A Modified Acyl-RAC Method of Isolating Retinal Palmitoyl Proteome for Subsequent Detection through LC-MS/MS

Abstract

Palmitoylation is a unique and reversible posttranslational lipid modification (PTM) that plays a critical role in many cellular events, including protein stability, activity, membrane association, and protein–protein interactions. The dynamic nature of palmitoylation dictates the efficient sorting of various retinal proteins to specific subcellular compartments. However, the underlying mechanism through which palmitoylation supports efficient protein trafficking in the retina remains unclear. Recent studies show that palmitoylation can also function as a signaling PTM, underlying epigenetic regulation and homeostasis in the retina. Efficient isolation of retinal palmitoyl proteome will pave the way to a better understanding of the role(s) for palmitoylation in visual function. The standard methods for detecting palmitoylated proteins employ 3 H- or 14 C-radiolabeled palmitic acid and have many limitations, including poor sensitivity. Relatively recent studies use thiopropyl Sepharose 6B resin, which offers efficient detection of palmitoylated proteome but is now discontinued from the market. Here, we describe a modified acyl resin–assisted capture (Acyl-RAC) method using agarose S3 high-capacity resin to purify palmitoylated proteins from the retina and other tissues, which is greatly compatible with downstream processing by LC-MS/MS. Unlike other palmitoylation assays, the present protocol is easy to perform and cost-effective. Graphical abstract

CysteineS-acetylation is a post-translational modification involved in metabolic regulation

“…These studies have repeatedly identified many sites of modification that are not positively identified as being palmitoylated by MS. Short-chain acyl modifications to cysteine were not known in the literature and not searched for and hence could not be observed even if present. 24,33,34 The presence of thioester bound acyl-cysteine sites that did not map to known sites of palmitoylation hinted at the existence of short-chain cysteine acylomes.…”

“…Previous studies characterizing the long-chain cysteine acylome have used a variety of chemical approaches to achieve enrichment. 24,33,34 Unfortunately, these approaches all fail to differentiate between long-chain and short-chain acylation. The ability to either enrich for specific S-acyl modifications, or match specific enriched sites to specific acyl modifications will be key to better understanding of the physiological role of short-chain acyl modifications of cysteine.…”