NIH P01 HL112730 (RAG, JVE), NIH R01 HL132075 (RAG, JVE), Barbra Streisand Women&#39;s Heart Center (RAG, JVE), Dorothy and E. Phillip Lyon Chair in Molecular Cardiology (RAG), Erika Glazer Endowed Chair in Woman&#39;s Heart Health (JVE) and Czech Academy of Sciences Institutional Support RVO: 68081715 (MS).

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
<ol>
	<li>Langendorff perfusion of mouse heart with ischemia and reperfusion 
	<ol>
		<li>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.</li>
		<li>Anticoagulate with intraperitoneal heparin 500 U/kg.</li>
		<li>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).</li>
		<li>Cannulate the aorta with a blunt-tip needle and perfuse the heart retrogradely with Krebs solution in constant pressure mode.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Store at -80 &#176;C until use</li>
	</ol>
	</li>
</ol>
2. Tissue Homogenization, Solubilization, and Sucrose Density Gradient Sedimentation
<ol>
	<li>Gradient preparation
	<ol>
		<li>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</li>
		<li>Filter each solution (0.22 &#181;m filters)</li>
		<li>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 &#181;g/mL
		<ol>
			<li>Use a gradient maker to prepare a mix between the 15% and 50% sucrose solutions</li>
			<li>Fill the left compartment with 5 mL of the 15% sucrose gradient</li>
			<li>Fill the right compartment with the 50% sucrose gradient</li>
			<li>Open the tap and switch on the pump to stir the two small compartments with magnetic stirrer</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Keep the gradients at 4 &#176;C (overnight if needed, but not longer)</li>
	</ol>
	</li>
	<li>Homogenization of heart tissue 
	<ol>
		<li>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 &#181;g/mL to maintain polysome structure, 0.1 U RNase inhibitor and protease inhibitor cocktail (1 tablet for 50 mL).</li>
		<li>Incubate the lysate on ice 15 min.</li>
		<li>Centrifuge at 18,000 x g for 15 min at 4 &#176;C to remove insoluble material and collect the supernatant.</li>
		<li>Reserve 50 &#181;L for protein determination, RNA isolation and RNA integrity analysis (see Figure 2).</li>
		<li>Layer the supernatant (500&#8211;100 &#181;L) on top of the gradients and balance the tubes with lysis buffer.</li>
	</ol>
	</li>
	<li>Sedimentation and collection of fractions
	<ol>
		<li>Perform ultracentrifugation for 120 min at 37,000 rpm at 4 &#176;C in a swinging bucket rotor. According to the manufacturer, this corresponds to 228,000 x g at maximal radius.</li>
		<li>Collect each gradient as 17 fractions (around 600 &#181;L each fraction) in microcentrifuge tubes with continuous monitoring of absorbance at 254 nm (OD254).</li>
		<li>Program the fraction collector as follows:
		<ol>
			<li>Discard the first 3 min of collection (corresponding to the liquid contained in the tubes before the gradient)</li>
			<li>From 4 to 19 min, collect the fractions at the speed of 1 mL/min in 17 fractions.</li>
		</ol>
		</li>
		<li>Place the fractions on ice as soon as each is collected. Freeze samples at -80 &#176;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.</li>
	</ol>
	</li>
</ol>
3. Isolation and Analysis of mRNAs from Polysome and Nontranslating Fractions
<ol>
	<li>Extraction of mRNA
	<ol>
		<li>Thaw the fractions on ice, if they were flash-frozen after collection</li>
		<li>Transfer 200 &#181;L of each fraction to a fresh tube 
		NOTE:&#160;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&#39;t get mixed.</li>
		<li>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.</li>
		<li>Add 700 &#181;L 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).</li>
		<li>Add 140 &#181;L chloroform and vortex for 15 s. Incubate for 2-3 min at RT.</li>
		<li>Centrifuge the samples for 15 min at 12,000 x g at 4 &#176;C.</li>
		<li>Carefully transfer the upper aqueous phase to a new tube.</li>
		<li>Since the RNA content of the fractions is not high, add 10 &#181;g of glycogen as carrier to each tube.</li>
		<li>Add 350 &#181;L isopropanol to each tube. Mix well and incubate at -20 &#176;C for 1 h to increase the yield.</li>
		<li>Centrifuge for 15 min at 12,000 x g at 4 &#176;C.</li>
		<li>Discard the supernatant and add 700 &#181;L ethanol 75% to wash the RNA pellet.</li>
		<li>Centrifuge for 5 min at 7,500 x g at 4 &#176;C.</li>
		<li>Remove ethanol and let the pellet air dry for 10&#8211;15 min. 
		NOTE: Over-drying the RNA pellet will prevent its complete solubilization in water.</li>
		<li>Resuspend the RNA pellet in 50 &#956;L of RNase-free ultrapure H2O.</li>
		<li>Incubate the samples for 10 min at 55 &#176;C.</li>
		<li>Use 2 &#956;L of each sample to measure the quality and quantity of the extracted RNA and store the remainder at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Reverse transcription and qPCR
	<ol>
		<li>Use 4 &#956;L of reverse transcription cDNA synthesis kit (see Table of Materials) for a 20 &#956;L reaction. Prepare the supermix according to the number of samples. Transfer the required volume to each tube and add 5 &#956;L of the input RNA to each tube. This volume can be corrected to be relevant for the range of the Taq polymerase working conditions.</li>
		<li>Incubate the tubes in a thermal cycler with the following program recommended by the manufacturer: 5 min at 25 &#176;C, 20 min reverse transcription for 20 min at 46 &#176;C, and reverse transcriptase inactivation for 1 min at 95 &#176;C.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Analysis of the results
	<ol>
		<li>Use the Ct value of the interested gene and the luciferase to calculate the delta Ct values</li>
		<li>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)</li>
	</ol>
	</li>
</ol>
4. Isolation and Analysis of miRNAs from Polysome Fractions and Nontranslating Fractions
<ol>
	<li>Extraction of mRNA and miRNA 
	NOTE: This protocol follows manufacturer&#8217;s instructions, with a few changes.
	<ol>
		<li>Thaw the glycogen and the spike-in control. Keep on ice.</li>
		<li>Add 700 &#181;L of miRNA extraction reagent to 200 &#181;L of pooled sample.</li>
		<li>Add 1 &#181;g/&#181;L of glycogen and homogenize it using the pipette. Incubate it for 5 min at room temperature.</li>
		<li>Add 3.5 &#181;L of 1.6 x 108 spike-in control and mix it.</li>
		<li>Add 200 &#181;L of chloroform and vortex it for at least 15 sec.</li>
		<li>Incubate for 3 min at room temperature.</li>
		<li>Centrifuge at 12,000 x g for 15 min at 4 &#176;C.</li>
		<li>Using a pipette, transfer the supernatant to a new 1.5 mL tube. Discard the middle and bottom phases.</li>
		<li>Add 1.5x volume of 100% ethanol to the supernatant and mix it.</li>
		<li>Transfer 700 &#181;L 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.</li>
		<li>Add 700 &#181;L of buffer 1 to the column and centrifuge it at full speed for 15 s at RT; discard the flow-through.</li>
		<li>Add 500 &#181;L of buffer 2 to the column and centrifuge it at full speed for 15 s at RT; discard the flow-through.</li>
		<li>Add 500 &#181;L of 80% ethanol to the column and centrifuge it at full speed for 2 min at RT; discard the flow-through.</li>
		<li>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.</li>
		<li>Add 16 &#181;L 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 &#176;C for future analysis. 
		NOTE: Two microliters of each sample can be used for OD analysis.</li>
	</ol>
	</li>
	<li>Reverse Transcription
	<ol>
		<li>Prepare on ice. For each 20 &#181;L RT-PCR reaction, add 4 &#181;L of the miRNA RT buffer, 2 &#181;L of nucleic acids, 9 &#181;L of total RNA, 2 &#181;L of enzyme and 3 &#181;L of RNAse-free water; mix it and spin it down briefly.</li>
		<li>Set the Thermal Cycler as follows: 
		37 &#176;C for 60 min; 
		95 &#176;C for 5 min; 
		4 &#176;C hold.</li>
		<li>Remove the tubes from the thermal cycler and add 200 &#181;L of RNAse-free water. Store at -20 &#176;C or proceed to the real-time PCR.</li>
	</ol>
	</li>
	<li>Real-time PCR 
	NOTE: This protocol follows 96 well plate format. Prepare the reaction mix on ice.
	<ol>
		<li>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.</li>
		<li>Add a total of 25 &#181;L of PCR mix + cDNA in each well.</li>
		<li>Seal the plate using an optical plate seal.</li>
		<li>Centrifuge the plate for 1 min at 1,000 x g at room temperature.</li>
		<li>Program the real-time cycler as follows: 
		Initial activation step at 95 &#176;C for 15 min; 
		3-step cycling: denaturation at 94 &#176;C for 15 s; annealing at 55 &#176;C for 30 s; extension at 70 &#176;C for 30 s (perform fluorescence data collection); add 40 cycles; 
		Melting-curve according to cycler default.</li>
	</ol>
	</li>
	<li>Comparative analysis 
	<ol>
		<li>Example of normalization (see Table 2):
		<ol>
			<li>Find the SCM minimum value.</li>
			<li>Normalize all the SCM values to this minimum.</li>
			<li>Subtract this value from the Ct values of miRNAs of interest (MOI) and reference gene (REF).</li>
			<li>Analyze data using the 2-&#916;Ct formula.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Proteomic Analysis
<ol>
	<li>Extraction of proteins from polysome fractions 
	<ol>
		<li>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.</li>
		<li>Prepare 100% stock of trichloroacetic acid (TCA) and store it at 4 &#176;C in dark. Mix 0.5 mL of sample with 60 &#181;L of 100% TCA and incubate at -20 &#176;C overnight in the dark. 
		NOTE: Use 1.5 mL low protein retention tubes (see Table of Materials).</li>
		<li>After overnight incubation, thaw the sample on ice and centrifuge at 15,000 x g, 4 &#176;C for 30 min. Discard supernatant. 
		NOTE: 100 &#181;g of total protein in sample gives visible protein pellet at the tube bottom.</li>
		<li>Pre-chill acetone in -20 &#176;C freezer. Add 0.5 mL of cold acetone to the protein pellet and centrifuge at 15,000 x g, 4 &#176;C for 15 min. Discard supernatant. Repeat this step twice.</li>
		<li>Air dry the pellet. Resuspend the pellet in 360 &#181;L of 50 mM Tris-HCl buffer (pH 8). 
		NOTE: If needed, use a pulse sonicator to resuspend the pellet.</li>
		<li>Add 40 &#181;L of 0.1 M dithiothreitol (final concentration 10 mM) and incubate at 55 &#176;C on shaker for 45 min. Add 50 &#181;L of 0.15 M iodoacetamide (final concentration 17 mM) and incubate at room temperature on shaker for 30 min in dark.</li>
		<li>Prepare trypsin solution: resuspend 20 &#181;g of trypsin in 100 &#181;L 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 &#176;C overnight. 
		NOTE: Add 1 M ammonium bicarbonate to bring pH to 8.</li>
		<li>After trypsin digestion, cool the sample, centrifuge briefly in bench centrifuge, add 10% formic acid to pH 2&#8211;3 to quench the trypsin activity, and proceed with sample desalting. 
		NOTE: Use &#181;-elution plate for sample desalting.</li>
		<li>Condition the sorbent in the wells of 96 well plate with 200 &#181;L of methanol using vacuum three times followed by conditioning by 200 &#181;L of 0.1% formic acid three times. Load sample and perform a sample wash using 200 &#181;L of 0.1% formic acid three times. Elute the sample with 100 &#181;L 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.</li>
		<li>Evaporate sample eluate to dryness using concentrator and either directly carry out mass spectrometry analysis or store at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>Mass spectrometry analysis
	<ol>
		<li>Reconstitute each pellet (consisting of the tryptic peptides) in 50&#8211;100 &#181;L and inject 1 &#181;L 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.</li>
		<li>Use a trap column C18 (300 &#181;m i.d. x 5 mm, 5 &#181;m, 100&#197;) followed by peptide separation using C18 column (75 &#181;m i.d. x 250 mm, 2 &#181;m, 100&#197;) with a flow rate setting at 300 nL/min. Set the nano-source capillary temperature to 275 &#176;C and the spray voltage to 2 kV.</li>
		<li>Apply a linear gradient of 5&#8211;35% B for 90 min, 35&#8211;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.</li>
	</ol>
	</li>
	<li>Protein identification/quantitation
	<ol>
		<li>After mass spectrometry analysis, convert the raw spectra files into compatible mzXML files using MSConvert software (http://proteowizard.sourceforge.net/tools/msconvert.html).</li>
		<li>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.</li>
		<li>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).</li>
		<li>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 &#8220;similar to&#8221; or hypothetical proteins should undergo comparison to see if the observed peptides correspond to a known protein.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Pourpirali, S., Valacca, C., Merlo, P., Rizza, S., D&#8217;Amico, S., Cecconi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+pseudohypoxia+targets+ambra1+mrna+to+p-bodies+for+translational+repression.">Prolonged pseudohypoxia targets ambra1 mrna to p-bodies for translational repression.</a> PLoS ONE. 10, e0129750 (2015).</li><li>Andres, A. M., Tucker, K. C., Thomas, A., Taylor, D. J., Sengstock, D., Jahania, S. M., Dabir, R., Pourpirali, S., Brown, J. A., Westbrook, D. G., Ballinger, S. W., Mentzer, R. M., Gottlieb, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+and+mitochondrial+biogenesis+in+atrial+tissue+of+patients+undergoing+heart+surgery+with+cardiopulmonary+bypass.">Mitophagy and mitochondrial biogenesis in atrial tissue of patients undergoing heart surgery with cardiopulmonary bypass.</a> JCI Insight. 2, e89303 (2017).</li><li>Kislinger, T., Cox, B., Kannan, A., Chung, C., Hu, P., Ignatchenko, A., Scott, M. S., Gramolini, A. O., Morris, Q., Hallett, M. T., Rossant, J., Hughes, T. R., Frey, B., Emili, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+survey+of+organ+and+organelle+protein+expression+in+mouse:+Combined+proteomic+and+transcriptomic+profiling.">Global survey of organ and organelle protein expression in mouse: Combined proteomic and transcriptomic profiling.</a> Cell. 125, 173-186 (2006).</li><li>Kren, B. T., Wong, P. Y., Shiota, A., Zhang, X., Zeng, Y., Steer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+trafficking+of+transcripts+and+micrornas+in+regenerating+liver+after+partial+hepatectomy.">Polysome trafficking of transcripts and micrornas in regenerating liver after partial hepatectomy.</a> American Journal of Physiology. Gastrointestinal and Liver Physiology. 297, G1181-G1192 (2009).</li><li>Gottlieb, R. A., Pourpirali, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+Mirnas+and+mrnas+in+ischemic+preconditioning+and+ischemia/reperfusion+injury.">Lost in translation: Mirnas and mrnas in ischemic preconditioning and ischemia/reperfusion injury.</a> Journal of Molecular and Cellular Cardiology. 95, 70-77 (2016).</li><li>Coudert, L., Adjibade, P., Mazroui, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+translation+initiation+during+stress+conditions+by+polysome+profiling.">Analysis of translation initiation during stress conditions by polysome profiling.</a> Journal of Visualized Experiments. , (2014).</li><li>Lorsch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+enzymology.+Laboratory+methods+in+enzymology:+Rna.+Preface.">Methods in enzymology. Laboratory methods in enzymology: Rna. Preface.</a> Methods in Enzymology. 530, xxi (2013).</li><li>Kuzniewska, B., Chojnacka, M., Milek, J., Dziembowska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+polysomal+fractions+from+mouse+brain+synaptoneurosomes+and+analysis+of+polysomal-bound+mrnas.">Preparation of polysomal fractions from mouse brain synaptoneurosomes and analysis of polysomal-bound mrnas.</a> Journal of Neuroscience Methods. 293, 226-233 (2018).</li><li>He, S. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+analysis+of+mammalian+cells.">Polysome analysis of mammalian cells.</a> Methods in Enzymology. 530, 183-192 (2013).</li><li>Molotski, N., Soen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+association+of+micrornas+with+polysomes+reflects+distinct+strengths+of+interactions+with+their+mrna+targets.">Differential association of micrornas with polysomes reflects distinct strengths of interactions with their mrna targets.</a> RNA. 18, 1612-1623 (2012).</li><li>Maroney, P. A., Yu, Y., Fisher, J., Nilsen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+micrornas+are+associated+with+translating+messenger+rnas+in+human+cells.">Evidence that micrornas are associated with translating messenger rnas in human cells.</a> Nature Structural and Molecular Biology. 13, 1102-1107 (2006).</li><li>Paul, P., Chakraborty, A., Sarkar, D., Langthasa, M., Rahman, M., Bari, M., Singha, R. S., Malakar, A. K., Chakraborty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+mirnas+and+human+diseases.">Interplay between mirnas and human diseases.</a> Journal of Cellular Physiology. 233, 2007-2018 (2018).</li><li>Rupaimoole, R., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microrna+therapeutics:+Towards+a+new+era+for+the+management+of+cancer+and+other+diseases.">Microrna therapeutics: Towards a new era for the management of cancer and other diseases.</a> Nature Reviews Drug Discovery. 16, 203-222 (2017).</li><li>Nelson, P. T., Hatzigeorgiou, A. G., Mourelatos, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mirnp:Mrna+association+in+polyribosomes+in+a+human+neuronal+cell+line.">Mirnp:Mrna association in polyribosomes in a human neuronal cell line.</a> RNA. 10, 387-394 (2004).</li><li>Nottrott, S., Simard, M. J., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+let-7a+mirna+blocks+protein+production+on+actively+translating+polyribosomes.">Human let-7a mirna blocks protein production on actively translating polyribosomes.</a> Nature Structural and Molecular Biology. 13, 1108-1114 (2006).</li><li>Kim, J., Krichevsky, A., Grad, Y., Hayes, G. D., Kosik, K. S., Church, G. M., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+many+micrornas+that+copurify+with+polyribosomes+in+mammalian+neurons.">Identification of many micrornas that copurify with polyribosomes in mammalian neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. , 360-365 (2004).</li><li>Androsavich, J. R., Chau, B. N., Bhat, B., Linsley, P. S., Walter, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-linked+microrna-21+exhibits+drastically+reduced+mrna+binding+and+silencing+activity+in+healthy+mouse+liver.">Disease-linked microrna-21 exhibits drastically reduced mrna binding and silencing activity in healthy mouse liver.</a> RNA. 18, 1510-1526 (2012).</li><li>Kraushar, M. L., Thompson, K., Wijeratne, H. R., Viljetic, B., Sakers, K., Marson, J. W., Kontoyiannis, D. L., Buyske, S., Hart, R. P., Rasin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+defined+neocortical+translation+and+polysome+assembly+are+determined+by+the+rna-binding+protein+hu+antigen+r.">Temporally defined neocortical translation and polysome assembly are determined by the rna-binding protein hu antigen r.</a> Proceedings of the National Academy of Sciences of the United States of America. , E3815-E3824 (2014).</li><li>McClatchy, D. B., Ma, Y., Liu, C., Stein, B. D., Martinez-Bartolome, S., Vasquez, D., Hellberg, K., Shaw, R. J., Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+azidohomoalanine+labeling+in+mammals+(palm)+detects+changes+in+liver-specific+lkb1+knockout+mice.">Pulsed azidohomoalanine labeling in mammals (palm) detects changes in liver-specific lkb1 knockout mice.</a> Journal of Proteome Research. 14, 4815-4822 (2015).</li><li>Schiapparelli, L. M., McClatchy, D. B., Liu, H. H., Sharma, P., Yates, J. R., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+of+biotinylated+proteins+by+mass+spectrometry.">Direct detection of biotinylated proteins by mass spectrometry.</a> Journal of Proteome Research. 13, 3966-3978 (2014).</li><li>Wi&#347;niewski, J. R., Zougman, A., Nagaraj, N., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+sample+preparation+method+for+proteome+analysis.">Universal sample preparation method for proteome analysis.</a> Nature Methods. 6, 359-362 (2009).</li><li>Rajalingam, D., Loftis, C., Xu, J. J., Kumar, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichloroacetic+acid-induced+protein+precipitation+involves+the+reversible+association+of+a+stable+partially+structured+intermediate.">Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate.</a> Protein Science. 18, 980-993 (2009).</li><li>Fic, E., Kedracka-Krok, S., Jankowska, U., Pirog, A., Dziedzicka-Wasylewska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+protein+precipitation+methods+for+various+rat+brain+structures+prior+to+proteomic+analysis.">Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis.</a> Electrophoresis. 31, 3573-3579 (2010).</li><li>Sashital, D. G., Greeman, C. A., Lyumkis, D., Potter, C. S., Carragher, B., Williamson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+quantitative+mass+spectrometry+and+electron+microscopy+analysis+of+ribosomal+30s+subunit+assembly+in+e.+Coli.">A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30s subunit assembly in e. Coli.</a> Elife. 3, (2014).</li><li>Liang, S., Bellato, H. M., Lorent, J., Lupinacci, F. C. S., Oertlin, C., van Hoef, V., Andrade, V. P., Roff&#233;, M., Masvidal, L., Hajj, G. N. M., Larsson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome-profiling+in+small+tissue+samples.">Polysome-profiling in small tissue samples.</a> Nucleic Acids Research. 46, e3 (2018).</li></ol>

The authors have nothing to disclose.

Polysome profile analysis allows for the study of protein translation by analyzing the translational state of a specific mRNA or the whole transcriptome6,7. It is also of great help when local translation needs to be studied such as synaptosomes8. Traditionally, this method involves the separation of mono- and polyribosomes and the associated mRNAs on a sucrose gradient which could be coupled with genomic or proteomic techniques to obtain the intended results6,9. For instance, a recent study performed by our group has revealed a post-transcriptional regulatory mechanism executed in the heart of patients undergoing CPB, which mimics ischemia/reperfusion injury. Taking advantage of polysome profile analysis, we have shown an increase in translation of nuclear-encoded mitochondrial proteins in heart after CPB procedure2.
The dynamic proteome polyribosomal profiling approach presented here can reveal changes in the population of mRNAs associated with polyribosomes and the proteins that are being actively translated. As translation of mRNAs is governed in part by miRNAs, analysis of miRNAs in these fractions can reveal additional insights. In fact, several studies have modified this method and proved it to be a suitable and reliable approach to investigate the miRNA mode of regulation10,11.
Many studies have been conducted in the last decade delving into the role of miRNAs, considering their importance in various biological functions12,13. Since miRNAs act through base-pairing with counterpart mRNAs so as to mark them for degradation, polysome profile analysis has been performed and shown that miRNAs are in fact found in the polysome fractions, targeting translating mRNAs14,15,16. miR-21 is a good example where it is associated with a low repression and weak polysomes binding in normal cells, while in cancer cells, the association with polysomes is increased and the repressive effect is much stronger17.
In this study, Real-time PCR analysis was done using the Ct values of the miRNAs of interest (MOI), the Ct values of the spike-in control miRNA (SCM) to reduce technical variations and the Ct value of a reference gene or miRNA (REF) which is stable among samples. The first step was to normalize the Ct values of the MOI and the REF to the Ct values of the SCM. Then there was a second step normalization of the MOI to the REF and the resulting value was used to calculate the 2-&#916;Ct formula. These values or fold-change values can be used to demonstrate differences between groups.
Since the extraction of proteins is difficult from polysome fractions, most of the studies performed until now, have used the fractions directly for western blot analysis. They simply sediment the protein content of each fraction and mix it with SDS-loading buffer to use for SDS-PAGE analysis8,18. Here, we have developed a method which allows us to not only extract mRNAs and miRNAs after fractionation, but also to obtain peptides and proteins with a high quality to proceed with Mass Spectrometry analysis.
This approach could be extended further by actively measuring the newly synthesized proteins using metabolic labeling such as biorthogonal amino acid homolog, such as azidohomoalanine (AHA) for methionine19,20. AHA incorporation followed by click chemistry allows for incorporation of a biotin tag followed by streptavidin purification of all proteins that incorporated AHA. This allows definitive identification of newly synthesized proteins &#8212; the other part of the dynamic proteome. Detection of miRNAs that redistribute among the translating and nontranslating fractions in response to a stimulus (here with I/R) allows interrogation of the dynamic regulation of protein translation. However, the factors that recruit miRNAs to the vicinity of ribosome-bound mRNA remain to be identified and systematically investigated.
In our study, in addition to TCA precipitation, we tested three other methods for protein extraction from polysome fractions: i) FASP (filter-aided sample preparation)21, ii) acetone precipitation, and iii) chloroform/methanol precipitation. The suitability of the methods was evaluated based on both the ability to process fractions with high concentration of sucrose (up to 50%) and number of proteins identified by mass spectrometry (Figure 7 and Table 3).
For FASP method, we followed the protocol of sample preparation given by a FASP Protein Digestion Kit that employed an ultrafiltration unit with a relative molecular mass cut-off of 30 kDa. This filter unit served as a tool for detergent removal, buffer exchange, protein digestion and peptide elution with the ability to retain high-molecular-weight substances (proteins and DNA) that would otherwise interfere with subsequent peptide separation21. Briefly, the sample was mixed with urea-containing buffer and loaded on unit spin filter with spinning steps as iodoacetamide solution, trypsin solution and sodium chloride solution (elution) were subsequently added. Final filtrate that contained digested peptides was acidified by TFA, desalted and stored for mass spectrometry analysis. Using this method, however, the bulk of precipitated sucrose steadily accumulated on the top of the filter, rendering the centrifugation and washing steps difficult.
For acetone precipitation and chloroform/methanol precipitation, multiple volumes of solvents were needed to precipitate proteins from a single volume of sample. This limited the size of sample volume applied when the precipitation was performed in 1.5 mL tubes (protein low-retention tubes). As well, the viscous sludge of sucrose accumulated at the bottom of tubes after precipitation for both methods and there were no visible pellets at the tube bottom and at the liquid interface after acetone precipitation and chloroform/methanol precipitation, respectively.
Table 3 and Figure 7 show the comparison of four protein extraction methods we used to optimize our experimental workflow. The red numbers given at each method (Figure 7) represent the number of proteins that were identified by mass spectrometry (see Section 5.2 and 5.3). Although sample volumes used for each method were different, chloroform/methanol and TCA precipitations resulted in highest number of proteins identified. However, due to disadvantages mentioned above (sample volume limitation and sucrose precipitation), we opted for TCA precipitation for subsequent experiments.
To find the optimum conditions for the sample processing, TCA precipitation was carried out at various temperatures and TCA concentrations22,23,24. Thus, we performed a precipitation with 10.7% TCA at 4 &#176;C and -20 &#176;C and further precipitated the supernatants with additional 60 &#181;L of TCA (final 19.4% TCA) after pellets were collected. This resulted in four pellets that were analyzed separated by mass spectrometry. In addition, we repeated the whole protocol twice to ensure that obtained results were reproducible. The workflow and the number of proteins identified in the pellets at both 4 &#176;C and -20 &#176;C conditions with two repeated experiments are shown in Figure 7 (numbers in red). Although the analysis of the pellets derived from the supernatants yielded an additional number of proteins (85 and 60 at 4 &#176;C; 81 and 55 at -20 &#176;C); a majority of the proteins were already identified in first pellets and thus, we opted to use 10.7% TCA for all subsequent experiments. Although chloroform/methanol precipitation resulted in a similar number of proteins identified compared to TCA precipitation, we preferred TCA precipitation due to several advantages: i) small volume of solvent needed to precipitate the proteins (60 &#181;L of TCA vs 1.28 mL of chloroform/methanol/water) enabling the use of larger sample volumes if needed while conveniently using 1.5 mL tubes for precipitation, ii) visibility of the pellet at the tube bottom, and iii) no accumulation of sucrose during precipitation steps.
Despite offering in-depth functional information regarding the translational state, a major drawback of this method is the need for fresh and large starting material. Many studies have tried to optimize the conditions using small volume of cells or tissues or add steps to increase the efficiency of extracting RNA and proteins25. Still, tissues obtained from different animals under the same experimental conditions could be pulled together, if need be.
In conclusion, this protocol allows for simultaneous analysis of mRNA, miRNA, and protein from fractions obtained from polysome profiling to study the regulatory mechanisms involved in ischemia/reperfusion. However, further studies are required to see whether the translating mRNAs are induced after ischemia or reperfusion and if they will be turned off upon the removal of stress.

The heart responds to the injury of ischemia (I) and reperfusion (R) in a dynamic fashion. However, there is little insight into acute changes in protein synthesis during the response. To address this, we took advantage of the well-established method of polysome profiling1 to identify changes in protein abundance that reflect redistribution of ribosomes and translational regulatory factors from cytosol to polysomes, and the increase in newly synthesized proteins (NSPs). In the setting of I/R, the increase in new protein synthesis occurs in a time frame that is inconsistent with transcription of new mRNAs2; moreover, discordance between mRNA expression levels and protein abundance has been reported3. For these reasons, we chose to analyze the changes in the dynamic proteome as reflected by protein translation. To do this, we quantify mRNA in the polysome fractions, and analyze the protein composition in the polysome fractions. Finally, because microRNAs (miRs) regulate availability of mRNAs for translation and can interfere with efficiency of protein translation4,5, we examined the distribution of miRs in the polysome fractions, focusing on the response to I/R.
We chose to use the isolated mouse Langendorff perfusion model and harvested tissue under basal conditions of continuous perfusion, after 30 min global no-flow ischemia, and after 30 min of ischemia followed by 30 min of reperfusion. We then solubilized the heart tissue and separated polysomes over a sucrose gradient, followed by proteomic analysis and selective detection of mRNAs and miRNAs by PCR and microarray, respectively. This combination of methods represents a powerful approach to understanding the dynamic proteome, enabling simultaneous detection of mRNA, miRNA, and NSPs, as well as the redistribution of regulatory proteins, miRNA, and mRNA between nontranslating fractions, low-efficiency polysomes, and high-efficiency polysomes (see Figure 1). Insights into the dynamic regulation of this process will be extended by further analysis of phosphorylation of key regulatory factors such as eIF2&#945; or mTOR. These individual steps are now described in detail.

<table>
	<tbody>
		<tr>
			<td>Pentobarbital</td>
			<td>Vortech Phamaceuticals</td>
			<td>9373</td>
			<td>for euthansia</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent</td>
			<td>103424</td>
			<td>used in langendorff preparation</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fine Science Tools</td>
			<td>91110-10</td>
			<td>used to hang the heart</td>
		</tr>
		<tr>
			<td>Langendorff system</td>
			<td>Radnoti + home made</td>
			<td>n/a</td>
			<td>A &#39;four heart&#39; system consisting of custom blown glass, tubing and water baths</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653-5KG</td>
			<td>Krebs buffer and Sucrose gradient</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td>Krebs buffer and Lysis buffer</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P-5504</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M7774-500G</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G5767</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-500G</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>Sucrose powder</td>
			<td>Sigma</td>
			<td>S0389-1KG</td>
			<td>Sucrose gradient</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>208337</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>Xylene Cyanole</td>
			<td>Sigma</td>
			<td>X-4126</td>
			<td>Sucrose gradient</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-aldrich</td>
			<td>239763</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>RNaseOUT</td>
			<td>Life Technologies</td>
			<td>C00019</td>
			<td>RNAse inhibitor for Lysis buffer</td>
		</tr>
		<tr>
			<td>Igepal CA-360 (NP40)</td>
			<td>Sigma</td>
			<td>I3021</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail tablets, EDTA free</td>
			<td>Roche</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>LE-80K</td>
			<td>Ultracentrifugation of the gradients</td>
		</tr>
		<tr>
			<td>Rotor</td>
			<td>Beckman</td>
			<td>SW41</td>
			<td>Ultracentrifugation of the gradients</td>
		</tr>
		<tr>
			<td>Biologic LP (pump)</td>
			<td>Biorad</td>
			<td>731-8300</td>
			<td>Fractionation of the gradients</td>
		</tr>
		<tr>
			<td>BioFrac</td>
			<td>Biorad</td>
			<td>741-0002</td>
			<td>Fractionation of the gradients</td>
		</tr>
		<tr>
			<td>Eppendorf RNA/DNA LoBind microcentrifuge tubes, 2 mL tube</td>
			<td>Sigma</td>
			<td>Z666513-100EA</td>
			<td>Gradient fraction and RNA extraction</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Life technologies</td>
			<td>AM9738</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Luciferase Control RNA</td>
			<td>Promega</td>
			<td>L4561</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C606-4</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Glycogen, RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0551</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>I9516</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>E7023-1L</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>BioRad</td>
			<td>170-8891</td>
			<td>Reverse transcription</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>175-5122</td>
			<td>Quantative PCR</td>
		</tr>
		<tr>
			<td>miRNeasy Micro Kit (50)</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td>Kit for total RNA isolation</td>
		</tr>
		<tr>
			<td>miScript II RT Kit (50)</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Kit for miRNA reverse transcription</td>
		</tr>
		<tr>
			<td>miScript Sybr Green PCR Kit (200)</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Kit for real-time PCR expression analysis of miRNAs</td>
		</tr>
		<tr>
			<td>Centrifuge 5424R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For centrifugation of 1.5ml or 2.0ml tubes at different temperatures. Max speed - 21130g</td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For real-time PCR plate centrifugation at different temperatures. Max speed - 2039g</td>
		</tr>
		<tr>
			<td>My Cycler Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For reverse transcription</td>
		</tr>
		<tr>
			<td>CFX96 Real-Time System/C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in Control</td>
			<td>Qiagen</td>
			<td>219610</td>
			<td>For quality control of RNA isolation</td>
		</tr>
		<tr>
			<td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear</td>
			<td>Bio-Rad</td>
			<td>HSP9641</td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td>For real-time PCR plate sealing</td>
		</tr>
		<tr>
			<td>Research plus Single-Channel Pipette, Gray; 0.5-10 &#181;L</td>
			<td>Eppendorf</td>
			<td>UX-24505-02</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic Pipets, P20</td>
			<td>Gilson</td>
			<td>F123600G</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic Pipets, P200</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Rainin L-1000XLS Pipet-Lite XLS LTS Pipette 100-1000 &#181;L</td>
			<td>Gilson</td>
			<td>17011782</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Glycogen, RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0551</td>
			<td>Improves total RNA isolation efficiency</td>
		</tr>
		<tr>
			<td>Posi-Click 1.7 mL Tubes, natural color</td>
			<td>Denville</td>
			<td>C2170</td>
			<td>RNA isolation and storage; reagent mix</td>
		</tr>
		<tr>
			<td>Thermal Cycling Tubes -0.2 mL Individual Caps, Standard 0.2 mL tubes with optically</td>
			<td>Denville</td>
			<td>C18098-4 (1000910)</td>
			<td>Reverse transcription reaction</td>
		</tr>
		<tr>
			<td>Sharp 10 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1096-FR</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Sharp 20 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1121</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Sharp 200 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1122</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Tips LTS 1 mL Filter</td>
			<td>Rainin</td>
			<td>RT-L1000F</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (200)</td>
			<td>Qiagen</td>
			<td>(it changes according to the miRNA)</td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>Gradient Master ver 5.3 Model 108</td>
			<td>BioComp Instruments</td>
			<td></td>
			<td>For preparation of sucrose gradients</td>
		</tr>
		<tr>
			<td>trichloroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T6399</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Sigma Aldrich</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Amresco</td>
			<td>M108</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Fisher Scientific</td>
			<td>BP172</td>
			<td></td>
		</tr>
		<tr>
			<td>iodoacetamide</td>
			<td>Gbiosciences</td>
			<td>RC-150</td>
			<td></td>
		</tr>
		<tr>
			<td>sequencing grade modified trypsine, porcine</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium bicarbonate</td>
			<td>BDH</td>
			<td>BDH9206</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A117</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A454</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A996</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 0.5 mL</td>
			<td>Eppendorf AG</td>
			<td>22431064</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 1.5 mL</td>
			<td>Eppendorf AG</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>HLB &#181;Elution plate 30 &#181;m</td>
			<td>Oasis</td>
			<td>186001828BA</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac concentrator</td>
			<td>Thermo Scientific</td>
			<td>Savant SPD2010</td>
			<td></td>
		</tr>
		<tr>
			<td>sonicator</td>
			<td>Qsonica</td>
			<td>Oasis180</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Sorvall Legend micro 21R</td>
		</tr>
		<tr>
			<td>LC trap column PepMap 100 C18</td>
			<td>Thermo Scientific</td>
			<td>160454</td>
			<td></td>
		</tr>
		<tr>
			<td>LC separation column PepMap RSLC C18</td>
			<td>Thermo Scientific</td>
			<td>164536</td>
			<td></td>
		</tr>
		<tr>
			<td>mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Orbitrap Elite ion trap mass spectrometer</td>
		</tr>
		<tr>
			<td>MSConvert software</td>
			<td>ProteoWizard Toolkit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorcerer-SEQUEST software</td>
			<td>Sage-N Research, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic proteomic and mirna analysis of polysomes from isolated mouse heart after langendorff perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

mRNA analysis 
mRNA results can be expressed as a distribution of a particular mRNA in each fraction (Figure 3A); for quantification, combine polyribosomal translating fractions and compare to the non-translating fraction (Figure 3B), presenting a ratio of mRNA abundance in translating to nontranslating fractions. Additional information is gained by examining the high efficiency polysome fractions separately from low-efficiency polysome fractions (and separate from nontranslating fractions). This is particularly important when analyzing miRNAs, which are enriched in the low-efficiency fractions (where they interfere with protein translation of their target mRNAs).
Representative results for a particular mRNA that changes its distribution across the translating and nontranslating fractions after the mouse hearts were subjected to ischemia and ischemia/reperfusion compared to sham are shown in Figure 4.
miRNA array analysis 
Representative results from a miRNA array analysis are shown in Figure 5. Here, a subset of miRNAs (custom-designed array plate) were analyzed in the pooled heavy fractions indicating that 22 miRNAs were associated with polysomes during ischemia, 9 miRNas during ischemia and reperfusion, with 7 that were common to both conditions. This shows that the association of miRNAs is dynamic in response to the stresses of ischemia and reperfusion.
Proteomic analysis 
Analysis of heavy, light and non-translating fractions of sham (control) sample: 
881 of total proteins were identified by mass spectrometry; 3, 46 and 208 proteins of total were unique to heavy, light and non-trans fractions, respectively (Figure 6A). The majority (88%) of mitochondrial ribosomal proteins (28S and 39S) were identified in light fraction (36 out of 41) and a majority (88%) of cytosolic ribosomal proteins (40S and 60S) were identified commonly in both heavy and light fractions (53 out of 60) confirming efficient separation and proteomic ability to identified heavier cytosolic vs lighter mitochondrial ribosomal proteins. The subset of identified mitochondrial and cytosolic ribosomal proteins is shown in Figure 6B.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58079/58079fig1.jpg" /> 
Figure 1: Typical UV absorbance profile of polysome fractions on sucrose gradient. The first three fractions represent void volume in the tubing. The high efficiency (high molecular weight, HMW) fractions are collected in fractions 4-7, the low efficiency (low molecular weight, LMW) fractions in 8-11, and the non-translating (and remaining cytosolic material) is collected in fractions 12-15 (nontranslating, NTR). The red line indicates conductivity, and the blue trace indicates UV absorbance at 254 nm. On the right is a cartoon of a test tube with sucrose gradient, showing distribution of polysomes after sedimentation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58079/58079fig2.jpg" /> 
Figure 2: RNA integrity control analysis on Bioanalyzer. RNA integrity number (RIN) of total RNA isolated from heart whole lysate. RIN is calculated according to 18S and 28S peaks. RIN range: 1-10, and RIN = 10 represents a very good integrity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58079/58079fig3.jpg" /> 
Figure 3: Representative profile of mRNA distribution in sucrose gradient. Hearts were subjected to basal Langendorff perfusion or ischemia and reperfusion (I/R). (A) Heart lysates were resolved on sucrose gradient and mRNA content for GAPDH (left) and COX4 (right) were determined in each fraction. Plot shows relative mRNA abundance in each fraction (fraction number on x-axis) for basal perfusion (sham, red line and diamonds) and ischemia/reperfusion (I/R, blue line and squares). Superimposed on the plot are UV absorbance indicating total mRNA content (light gray curve) and conductance (straight gray sloping line). Boxes indicate how fractions were pooled. (B) Bar graph plot shows ratio of mRNA abundance in translating (polysomes) vs nontranslating (NT) fractions for GAPDH (left) and COX4 (right) mRNAs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58079/58079fig4.jpg" /> 
Figure 4: Representative mRNA results. Quantitation of one mRNA in the pooled fractions (HMW, LMW, NTR) showing changes according to the experimental group. Polysome fractionation of mouse hearts was performed after Langendorff perfusion. Results are plotted as % of total mRNA (upper panel) and as ratio of polysomes (HMW+LMW) to nontranslating fraction (NTR). Error bars represent results from 5 hearts per group (sham, ischemia, or I/R). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58079/58079fig5.jpg" /> 
Figure 5: Representative miRNA results. Venn Diagram of a pathway focused miRNA analysis showing up and downregulated miRNAs in heavy fraction pools of mice samples after heart ischemia or ischemia and reperfusion. Cut-off value: 1.5 fold. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58079/58079fig6.jpg" /> 
Figure 6: Proteins identified by mass spectrometry. (A) Venn diagram of the proteins common and unique to heavy, light and non-trans fractions. (B) Subset of mitochondrial and cytosolic ribosomal proteins identified. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58079/58079fig7.jpg" /> 
Figure 7: Schematic workflows for protein extraction methods tested. The protein extraction methods were tested by using sham (control) sample and heavy (high efficiency polysomes) fraction with highest sucrose content. Numbers in red indicate the number of proteins that were identified by mass spectrometry; where two numbers are shown, they are from two separate experiments. The detailed description is given in the text. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58079/58079fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FRACTIONS</td>
			<td>GENE Ct</td>
			<td>LUCIFERASE Ct</td>
			<td>&#916;Ct</td>
			<td>2&#916;Ct</td>
			<td>SUM OF ALL FRACTIONS</td>
			<td>% OF EACH FRACTION</td>
			<td>Polysome/NTR Ratio</td>
		</tr>
		<tr>
			<td>High efficiency (HMW)</td>
			<td>28.75</td>
			<td>22.79</td>
			<td>5.96</td>
			<td>0.016</td>
			<td rowspan="3">0.046</td>
			<td>34.64</td>
			<td rowspan="3">2.77</td>
		</tr>
		<tr>
			<td>Low efficiency (LMW)</td>
			<td>28.43</td>
			<td>22.63</td>
			<td>5.80</td>
			<td>0.018</td>
			<td>38.81</td>
		</tr>
		<tr>
			<td>Non Translating (NTR)</td>
			<td>29.12</td>
			<td>22.77</td>
			<td>6.35</td>
			<td>0.012</td>
			<td>26.55</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>Polysome/NTR Ratio = (HMW + LMW)/NTR</td>
		</tr>
	</tbody>
</table>
Table 1: Sample analysis of mRNA. Method of analysis of one mRNA is shown for the different pooled fractions (HMW, LMW, NTR) after quantitative PCR. &#916;Ct = gene Ct - luciferase Ct, and the 2&#916;Ct is 2&#916;Ct.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Find the SCM minimum value: 22.77</td>
		</tr>
		<tr>
			<td></td>
			<td>Sample 1</td>
			<td>Sample 2</td>
			<td>Sample 3</td>
		</tr>
		<tr>
			<td>Ct MOI</td>
			<td>22.92</td>
			<td>22.36</td>
			<td>22.58</td>
		</tr>
		<tr>
			<td>Ct SCM</td>
			<td>22.81</td>
			<td>22.77</td>
			<td>22.9</td>
		</tr>
		<tr>
			<td>Ct REF</td>
			<td>23.27</td>
			<td>23.55</td>
			<td>23.19</td>
		</tr>
		<tr>
			<td colspan="4">Normalize all the SCM values to this minimum</td>
		</tr>
		<tr>
			<td></td>
			<td>Sample 1</td>
			<td>Sample 2</td>
			<td>Sample 3</td>
		</tr>
		<tr>
			<td>Ct SCM</td>
			<td>0.05</td>
			<td>0</td>
			<td>0.13</td>
		</tr>
		<tr>
			<td colspan="4">Subtract these values from the Ct values of MOI and REF</td>
		</tr>
		<tr>
			<td></td>
			<td>Sample 1</td>
			<td>Sample 2</td>
			<td>Sample 3</td>
		</tr>
		<tr>
			<td>Ct MOI</td>
			<td>22.87</td>
			<td>22.36</td>
			<td>22.45</td>
		</tr>
		<tr>
			<td>Ct REF</td>
			<td>23.22</td>
			<td>23.55</td>
			<td>23.06</td>
		</tr>
	</tbody>
</table>
Table 2: Sample real-time PCR calculation for miRNA expression comparison in polysome and nontranslating fractions. miRNAs of interest (MOI) and reference gene (REF) were analyzed in the pooled heavy fractions of mice after ischemia or ischemia/reperfusion. The Ct values were first normalized to the spike-in control (SCM) Ct value, then the 2-&#916;Ct formula was used to compare miRNA expression.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Method</td>
			<td>Sample volume [mL]</td>
			<td># of proteins identified</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>FASP</td>
			<td>1</td>
			<td>227</td>
			<td>bulk of sucrose precipitated on filter</td>
		</tr>
		<tr>
			<td>acetone precipitation</td>
			<td>0.6</td>
			<td>372</td>
			<td>viscous sludge of sucrose at the tube bottom; 4:1 (reagent:sample) volumes; no visible protein pellet</td>
		</tr>
		<tr>
			<td>chloroform/methanol precipitation</td>
			<td>0.32</td>
			<td>389</td>
			<td>viscous sludge of sucrose at the tube bottom; 8:1 (reagent:sample) volumes; no visible protein pellet</td>
		</tr>
		<tr>
			<td>TCA precipitation (at 4 &#176;C)</td>
			<td>0.5</td>
			<td>405, 415</td>
			<td>no sucrose precipitation; 0.12:1 (reagent:sample) volumes; visible pellet at the tube bottom</td>
		</tr>
		<tr>
			<td>re-precipitation</td>
			<td></td>
			<td>85, 60</td>
			<td>additional TCA precipitation of supernatant after first pellet was collected</td>
		</tr>
		<tr>
			<td>TCA precipitation (at -20 &#176;C)</td>
			<td>0.5</td>
			<td>440, 430</td>
			<td>no sucrose precipitation; 0.12:1 (reagent:sample) volumes; visible pellet at the tube bottom</td>
		</tr>
		<tr>
			<td>re-precipitation</td>
			<td></td>
			<td>81, 55</td>
			<td>additional TCA precipitation of supernatant after first pellet was collected</td>
		</tr>
	</tbody>
</table>
Table 3: Comparison of protein extraction methods. FASP, acetone precipitation, chloroform/methanol precipitation, and TCA precipitation at 4 &#176;C and -20 &#176;C were evaluated. The goal was to maximize the number of proteins identified. TCA precipitations were evaluated on a second batch of material to confirm reproducibility.

Watch this Scientific Journal Video about Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion at JoVE.com

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

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

dynamic-proteomic-mirna-analysis-polysomes-from-isolated-mouse-heart

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.5K Views.  Cedars-Sinai Medical Center. Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

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

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

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

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

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

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible with a wide variety of detergents. Bottom-up proteomics workflows like FASP increasingly rely on LC-MS/MS methods performed in data-independent analysis (DIA) mode, a scanning method that allows deep proteome coverage and low incidence of missing values. 
In this report, we will provide the details of a workflow that combines a FASP protocol, a double StageTip purification step and LC-MS/MS in DIA mode for urinary proteome mapping. As a model sample, we analyzed expressed prostatic secretions (EPS)-urine, a sample collected after a digital rectal exam (DRE), which is of interest in prostate cancer biomarker discovery studies.

Here, we present an optimized on-filter digestion protocol with detailed information about the following: protein digestion, peptide purification and data independent acquisition analysis. This strategy is applied to the analysis of expressed prostatic secretions-urine samples and allows high proteome coverage and low missing value label-free profiling of the urinary proteome.

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. 
Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo F&#246;rster resonance energy transfer measurements, followed by data evaluation.

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...