Badrilla has developed a rapid, easy to use kit to capture proteins that are S-palmitoylated for further in depth study. Previously, the principle method for detecting S-palmitoylated proteins was to incubate cells with labelled [3H]-palmitate, and then visualise the proteins using autoradiography. However, this method lacked sensitivity and required long exposure times to detect the radioactive signal.
Next, the technique of acyl-biotin exchange (ABE) was developed, using the conversion of the thioester bond to a disulphide-linked biotin to identify S-palmitoylation. In ABE, the protein is treated with N-ethylmaleimide (NEM) to block any free thiol bonds. Then hydroxylamine is used to specifically cleave the thioester-linked fatty acid (palmitate in most cases) before the free thiol thus created is labelled with a biotin-conjugate. The biotinylated protein can then be captured using a streptavidin-agarose resin, which can be further analysed with mass spectrometry. Although this method is effective in detecting S-palmitoylated proteins, the procedure is complex and requires skill to develop and implement.
A rapid alternative to ABE is resin assisted capture (acyl-RAC). This method replaces the biotinylation step with direct conjugation to the resin containing, thiol-reactive groups. This results in the covalent capture of proteins on a resin, permitting more stringent treatment to harvest S-acylated proteins with ease and high purity.
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Introducing a novel method to identify S-palmitoylated proteins: The CAPTUREome™ S-Palmitoylated Protein Kit that utilises acyl-RAC in three simple steps:
Biological samples (1mg cell protein) are dissolved in blocking buffer, which covalently masks any free thiol groups on proteins in the sample. Proteins are then separated from the blocking reagent by acetone precipitation, washed and recovered as a pellet. The pellets are then re-dissolved in the binding buffer and divided into 5-paired samples.
S-palmitate groups are removed from the experimental samples via cleavage of thioester bonds, by incubation with the thioester cleavage reagent. This results in the creation of a few new free thiol groups on specific proteins (where the palmitic acid group used to be).
CAPTUREome™ resin bonds (covalently) to free thiols, capturing the previously S-palmitoylated proteins on the resin. The resin is washed and the proteins of interest eluted with a chemical reductant to permit analysis of the proteins released from the resin.
The CAPTUREome™ S-Palmitoylated Protein Kit will process 4x5 samples, each with a paired negative control that can be easily analysed using SDS-PAGE with protein staining, immunoblotting (for a specific protein) or by mass spectrometry.
The following data were obtained using CAPTUREomeTM S-Palmitoylated Protein Kit following the protocol described above. Caveolin-3 was identified as S-palmitoylated in adult rat ventricular myocytes. IF is the input fraction, cUF is the fraction that was cleaved but did not bind to the resin, cBF is cleaved and did bind to the resin (these are the S-palmitoylated proteins), pUF are the preserved (not cleave) and not bound to resin, pBF are the preserved (not cleaved) and bound to the resin. These data show that Caveolin-3 is almost completely recovered in cBF - meaning that each Caveolin-3 protein was S-palmitoylated. The absence of Caveolin-3 in the pBF shows that the blocking stage (step 1) was complete.
As demonstrated above, methods of detecting cysteine fatty acylation have advanced over time. However, techniques are currently being developed that aim to quantify the number of S-acylation sites found on proteins, via the use of mass-tag labelling. This will contribute to a greater understanding of how the quantity of actylation of a protein affects its biological function.
Authors: Naomi Abecassis and Eleanor Eisenstadt