This work was supported by the National Institutes of Health grants 5R01GM115446 and 1R01GM130840.

Mice used in this protocol were euthanized according to NIH guidelines. The Animal Welfare Committee at University of Texas Health Science Center in Houston approved all animal work.
1. Preparation of cell lysates
<ol>
	<li>Prepare lysis buffer as described in Table 1. To 10 mL of PBS, add 0.1 g of n-dodecyl &#946;-D-maltoside detergent (DDM) and rotate to dissolve. Add 100 &#181;L of phosphatase inhibitor cocktail 2, ML211 (10 &#181;M), PMSF (10 mM) and protease inhibitor c...

<ol>
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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+analysis+of+protein+S+-acylation+by+resin-assisted+capture.">Site-specific analysis of protein S -acylation by resin-assisted capture.</a> Journal of Lipid Research. 52 (2), 393-398 (2011).</li><li>Guo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Profiling+of+Cysteine-Based+Reversible+Modifications.">Proteomic Profiling of Cysteine-Based Reversible Modifications.</a> Nature Protocols. 9 (1), 64-75 (2014).</li><li>Zaballa, M. E., van der Goot, F. 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No conflicts of interest declared.

Here, we successfully utilized the acyl-RAC assay to detect S-acylation of selected proteins in both cultured human cells and primary cells derived from mouse tissue. This method is simple, sensitive, and can be easily performed with minimal equipment requirements using standard biochemistry techniques. This method has been shown to successfully identify novel S-acylated proteins such as the &#946;-subunit of the protein translocating system (Sec61b), ribosomal protein S11 (Rps11), and microsomal glutathione-S-t...

S-acylation is a reversible post-translational modification involving addition of a fatty acyl chain to an internal cysteine residue on a target protein via a labile thioester bond1. It was first reported as a modification of proteins with palmitate, a saturated 16-carbon fatty acid2, and therefore this modification is often referred to as S-palmitoylation. In addition to palmitate, proteins can be reversibly modified by a variety of longer and shorter saturated (myristate and stearate), monounsaturated (oleate) and polyunsaturated (arachidonate and eicosapentanoate) fatty acids3,4,5,6,7. In eukaryotic cells, S-acylation is catalyzed by a family of enzymes known as DHHC protein acyltransferases and the reverse reaction of cysteine deacylation is catalyzed by protein thioesterases, most of which still remain enigmatic8.
The lability of the thioester bond makes this lipid modification reversible, allowing it to dynamically regulate protein clustering, plasma membrane localization, intracellular trafficking, protein-protein interactions and protein stability9,10. Consequently, S-acylation has been linked to several disorders including Huntington&#8217;s disease, Alzheimer&#8217;s disease and several types of cancer (prostate, gastric, bladder, lung, colorectal), which necessitates development of reliable methods to detect this post-translational protein modification11.
Metabolic labeling with radioactive ([3H], [14C] or&#160;[125I]) palmitate was one of the first approaches developed to assay protein S-acylation12,13,14. However, radiolabeling-based methods present health concerns, are not very sensitive, time consuming, and only detect lipidation of highly abundant proteins15. A faster and nonradioactive alternative to radiolabeling is metabolic labeling with bioorthogonal fatty acid probes, which is used routinely to assay dynamics of protein S-acylation16. In this method, a fatty acid with a chemical reporter (alkyne or azide group) is incorporated into the S-acylated protein by a protein acyltransferase. Azide-alkyne Huisgen cycloaddition reaction (click chemistry)&#160;can then be used to attach a functionalized group, such as a fluorophore or biotin, to the integrated fatty acid allowing for detection of the S-acylated protein17,18,19.
Acyl-biotin exchange (ABE) is one of the extensively used biochemical methods for capture and identification of S-acylated proteins that bypasses some of the shortcomings of metabolic labeling such as unsuitability for tissue samples15. This method can be applied for analysis of S-acylation in a diverse range of biological samples, including tissues and frozen cell samples20,21. This method is based on selective cleavage of the thioester bond between the acyl group and the cysteine residue by neutral hydroxylamine. The liberated thiol groups are then captured with a thiol-reactive biotin derivative. The generated biotinylated proteins are then affinity-purified using streptavidin agarose and analyzed by immunoblotting.
An alternative approach termed acyl-resin assisted capture (Acyl-RAC) was later introduced to replace the biotinylation step with direct conjugation of free cysteines by a thiol-reactive resin22,23. This method has fewer steps compared to ABE and similarly can be used to detect protein S-acylation in a wide range of samples1.
Acyl-RAC consists of 4 main steps (Figure 1), 
1. Blocking of free thiol groups; 
2. Selective cleavage of the cysteine-acyl thioester bond with neutral hydroxylamine (HAM) to expose cysteine thiol groups; 
3. Capturing of the lipidated cysteines with a thiol-reactive resin; 
4. Selective enrichment of the S-acylated proteins after elution with reducing buffer.
The captured proteins can then be analyzed by immunoblotting or subjected to mass spectrometry (MS) based proteomics to assess the S-acylated proteome in a varied range of species and tissues22,24,25. Individual S-acylation sites can also be identified by trypsin digestion of the captured proteins and analysis of the resulting peptides by LC-MS/MS22. Here, we demonstrate how acyl-RAC can be used for simultaneous detection of S-acylation of multiple proteins in both a cell line and a tissue sample.

<table border="0" cellpadding="0" cellspacing="0" style="width:745px;" width="746">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cOmplete Protease Inhibitor Cocktail tablets</td>
			<td style="width:244px;">Sigma</td>
			<td align="right" style="width:119px;">11836170001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf Centrifuge 5424</td>
			<td>Eppendorf</td>
			<td align="right" style="width:119px;">22620444</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydroxylamine (HAM)</td>
			<td>Sigma</td>
			<td align="right" style="width:119px;">159417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methyl methanethiosulfonate (MMTS)</td>
			<td>Sigma</td>
			<td align="right" style="width:119px;">64306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini tube rotator</td>
			<td>LabForce</td>
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			<td height="21" style="height:21px;">ML211</td>
			<td>Cayman</td>
			<td align="right" style="width:119px;">17630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-Therm Cool-Heat-Shake</td>
			<td>Benchmark Scientific</td>
			<td style="width:119px;">H5000-HC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Dodecyl &#946;-D-maltoside (DDM)</td>
			<td>Sigma</td>
			<td style="width:119px;">D641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphatase Inhibitor Cocktail 2</td>
			<td>Sigma</td>
			<td style="width:119px;">P5726</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiopropyl-Sepharose 6B (TS)</td>
			<td>Sigma</td>
			<td style="width:119px;">T8387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasonics Quantrex Sonicator</td>
			<td>L &#38; R</td>
			<td style="width:119px;"></td>
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</table>

detection of protein s-acylation using acyl-resin assisted capture

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.

Following the protocol described above, we first used acyl-RAC to simultaneously detect S-acylation of several proteins in Jurkat cells, an immortalized T cell line originally derived from the peripheral blood of a T cell leukemia patient27. Regulatory T cell proteins previously identified as S-acylated9,28,29 were chosen to demonstrate the utility of this method. As shown in Figure 2...

Watch this Scientific Journal Video about Detection of Protein S-Acylation using Acyl-Resin Assisted Capture at JoVE.com

Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

Acyl-RAC (Acyl-Resin Assisted Capture) is a highly sensitive, reliable and easy to perform method to detect reversible lipid modification of cysteine residues (S-acylation) in a variety of biological samples.

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty ...

Acyl-resin assisted capture or Acyl-RAC is a sensitive and reliable method that can be used to detect protein S-acylation in a variety of biological samples. This protocol bypasses some of the limitations of metabolic labeling and radio labeling, and allows the simultaneous detection of S-acylation of multiple proteins. Not only live cells but also primary tissues and frozen samples.S-acylation can regulate a variety of cellular processes such as protein trafficking, plasma membrane targeting, signal transduction, iron transport, and protein-protein interactions. It is important to use freshly prepared hydroxylamine solution with carefully adjusted pH for each experiment to ensure an efficient and specific cleavage of the thioester bond. With a demonstration of this method it's critical for steps like chloroform, methanol precipitation.The pancake shape pellet obtained during this step should be handle very carefully. It can be fragile and partial loss of the sample during the step can lead to uneven sample recovery. Demonstrating the procedure will be Savannah West, a graduate student from our laboratory.To obtain cell lysates, collect the cells of interest into a conical centrifuge tube. And remove any cell debris by centrifugation. Wash the pellet in 5 milliliters of PBS.And immediately resuspend the cells in 600 microliters of freshly prepared lysis buffer. Agitate the sample at 1, 500 revolutions per minute in a thermo shaker for 30 minutes at four degree Celsius. Before clearing the lysates, pellet detergent and soluble material by centrifugation.At the end of centrifugation, collect the cleared lysate in a pre-cooled 1.5 milliliter microcentrifuge tube on ice. And perform a Bradford or bicinchoninic acid assay to estimate the protein concentration according to standard protocols. Add methanol and chloroform to lysate at a 2:1 ratio.Shake rigorously to create a homogenous suspension and centrifuge the sample to collect a protein pellet at the interface between the aqueous and organic phases. Tilting the tube, use a needle or a gel loading tip to aspirate as much solvent as possible. Air dry the protein pellet for a few minutes.Before gently mixing the tube contents with 600 microliters of methanol, taking care not to break up the pellet. After carefully removing the methanol wash, dry the protein pellet on a bench top for approximately five minutes. Next, resuspend the protein pellet in 200 microliters of 2SHB buffer.And vortex at 42 degree Celsius and 1, 500 revolutions per minute in a thermo shaker. When the pellet is dissolved, add 200 microliters of 0.2%MMTS in 2SHB to the sample. And incubate the protein for 15 minutes at 42 degree Celsius and 1, 500 revolutions per minute in a thermo shaker.At the end of the incubation, perform 3:4 chloroform-methanol precipitations as demonstrated. To remove the MMTS, dissolve the pellet in 100 microliters of fresh 2SHB buffer with vortexing. And dilute the sample with 300 microliters of buffer A after each precipitation.After the final precipitation, dissolve the samples in 200 microliters of 2SHB buffer and dilute with 240 microliters of buffer A.Measure the protein concentration again and set aside a 40 microliters from each sample as input controls. Split samples into two equal volumes of 200 microliters. And mark the tubes as plus hydroxylamine and minus hydroxylamine.Add 50 microliters of freshly prepared neutral two molar hydroxylamine to a final concentration of 400 millimolar to the plus hydroxylamine tube. And 50 microliters of neutral two molar sodium chloride to the negative control, a minus hydroxylamine tube. Then add 30 microliters of TS bead slurry to each tube.And rotate the tubes for one to two hours at room temperature. At the end of the incubation wash the beads four times with 1%SDS in buffer A to remove any residual hydroxylamine. After the last wash, wash all of the bead samples with three gentle one minute microcentrifugations.Carefully aspirating the supernatants and resuspending the beads in 500 microliters of 1%SDS in buffer A at each wash. After the last wash, gently spin down the beads as demonstrated. And aspirate as much supernatant as possible without disturbing the beads.To recover the proteins from the beads, add 50 microliters of 4%SDS sample buffer to each tube and incubate the samples at 80 degree Celsius and 1, 500 revolutions per minute for 15 minutes in a thermo shaker. At the end of the incubation, let the samples cool before centrifuging to completely pellet the beads. Then use a gel loading tip to transfer the eluted proteins into new 1.5 milliliter tubes.And run the samples on an SDS-PAGE gel to analyze the S-acylation of the proteins of interest by Western blotting. Tyrosine kinase Lck can be detected in lysates treated with neutral two molar hydroxylamine. to cleave the thioester bond between cysteine residues and the fatty acid moiety.Stripping and reprobing with antibodies against proteins Fyn and LAT demonstrate that acyl-resin assisted capture assay can be used to analyze S-acylation of multiple proteins at the same time. In addition, S-acylation of Lck, Fyn, and LAT can be readily detected in primary mouse splenocytes. Indicating that this modification is conserved between two species.In addition to the identification of novel S-acylated proteins, this method can also be used to assess changes in protein S-acylation under different biological conditions. The number of newly identified S-acylated proteins has increased tremendously after the double up meant a fast, and reliable technique such as Acyl-RAC. In addition to the identification of novel S-acylated proteins, this method can also be used to assess changes in protein S-acylation under different biological conditions.

detection-of-protein-s-acylation-using-acyl-resin-assisted-capture

Research

JoVE Journal

Biochemistry

9.6K visualizações.  McGovern Medical School at UT Health. Captura assistida por acicl-resina ou Acyl-RAC é um método sensível e confiável que pode ser usado para detectar aciilação de proteínas em uma variedade de amostras biológicas. Este protocolo contorna algumas das limitações da rotulagem metabólica e rotulagem de rádio, e permite a detecção simultânea da S-acylation de múltiplas proteínas. Não só células vivas, mas também tecidos primários e amostras congeladas.A aciilação de S pode regular uma variedade de proce...