 40 µL of 0.1 M dithiothreitol (final concentration 10 mM) and incubate at 55 °C on shaker for 45 min. Add 50 µL of 0.15 M iodoacetamide (final concentration 17 mM) and incubate at room temperature on shaker for 30 min in dark.
   7. Prepare trypsin solution: resuspend 20 µg of trypsin in 100 µL of 50 mM ammonium bicarbonate. Add corresponding volume of trypsin solution to sample at 1:50 (w/w) trypsin:protein ratio, ensure that pH is 8 and incubate on shaker at 37 °C overnight.
      NOTE: Add 1 M ammonium bicarbonate to bring pH to 8.
   8. After trypsin digestion, cool the sample, centrifuge briefly in bench centrifuge, add 10% formic acid to pH 2–3 to quench the trypsin activity, and proceed with sample desalting.
      NOTE: Use µ-elution plate for sample desalting.
   9. Condition the sorbent in the wells of 96 well plate with 200 µL of methanol using vacuum three times followed by conditioning by 200 µL of 0.1% formic acid three times. Load sample and perform a sample wash using 200 µL of 0.1% formic acid three times. Elute the sample with 100 µL of 50% acetonitrile/0.1% formic acid twice into low protein retention 0.5 mL tube.
      NOTE: Elute the sample slowly at first using gravitation followed by low vacuum on.
   10. Evaporate sample eluate to dryness using concentrator and either directly carry out mass spectrometry analysis or store at -80 °C until use.
2. Mass spectrometry analysis
   1. Reconstitute each pellet (consisting of the tryptic peptides) in 50–100 µL and inject 1 µL into mass spectrometer.
      Note: Optimize reconstitution and injection volumes to obtain maximum intensity LC/MS/MS signal. In our case, the maximum signal (total ion current; TIC) in the heart of the scan was in range of 1E8 to 2E9.
   2. Use a trap column C18 (300 µm i.d. x 5 mm, 5 µm, 100Å) followed by peptide separation using C18 column (75 µm i.d. x 250 mm, 2 µm, 100Å) with a flow rate setting at 300 nL/min. Set the nano-source capillary temperature to 275 °C and the spray voltage to 2 kV.
   3. Apply a linear gradient of 5–35% B for 90 min, 35–95% B for 3 min, holding at 95% B for 7 min and equilibration at 5% B for 25 min (A: 0.1% formic acid/water and B: 0.1% formic acid in acetonitrile). Acquire MS2 spectra for the 15 highest intensity ions from each MS1 scan using CID mode. Use 3 mass spectrometry replicates for each sample analyzed.
      NOTE: Optimize and use the experimental conditions and parameters that are suitable for your samples on the particular mass spectrometry instrument. The conditions/parameters mentioned in section 5.2. were used and optimized in our laboratory.
3. Protein identification/quantitation
   1. After mass spectrometry analysis, convert the raw spectra files into compatible mzXML files using MSConvert software (http://proteowizard.sourceforge.net/tools/msconvert.html).
   2. Submit the converted files into SEQUEST search engine with the search criteria, e.g., UniProtKB/Swiss-Prot mouse database (most up to date canonical reviewed); semi-enzyme digest using trypsin (after KR/-); with up to 2 missed cleavages; precursor mass range: 400 to 4,500 amu; static modification: carbamidomethyl (C) 57.021465 amu; peptide mass tolerance: 50 ppm; fragment mass type: monoisotopic.
      NOTE: Other protein database search engines and data pipelines can be used. If using database for e.g. rat, which is less complete than databases for mouse and human, you may need to search against mouse (and/or human) databases to increase proteome coverage. In this case, redundancy in protein names will need to be removed.
   3. Perform a post-search analysis. Set the filters for viewing the data. For example, set a minimum protein probability (protein threshold) as 95% or 99%, i.e., only the proteins for which a statistical analysis implies 95% or 99% probability of being present in the sample will be displayed. Set the filter for peptide threshold (e.g., 95%), i.e., a minimum probability that will be used in determining whether a spectrum identifies a peptide. Select the minimum number of peptides that will determine if a protein is present (2 peptides as a minimum for protein identification is recommended).
   4. Evaluate identified proteins for quality/quantity. Ensure that proteotypic peptides are used for quantification. If isoform is identified ensure it is bases on observation of a tryptic peptide comprised of an amino acid sequence unique to the specific isoform. Any “similar to” or hypothetical proteins should undergo comparison to see if the observed peptides correspond to a known protein.