We thank Yutian Gan for use of Figure 3. This work was supported by National Science Foundation 0420971; the Smith College Blakeslee, Wilens and Holmes funds; and the Howard Hughes Medical Institute.

1. Sample preparation
<ol>
	<li>Use fresh, flash-frozen or RNAlater-treated tissue from mice as starting material. Blot away any excess preservative with a Kimwipe before proceeding.</li>
	<li>Remove all tendon and fascia around the muscle and mince the tissue with single edged razor blades for 2 min on chilled glass plates in a cold room (4&deg;C) until the tissue is well minced or powdery. Collect the sample in a pre-weighed microfuge tube and determine the weight of the tissue. Use no more than 10 mg of the tissue as your starting material.</li>
	<li>Add 45 &mu;L extraction buffer (8 M urea, 50 mM DTT, 4% CHAPS, 0.2% carrier ampholytes 5/8, 0.0002% bromophenol blue) per 1 mg minced tissue, at room temperature. If the volume is greater than 350 &mu;L, add 350 &mu;L initially and homogenize (step 1.4), then add the rest of the buffer to minimize splashing.</li>
	<li>Homogenize the tissue seven times with a motorized pestle (Kontes Corp) for 15 seconds at 4&deg;C, with two minutes cooling on ice between each homogenization.</li>
	<li>Centrifuge the samples at 15,600 x g for 30 minutes at 4&deg;C. Save the supernatant and measure its volume. The cell debris pellet is discarded.</li>
	<li>Precipitate the proteins in the supernatant with ice-cold reagent-grade acetone (3 times v:v ratio). Vortex the tubes for 15 seconds. Centrifuge for 15,600 x g for 10 seconds to form pellets, and resuspend in extraction buffer with the Kontes motor and polypropylene stirring rods (BioSpec Prod)</li>
	<li>Repeat step 1.6.</li>
	<li>Either proceed with protein concentration estimation of the samples or store them at -20&deg;C.</li>
</ol>
2. Protein concentration estimation
Use the Lowry assay7 or the BCA assay8 (Pierce); aim for about 4.5 mg ml-1.
3. Isoelectric focusing
<ol>
	<li>Remove a ReadyStrip IPG strip (11 cm, pH 5-8, Bio-Rad) from -20&deg;C and allow it to equilibrate at RT for 10-15 min.</li>
	<li>Vortex the sample and pipette 600-800 &mu;g (total volume 200 &mu;L) of protein sample in a straight line at the back edge of a channel in the sample rehydration tray, leaving about 1 cm at each end.</li>
	<li>Using forceps, peel off the plastic cover of the IPG strip - make sure to handle only the ends of the strip and avoid any contact with the gel. Note the basic end of the strip and position it at the left side of the tray. Place the IPG strip gel-side down on top of the sample, avoid trapping bubbles underneath. Allow it to rehydrate for one hour.</li>
	<li>Overlay with 2.5 mL mineral oil (BioRad). Cover the tray with the lid, and leave to rehydrate overnight at room temperature.</li>
	<li>Place a paper wick at both ends of the focusing tray channel and wet each one with 10 &mu;L Millipore water.</li>
	<li>Take out IPG strip and hold vertically for about 10 seconds the drain the oil. Blot the plastic backing with a Kimwipe.</li>
	<li>Place the IPG strip gel side down and with basic end to the left in the focusing tray. Cover the strip with 2.5 mL mineral oil.</li>
	<li>Place the tray into the PROTEAN IEF cell (Bio-Rad) and program a 3-step protocol (Table 1) at default cell temperature of 20&deg;C, a maximum current of 50 &mu;A/strip, and no rehydration time.
	<table border="1">
		<tbody>
			<tr>
				<td>&nbsp;</td>
				<td>Voltage</td>
				<td>Time</td>
				<td>Volt-Hrs</td>
				<td>Ramp</td>
			</tr>
			<tr>
				<td>Step 1</td>
				<td>250</td>
				<td>20 min</td>
				<td>&nbsp;</td>
				<td>Linear</td>
			</tr>
			<tr>
				<td>Step 2</td>
				<td>8,000</td>
				<td>2.5 hr</td>
				<td>&nbsp;</td>
				<td>Linear</td>
			</tr>
			<tr>
				<td>Step 3</td>
				<td>8,000</td>
				<td>&nbsp;</td>
				<td>40,000</td>
				<td>Rapid</td>
			</tr>
			<tr>
				<td>Total</td>
				<td>&nbsp;</td>
				<td>6.5 hr</td>
				<td>40,000</td>
				<td>&nbsp;</td>
			</tr>
		</tbody>
	</table>
	 Table 1. Three-step protocol of PROTEAN IEF cell for 11 cm IPG strips.</li>
	<li>Take the IPG strips out of the IEF cell using forceps. Blot the plastic backing with a Kimwipe and place the IPG strip gel side up in a clean rehydration tray. You can cover the rehydration tray and wrap it with plastic wrap and store at - 80&deg;C or proceed.</li>
</ol>
4. SDS polyacrylamide gel electrophoresis
<ol>
	<li>Transfer the strip (gel side up) into an 11 cm equilibration tray.</li>
	<li>Add 4 mL of reduction buffer (6 M urea, 2% SDS, 0.05 M Tris/HCl pH 8.8, 20% glycerol, 2% DTT) to the channel and equilibrate the strip for 10 min with mild shaking.</li>
	<li>Remove the reduction buffer, add 4 mL of alkylation buffer (6 M urea, 2% SDS, 0.05 M Tris/HCl pH 8.8, 20% glycerol, 2.5% iodoacetamide) and equilibrate the strip for 10 minutes with mild shaking.</li>
	<li>Rinse the IPG strip by dipping the strip briefly in 1 X SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.2).</li>
	<li>Lay the strip gel side up onto the back plate of the 11 cm Criterion pre-cast 10.5%-14% Tris-HCl SDS polyacrylamide gel (Bio-Rad) and push it gently in so that it makes full contact with the SDS gel; make sure no bubbles are trapped between the two gel surfaces.</li>
	<li>Overlay the IPG strip with 1 mL of molten agarose solution (0.5% agarose, 25 mM Tris, 192 mM glycine, 0.1% SDS, bromophenol blue) and let the agarose solidify for 5 minutes.</li>
	<li>Load 5 &mu;L of protein marker 10-225 kDa (USB, P/N: 76740) into the single well as molecular weight standards.</li>
	<li>Run the gel in SDS buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.2) at 200 V at room temperature or in a cold room (4&deg;C) using the bromophenol blue dye front migration to monitor the electrophoresis.</li>
	<li>Remove the gel from the plastic cast and stain overnight by shaking gently in Coomassie Brilliant Blue R-250 stain (45.5% methanol, 45.5% dH2O, 9.0% acetic acid, 0.25% Coomassie Brilliant Blue R-250).</li>
	<li>Shake the gel in Destain 1 solution (45.5% methanol, 45.5% dH2O, 9.0% acetic acid) twice for 3 hours each and in Destain 2 (5% methanol, 90% dH2O, 5% acetic acid) overnight. Store the gel in 7% acetic acid for analysis.</li>
</ol>
5. Gel imaging and analysis
<ol>
	<li>Capture images of the gels using the Quantity One software program that operates the VersaDoc imaging system (BioRad). Crop the image areas uniformly for all gels in your experiment.</li>
	<li>Load the cropped images on the PDQuest 8.0 software program to create an experiment set. Follow the Experiment setup wizard to define the parameters for spot detection and spot matching across the gels. Inspect and refine the spot matching results manually if needed.</li>
	<li>Perform quantitative and statistical analysis of the gels to detect matched protein spots with two-fold or higher statistically significant differences in expression between the two gel groups. Create an analysis set with these spots of interest and cut them out using an EXQuest spot cutter (BioRad) for trypsin digestion and LC-MS-based protein identification.</li>
</ol>
6. In-gel tryptic digestion
For this step a modified Pierce In-Gel Tryptic Digest Kit (#89871X, Pierce) protocol is used.
<ol>
	<li>Add 200 &mu;L of destaining solution (25 mM ammonium bicarbonate, 50% acetonitrile) to the gel plugs. Vortex and incubate the samples at 37&deg;C for 30 minutes with shaking. Carefully remove and discard the solution.</li>
	<li>Repeat step 6.1.</li>
	<li>Add 30 &mu;L of freshly prepared reducing buffer (50 mM Tris[2-carboxyethyl] phosphine, 22.5 mM ammonium bicarbonate) to the tubes containing the gel plugs and incubate at 60&deg;C for 10 minutes.</li>
	<li>Allow samples to cool; then remove and discard the reducing buffer.</li>
	<li>Add 30 &mu;L of alkylation buffer (100 mM iodoacetamide, 20 mM ammonium bicarbonate, prepared immediately before use in foil-wrapped tubes. Incubate the samples in the dark at room temperature for 1 hour.</li>
	<li>Remove and discard the alkylation buffer. Wash the gel plugs by adding 200 &mu;L of destaining solution to each tube. Incubate the samples at 37&deg;C for 15 minutes.</li>
	<li>Remove and discard the destaining solution and repeat the wash.</li>
	<li>Add 50 &mu;L of acetonitrile to the gel plugs and incubate them for 10 minutes at room temperature to allow them to shrink dry, then carefully remove the acetonitrile.</li>
	<li>Repeat step 6.8 once more. Gel pieces should look white and small.</li>
	<li>Allow gel pieces to dry in a Centrivap Concentrator (Labconco, Model EX1245) for 10 minutes.</li>
	<li>Swell the gel pieces by adding 10 &mu;L of activated trypsin solution (10 ng/&mu;L trypsin, 25 mM ammonium bicarbonate). Incubate at room temperature for 15 minutes.</li>
	<li>Add 25 &mu;L digestion buffer (25 mM ammonium bicarbonate) to the tubes. Incubate the samples at room temperature overnight with shaking.</li>
	<li>Sonicate the samples for 10 minutes (Branson, bath sonicator model 2510) and spin them down before removing the supernatant to a fresh tube. Save the supernatant.</li>
	<li>To further extract peptides, add 10 &mu;L of 0.1% formic acid and incubate for 5 minutes at room temperature with shaking. Sonicate the samples for 10 minutes, collect the supernatant and combine with saved supernatant in step 6.13.</li>
</ol>
7. Microscale desalting of peptide extracts
<ol>
	<li>Remove ammonium bicarbonate and other salts in the peptide extracts with the help of PepClean C-18 Spin Columns (ThermoFisher) according to manufacturer instructions.</li>
	<li>Elute peptides twice with 20 &mu;L 70% acetonitrile, evaporate the samples dry by centrifugation in the Centrivap Concentrator for 1 hour and resuspend the peptides in 10 &mu;L 0.1% formic acid for MS analysis.</li>
</ol>
8. Protein identification by HPLC-coupled mass spectrometry
<ol>
	<li>Separate 5 &mu;L of the peptide mixture on a high performance liquid chromatograph (HPLC) over 40 minutes, using a linear 2-50% acetonitrile gradient with 0.2% formic acid on a Pepswift monolithic PS-DVB column (Dionex) coupled to a Nanospray I source onto an ion trap mass spectrometer (LCQ Deca XP Max, Thermo Fisher Scientific) at a flow rate of 400 nL/min at the tip.</li>
	<li>Detect peptide MS1 signals between 400 - 1400 m/z and allow for the 3 most intense ion peaks to be isolated for MS2 sequencing by CID with dynamic exclusion.</li>
	<li>For protein identification, analyze the peptide results via a Sequest mouse reference database search (BioWorks software, Thermo-Fisher Scientific) with a static alkylated cysteine modification and dynamic modifications for phosphorylation (serine, threonine, tyrosine), methylation (histidine), oxidation (methionine), ADP-ribosylation (arginine), and N-terminal acetylation. Apply conservative matching criteria (e.g., peptide tolerance set to 2 AMU and 1.00 fragment tolerance, proteins contain at least 2 peptides passing an Xcorr vs Charge State filter [z=1/x=1.5, z=2/x=2.00, z=3/x=2.50, z=4/x=3.00] and a probability of p &lt; 0.05)5 and manually inspect the MS spectra for confident protein identification.</li>
</ol>
9. Representative Results:
Male and female unexercised, murine skeletal muscle (biceps brachii) was extracted and separated into a two-dimensional map5, the first level of the muscle comparative proteome (Figure 2). Once visualized using high-resolution digital imaging; about 800 protein spots were detected in each. Using the male proteome map as a baseline, it is clear that there are numerous spots that change in abundance in the female proteome. The spot intensities are measures of the change in the protein amounts (Figure 3). The identities of the protein spots that change more than two-fold (up or down) and are statistically significant (p &lt; 0.05) with an n=5 mice, are determined by amino acid sequence analysis using liquid chromatography-coupled mass spectrometry. These results show that females demonstrate an abundance decrease in sugar energy metabolism enzymes and in creatine kinase enzyme (CK) isoforms, a different energy supply system in muscles. Both humans and animals display higher male serum CK levels at rest and following exercise9,10, but serum CK levels do not necessarily correlate with the amount of myofibrillar disruption11,12. This gender dimorphism in the cellular abundance of CK in murine biceps brachii muscle is a novel finding5 and may answer the physiologically different serum CK levels.
Figures:
<img alt="Figure 1" src="/files/ftp_upload/3536/3536fig1.jpg" /> 
Figure 1. An overall schematic of the protocol for comparative proteomics. The protocol is subdivided into three groups: sample preparation; sample analysis; and, protein identification. The ends of each of these three groups of protocols are also reasonable pausing places, although the best results are garnered if the overall protocol is carried out without stopping.
<img alt="Figure 2" src="/files/ftp_upload/3536/3536fig2.jpg" /> 
Figure 2. Comparison of female murine biceps brachii protein spots relative to the identical spots in male biceps brachii (green circles o). Spots that increased greater than or equal to two-fold in females are circled red (o) and those that decreased less than or equal to two-fold are circled blue (o).
<img alt="Figure 3" src="/files/ftp_upload/3536/3536fig3.jpg" /> 
Figure 3. Gel Spot Intensity Analysis: X-axis, female pre-exercise (control, n=5); Y-axis, female single bout 0 hr time point group (n=5). Regression line: correlation coefficient = 0.919; slope = 0.976; intercept = -0.0293. Spots above the red line and below the blue line change more than +/- two-fold.

<ol>
	<li>Fehrenbach, E., Mooren, F. C., V&#246;lker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular and Cellular Exercise Physiology. , 199-217 (2005).</li><li>Mooren, F. C., Mooren, F. C., V&#246;lker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cell.">The Cell.</a> Molecular and Cellular Exercise Physiology. , 3-18 (2005).</li><li>Thompson, H. S., Clarkson, P. M., Scordilis, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+repeated+bout+effect+and+heat+shock+proteins:+intra-muscular+HSP27+and+HSP70+expression+following+two+bouts+of+eccentric+exercise+in+humans.">The repeated bout effect and heat shock proteins: intra-muscular HSP27 and HSP70 expression following two bouts of eccentric exercise in humans.</a> Acta. Physiol. Scand. 174, 47-56 (2002).</li><li>McHugh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+understanding+of+the+repeated+bout+effect:+the+protective+effect+against+muscle+damage+from+a+single+bout+of+eccentric+exercise.">Recent advances in the understanding of the repeated bout effect: the protective effect against muscle damage from a single bout of eccentric exercise.</a> Scand. J. Med. Sci. Sports. 13, 88-97 (2003).</li><li>Metskas, L. A., Kulp, M., Scordilis, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+Dimorphism+in+the+Exercise-Naïve+Murine+Skeletal+Muscle+Proteome.">Gender Dimorphism in the Exercise-Naïve Murine Skeletal Muscle Proteome.</a> Cell Molec. Biol. Lett. 15, 507-516 (2010).</li><li>Lopez, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+electrophoresis+in+proteome+expression+analysis.">Two-dimensional electrophoresis in proteome expression analysis.</a> J. Chromatogr. B. 849, 190-202 (2007).</li><li>Lowry, O. H., Rosenburg, N. J., Farr, A. L., Randall, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+measurement+with+the+Folin+phenol+reagent.">Protein measurement with the Folin phenol reagent.</a> J. Biol. Chem. 193, 265-275 (1951).</li><li>Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., Klenk, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Protein+Using+Bicinchoninic+Acid.">Measurement of Protein Using Bicinchoninic Acid.</a> Anal. Biochem. 150, 76-85 (1985).</li><li>Norton, J. P., Clarkson, P. M., Graves, J. E., Litchfield, P. L., Kirwan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+creatine+kinase+activity+and+body+composition+in+males+and+females.">Serum creatine kinase activity and body composition in males and females.</a> Hum. Biol. 57, 591-598 (1985).</li><li>Amelink, G. J., Kamp, H. H., B&#228;r, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creatine+kinase+isoenzyme+profiles+after+exercise+in+the+rat:+sex-linked+differences+in+leakage+of+CK-MM.">Creatine kinase isoenzyme profiles after exercise in the rat: sex-linked differences in leakage of CK-MM.</a> Pfl&#252;gers. Arch. 412, 417-421 (1988).</li><li>Kendall, B., Eston, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-induced+muscle+damage+and+the+potential+protective+role+of+estrogen.">Exercise-induced muscle damage and the potential protective role of estrogen.</a> Sports Med. 32 (2), 103-123 (2002).</li><li>Tiidus, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+oestrogen+influence+skeletal+muscle+damage,+inflammation,+and+repair?.">Can oestrogen influence skeletal muscle damage, inflammation, and repair?.</a> Br. J. Sports Med. 39, 251-253 (2005).</li></ol>

No conflicts of interest declared.

The LC/MS proteomics method presented here is a most reliable and reproducible protocol for a rapid analysis of the first level of the skeletal muscle proteome. It allows for a reasonably expedient comparison of gender specificity. Low abundance proteins would require a fractionation of the muscle sample to remove as many of the contractile proteins as possible, thereby increasing the low abundance proteins. Tailoring the proteome profile can be accomplished with differing pH range IPG strips and alternate percentage gradient gels if desired. More sensitive fluorescent stains exist, but we recommend that each laboratory determine the linearity of these stains in your system. For a more rigorous analysis of protein expression the DIGE system (two-Dimensional In-Gel Electrophoresis system, GE Healthcare Life Sciences) can be used, however this does add a level of complexity to the methodology. Further, the protocol herein described can be used with most animal tissues for which a genome has been sequenced, as well as de novo sequencing of a protein from any source, as long as it can be resolved on a two-dimensional gel, since overlapping enzymatic fragments can be generated using high purity enzymes in addition to trypsin. Samples from other tissue sources may require some alterations in the extraction methodology, especially if looking for membrane and hydrophobic proteins, however, we have had good results using this protocol with cardiac, liver, kidney and brain tissues. Other post-translational modifications may be detected by modifying the mass spectrum database search criteria. In sum, we have presented a reasonably detailed initial protocol for proteome profiling analysis.

<table border="1">
<tbody>
<tr>
<th>Name of the reagent</th>
<th>Type</th>
<th>Company</th>
<th>Catalogue number</th>
<th>Comments</th>
</tr>
<tr>
<td>PepClean C-18 Spin Columns</td>
<td>Consumable</td>
<td>ThermoFisher Scientific</td>
<td>PI-89870</td>
<td></td>
</tr>
<tr>
<td>Pepswift monolithic column (100um x 5 cm)</td>
<td>Consumable</td>
<td>Dionex</td>
<td>162348</td>
<td></td>
</tr>
<tr>
<td>Criterion pre-cast 10.5&#37;-14&#37; Tris-HCl SDS polyacrylamide gels</td>
<td>Consumable</td>
<td>BioRad</td>
<td>345-0106</td>
<td></td>
</tr>
<tr>
<td>Readystrip IPG strips</td>
<td>Consumable</td>
<td>BioRad</td>
<td>Varying pH ranges</td>
<td></td>
</tr>
<tr>
<td>Acetic acid</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>A465-1</td>
<td></td>
</tr>
<tr>
<td>Acetone</td>
<td>Reagent</td>
<td>Pharmco</td>
<td>329000</td>
<td></td>
</tr>
<tr>
<td>Acetonitrile </td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>A955</td>
<td></td>
</tr>
<tr>
<td>Agarose</td>
<td>Reagent</td>
<td>BioRad</td>
<td>163-2111</td>
<td>Overlay solution</td>
</tr>
<tr>
<td>Ammonium bicarbonate</td>
<td>Reagent</td>
<td>Fluka</td>
<td>40867</td>
<td></td>
</tr>
<tr>
<td>Bromophenol blue</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>BP114</td>
<td></td>
</tr>
<tr>
<td>Bio-Lyte Ampholytes</td>
<td>Reagent</td>
<td>BioRad</td>
<td></td>
<td>Varying pH ranges</td>
</tr>
<tr>
<td>CHAPS</td>
<td>Reagent</td>
<td>USB</td>
<td>13361</td>
<td></td>
</tr>
<tr>
<td>Commassie blue R-250</td>
<td>Reagent</td>
<td>ThermoFisher Scientific</td>
<td>20278</td>
<td></td>
</tr>
<tr>
<td>Dithiothreitol (DTT)</td>
<td>Reagent</td>
<td>USB</td>
<td>15395</td>
<td></td>
</tr>
<tr>
<td>Formic acid</td>
<td>Reagent</td>
<td>ThermoFisher Scientific</td>
<td>28905</td>
<td></td>
</tr>
<tr>
<td>Glycerol</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G6279</td>
<td></td>
</tr>
<tr>
<td>Glycine</td>
<td>Reagent</td>
<td>USB</td>
<td>16407</td>
<td></td>
</tr>
<tr>
<td>Iodoacetamide</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>I6125</td>
<td></td>
</tr>
<tr>
<td>Methanol</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>A452</td>
<td></td>
</tr>
<tr>
<td>Mineral oil</td>
<td>Reagent</td>
<td>BioRad</td>
<td>163-2129</td>
<td></td>
</tr>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>L6026</td>
<td></td>
</tr>
<tr>
<td>Tris base</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>93349</td>
<td></td>
</tr>
<tr>
<td>Tris[2-carboexyethyl] phosphine</td>
<td>Reagent</td>
<td>ThermoFisher Scientific</td>
<td>77720</td>
<td></td>
</tr>
<tr>
<td>Trypsin Endoproteinase, TPCK treated, MS grade</td>
<td>Reagent</td>
<td>Pierce</td>
<td>90055</td>
<td>modified</td>
</tr>
<tr>
<td>Urea</td>
<td>Reagent</td>
<td>USB</td>
<td>75826</td>
<td></td>
</tr>
<tr>
<td>Water</td>
<td>Reagent</td>
<td>Burdick&amp; Jackson</td>
<td>365</td>
<td></td>
</tr>
<tr>
<td>Centrivap Concentrator</td>
<td>Tool</td>
<td>Labconco</td>
<td></td>
<td></td>
</tr>
<tr>
<td>Exquest spot cutter</td>
<td>Tool</td>
<td>BioRad</td>
<td></td>
<td></td>
</tr>
<tr>
<td>LCQ Deca XP Max ion trap mass spectrometer</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td></td>
<td></td>
</tr>
<tr>
<td>Motorized pestle</td>
<td>Tool</td>
<td>Kontes Corp</td>
<td></td>
<td></td>
</tr>
<tr>
<td>Polypropylene stirring </td>
<td>Tool</td>
<td>BioSpec Prod</td>
<td></td>
<td></td>
</tr>
<tr>
<td>rods</td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td>PROTEAN IEF cell</td>
<td>Tool</td>
<td>BioRad</td>
<td></td>
<td></td>
</tr>
<tr>
<td>Sonicator</td>
<td>Tool</td>
<td>Branson</td>
<td></td>
<td></td>
</tr>
<tr>
<td>Surveyor Plus HPLC system</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td></td>
<td></td>
</tr>
<tr>
<td>VersaDoc imaging system</td>
<td>Tool</td>
<td>BioRad</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>

skeletal muscle gender dimorphism from proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

Watch this Scientific Journal Video about Skeletal Muscle Gender Dimorphism from Proteomics at JoVE.com

Skeletal Muscle Gender Dimorphism from Proteomics

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

skeletal-muscle-gender-dimorphism-from-proteomics

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Medicine

12.4K Views.  Smith College. A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Video: Skeletal Muscle Gender Dimorphism from Proteomics

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...