Name: Detection of Protein S-Acylation using Acyl-Resin Assisted Capture (Video) | JoVE Journal
Uploaded: 2020-04-10T15:00:00.000+00:00
Duration: 8 min 31 s
Description: Learn Acyl-RAC for detecting protein S-acylation in live cells, tissues, and frozen samples. This sensitive method enables simultaneous analysis of multiple proteins to study cellular trafficking and signaling.

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5 minutes

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 cocktail (1x) and chill the buffer on ice before use.</li>
	<li>Transfer required media containing cells from the incubator into 15 mL or 50 mL conical tubes and spin cells at 350 x g for 5 min and aspirate to get rid of any cell debris. 
	NOTE: We used 1 x 107 cells for each acyl-RAC reaction to be performed.</li>
	<li>Wash the pellet by resuspending it in 5 mL of PBS and spinning at 350 x g for 5 min. 
	NOTE: Perform step 1.3 quickly to avoid cell lysis due to extended incubation in PBS.</li>
	<li>Add 600 &#181;L of lysis buffer prepared in step 1.1 to the pellet and lyse it by shaking at 1500 rpm in a thermal shaker for 30 min at 4 &#176;C.</li>
	<li>Clear lysates by centrifugation at 20,000 x g at 4 &#730;C for 30 min to pellet detergent-insoluble materials. Collect cleared lysate in pre-cooled 1.5 mL microfuge tubes and keep them on ice.</li>
	<li>Perform a Bradford/BCA assay to estimate protein concentration. It is critical to ensure the same amount of protein across different samples prior to performing the experiment. We recommend using at least 500 &#181;g of protein per reaction. 
	NOTE: In the experiments described, we used cultured (Jurkat) cells and primary splenocytes from mice tissue. The lysis method described above can be adopted to other cell types as well. The average protein concentration obtained for the abovementioned cell types is approximately 500 &#181;g per 1 x 107 cells. Jurkat cells were maintained in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4,500 mg/L glucose, and 1,500 mg/L sodium bicarbonate supplemented with 10% FBS at 37 &#176;C with 5% CO2. We used 1 x 107 Jurkat cells per reaction. Primary splenocytes were isolated from mouse spleen tissue as described26. Briefly, spleen tissue was macerated on ice, followed by lysis of erythrocytes in hypotonic solution and separation from the lymphocytes by centrifugation. We used 1 x 107 primary cells per reaction.</li>
</ol>
2. Acyl-RAC: Blocking of free thiol groups
NOTE: All subsequent steps can be performed at room temperature (RT).
<ol>
	<li>Transfer lysate into a fresh 1.5 mL microfuge tube and perform chloroform-methanol (CM) precipitation as described below.
	<ol>
		<li>Add methanol (MeOH) and chloroform (CHCl3) to the lysate at a final ratio of lysate: MeOH: CHCl3 of 2:2:1 and shake vigorously to create a homogeneous suspension.</li>
		<li>Spin at 10,000 x g for 5 min to form a pellet (&#8220;pancake&#8221;) at the interphase between aqueous and organic phases.</li>
		<li>Tilt the tube and aspirate as much solvent as possible using a needle or a gel loading tip.</li>
		<li>Air dry the protein pellet for a few minutes and gently wash it by adding 600 &#181;L of MeOH and mixing gently to avoid breaking up the pellet</li>
		<li>Carefully remove the remaining MeOH and dry the protein pellet on a benchtop for approximately 5 min.</li>
		<li>(Optional) Perform an additional centrifugation step to spin down any broken pellet after the MeOH wash to avoid loss of sample. 
		NOTE: The experiment can be stopped after the CM precipitation step. Once the pellet is obtained, it may be stored in 500 &#181;L of MeOH in -20 &#176;C up to a week.
		<ol>
			<li>Dissolve the protein pellet in 200 &#181;L of 2SHB buffer by vortexing at 42 &#176;C/1500 rpm in a thermal shaker until the pellet is dissolved.</li>
		</ol>
		</li>
		<li>(Optional) Incubate for an additional 5&#8211;10 min in a sonicating water bath to dissolve the pellet. 
		NOTE: The length of sonication varies depending on solubility of the material. However, prolonged sonication can cause protein degradation.</li>
	</ol>
	</li>
	<li>Prepare 0.2% methyl methanethiosulfonate (MMTS) (v/v) in 2SHB by adding 2 &#181;L of MMTS to 998 &#181;L of 2SHB buffer. 
	NOTE: Use freshly prepared 0.2% MMTS for each experiment.</li>
	<li>Add 200 &#181;L of 0.2% MMTS in 2SHB to each tube to a final concentration of 0.1% MMTS. Incubate for 15 min at 42 &#730;C with shaking at 1500 rpm in a thermal shaker.</li>
</ol>
3. Acyl-RAC: Hydroxylamine (HAM) cleavage and capture of S-acylated proteins
<ol>
	<li>Repeat 3-4x CM precipitations as described above to remove MMTS. Removal of MMTS can be estimated by the lack of distinct odor of MMTS. After each precipitation, dissolve the pellet in 100 &#181;L of 2SHB buffer by vortexing at 42 &#730;C/1500 rpm in a thermal shaker until the pellet dissolves, and then dilute with 300 &#181;L of Buffer A.</li>
	<li>After final precipitation, dissolve samples in 200 &#181;L of 2SHB buffer as described above and dilute with 240 &#181;L of Buffer A.</li>
	<li>In case of comparing changes in S-acylation in response to several treatment conditions, measure protein concentration again and proceed with equal amount of protein for each condition.</li>
	<li>Retain 40 &#181;L from each sample as an input control.</li>
	<li>Split samples into two equal parts of 200 &#181;L and mark tubes as &#8220;+ HAM&#8221; and &#8220;- HAM&#8221;. Add 50 &#181;L of freshly prepared neutral 2 M HAM (pH 7.0-7.5) to a final concentration of 400 mM to one of the tubes (+ HAM) and 50 &#181;L of neutral 2 M NaCl to the second tube (- HAM), which will be used as a negative control. Proceed to addition of thiopropyl-Sepharose (TS) beads. 
	NOTE: The experiment can be stopped after any of the CM precipitation steps. Once the pellet is obtained, it may be stored in 500 &#181;L of MeOH in -20 &#176;C up to a week. Neutral pH of 2 M HAM ensures its selectivity for the acyl-cysteine thioester bond and should be carefully adjusted. Care should be taken when handling samples in 2SHB buffer to avoid loss of sample due to excessive foaming. All following steps are identical for - HAM and + HAM samples.</li>
	<li>Add 30 &#181;L of TS bead-slurry to each tube and rotate the tubes for 1-2 h at RT.</li>
	<li>Wash the TS beads 4x with 1% SDS in Buffer A to remove residual HAM.
	<ol>
		<li>Gently spin down all bead samples using a microfuge for 1 min and carefully aspirate the supernatant.</li>
		<li>Resuspend the beads in 500 &#181;L of 1% SDS in Buffer A.</li>
		<li>Repeat step 3.7.1&#8211;3.7.2 thrice. 
		NOTE: Activated thiopropyl-Sepharose (TS) is supplied freeze-dried in the presence of additives that must be washed away at neutral pH before coupling. Distilled water is recommended for swelling and washing. Weigh 0.1 g of beads and resuspend in 1 mL of distilled water and allow it to swell while rotating for 30 min-1 h at RT. Wash beads 1x with buffer A and prepare a slurry with buffer A, in a ratio of 50% settled medium to 50% buffer. Swollen TS may be stored at neutral pH in the presence of 20% ethanol at 4 &#176;C up to a week. Do not use sodium azide as a bacteriostatic agent since azide ions react with the 2-pyridyl disulfide groups. For higher efficiency, prepare fresh beads. While handling the beads, the tip of a P200 pipette tip can be cut slightly so as to prevent any damage. 
		NOTE: The experiment can be stopped at any stage after CM precipitation. The pellet may be store in 500 &#181;L of MeOH in -20 &#176;C up to a week.</li>
	</ol>
	</li>
</ol>
4. Elution and detection of S-acylated proteins
<ol>
	<li>After the last wash, gently spin down the beads as described above and aspirate as much supernatant as possible without disturbing the beads.</li>
	<li>Recover the proteins from beads with 4x SDS sample buffer with DTT.
	<ol>
		<li>Add 50 &#181;L of 4x SDS sample buffer to the beads and incubate at 80 &#730;C, 1500 rpm for 15 min in a thermal shaker. Let the tubes cool.</li>
		<li>Centrifuge the beads at 5,000 x g for 3 min to completely pellet the beads and transfer the eluted proteins to a fresh 1.5 mL tube using a gel loading tip.</li>
	</ol>
	</li>
	<li>Run SDS-PAGE and analyze S-acylation of the protein(s) of interest by western blotting.</li>
</ol>

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<li>Zhou, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-background+acyl-biotinyl+exchange+largely+eliminates+the+coisolation+of+non-+s-acylated+proteins+and+enables+deep+s-acylproteomic+analysis%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Low-background acyl-biotinyl exchange largely eliminates the coisolation of non- s-acylated proteins and enables deep s-acylproteomic analysis.</a> Analytical Chemistry. 91 (15), 9858-9866 (2019).</li>
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<li>Percher, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass-tag+labeling+reveals+site-specific+and+endogenous+levels+of+protein+S-fatty+acylation%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (16), 4302-4307 (2016).</li>
</ol>

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.

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

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...

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 ...

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

Research

JoVE Journal

Biochemistry

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Glycopeptide Capture for Cell Surface Proteomics

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Learn Acyl-RAC for detecting protein S-acylation in live cells, tissues, and frozen samples. This sensitive method enables simultaneous analysis of multiple proteins to study cellular trafficking and signaling.

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

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