Site-specific analysis of protein S-acylation by resin-assisted capture

Abstract

Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells.

Fig. 1.

A schematic overview of the acyl-RAC assay. Free thiols are first blocked with MMTS. Thioesters are then cleaved with neutral hydroxylamine (NH₂OH), and the newly liberated thiols are captured with thiol-reactive Sepharose resin. After being washed, captured proteins are eluted with reductant and analyzed by SDS-PAGE with either protein staining or immunoblotting. To identify individual sites of S-acylation, captured proteins are subjected to “on-resin” proteolysis (typically with trypsin), and resulting peptides are eluted and analyzed by mass spectrometry (LC-MS/MS). X, 2-thiopyridyl.

Fig. 2.

Detection of S-acylated H-Ras by acyl-RAC. A: H-Ras C terminus and associated posttranslational modifications. H-Ras undergoes S-acylation at Cys¹⁸¹ and Cys¹⁸⁴ (via thioester linkages), as well as S-prenylation at Cys¹⁸⁶ (via a thioether linkage). B: HEK293 cells were transfected with wild-type (WT) HA-H-Ras or the Cys^181/184Ser mutant of HA-H-Ras and subjected to acyl-RAC, and captured proteins were analyzed by immunoblotting for HA. C: HEK293 cells were transfected with WT HA-H-Ras for 18 h and then treated with 2-bromopalmitate (2-BP)for another 18 h. Cells were subjected to acyl-RAC and analyzed by immunoblotting for HA.

Fig. 3.

Identification of S-acylation sites by acyl-RAC coupled with mass spectrometry. A: Representative MS/MS spectrum of the N-terminal peptide from the heterotrimeric GTPase Gα_s containing Cys³, the known site of S-palmitoylation. The 117 amu peak corresponds to the reporter ion from the NH₂OH+ sample, whereas the 114 amu peak (control) was not detected. B: Representative list of known S-acylated sites identified by MS-coupled acyl-RAC (see supplementary Table I for the complete listing of sites identified). C: Validation of MS data by transfection of HEK293 cells with putative S-acylated proteins followed by acyl-RAC and immunoblotting for the specified individual proteins. In each case, the identified sites of S-acylation were mutated to serine as noted. For MGST3, the identified peptide contained two Cys residues (Cys¹⁵⁰ and Cys¹⁵¹); therefore, both single and double mutants (DM) were subjected to acyl-RAC.