Mass-Tag Labeling Using Acyl-PEG Exchange for the Determination of Endogenous Protein S-Fatty Acylation

Abstract

The covalent coupling of fatty acids to proteins provides an important mechanism of regulation in cells. In eukaryotes, cysteine fatty acylation (S-fatty acylation) has been shown to be critical for protein function in a variety of cellular pathways as well as microbial pathogenesis. While methods developed over the past decade have improved the detection and profiling of S-fatty acylation, these are hampered in their ability to characterize endogenous protein S-fatty acylation levels under physiological conditions. Furthermore, understanding the contribution of specific sites and levels of S-fatty acylation remains a major challenge. To evaluate S-fatty acylation of endogenous proteins as well as to determine the number of S-fatty acylation events, we developed the acyl-PEG exchange (APE) that utilizes cysteine-specific chemistry to exchange S-fatty acylation sites with mass-tags of defined size, which can be readily observed by western blotting. APE provides a readily accessible approach to investigate endogenous S-fatty acylation from any sample source, with high sensitivity and broad applicability that complements the current toolbox of methods for thioester-based post-translational modifications. © 2017 by John Wiley & Sons, Inc.

Keywords: PEGylation; mass-shift; post-translational modification quantification; s-fatty-acylation.

Figure 1

Mass-tag analysis of S-fatty acylation. With APE, cell lysates are reduced with TCEP, and free cysteine residues are capped with NEM. S-fatty acid groups are removed by NH₂OH, and the exposed cysteines are reacted with mPEG-Mal. Proteins are separated by SDS/PAGE and analyzed by western blot, enabling the detection of both unmodified and S-fatty acylated proteins.

Figure 2

APE enables mass-shift based detection of protein S-fatty acylation levels. Experimental replicate of Fig.2 from Percher et al. (2016)(Percher et al., 2016). HEK293T transfected with HA-HRas were lysed and total cell lysates were subjected to APE with NEM (25 mM), NH₂OH (0.75 M), and mPEG-Mal (1 mM), and compared with negative controls. Samples were analyzed by western blot using anti-HA and anti-CANX antibodies. The number of PEGylation events are indicated by asterisks (*). Apo refers to non-PEGylated protein.