Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. Prenylated proteins incorporate either C15 (farnesyl) or C20 (geranylgeranyl) isoprenoids derived from farnesyl (FPP) or geranylgeranyl diphosphate (GGPP), respectively (Fig. 1) [1]. The lipidation reaction is catalyzed by protein prenyltransferases, which transfer the isoprenoid to the thiol group of a cysteine residue located near the C-terminus of a protein substrate, resulting in the formation of a thioether bond (Fig. 1). Prenylation increases the hydrophobicity of proteins, resulting in their translocation to various membranes where they participate in signal transduction pathways. However, isoprenyl groups can serve to mediate protein–protein interactions and function in other roles as well [2].

Interest in prenylation initially resulted from the discovery that Ras proteins are farnesylated and that inhibition of farnesylation of oncogenic Ras variants reverses tumor formation [3]. Those observations facilitated the development of enzyme inhibitors that subsequently moved into clinical trials. Although none of these drug candidates have been approved for cancer therapy thus far, one of them, Tipifarnib received a Fast Track Designation from the FDA for the treatment of patients with head and neck squamous cell carcinoma in 2019 [4]. In addition, they have shown promise in other diseases. In 2020, the first farnesyltransferase inhibitor (FTI), Lonafarnib, was approved for treating progeria, a premature aging disease in children, and FTIs have been developed with antifungal activity [5], [6]. Beyond Ras-driven cancers, prenylated proteins have been implicated in the deployment of a wide variety of other diseases ranging from leukemia, Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS) [7], [8], [9].

While many prenylated proteins have been identified using traditional biochemical techniques, it is likely that all of them have yet to be detected. Additionally, when prenylated proteins are implicated in a disease process, typically deduced from experiments performed with a broad spectrum of prenyltransferase inhibitors, identifying the specific involved prenylated proteins is often more complex. As a complement to more traditional biochemical and genetic methods, chemists have developed a range of chemical probes that can be used to study these types of questions [10], [11]. Thus, to address those issues and develop general methods to globally visualize, identify, and monitor all prenylated proteins simultaneously, our laboratory and others have developed chemical proteomic approaches [12], [13], [14], [15], [16]. For prenylation, such methods generally rely on the metabolic incorporation of isoprenoid analogs bearing bio-orthogonal functionalities. After incorporation in cellulo, the lysate is subjected to a bio-orthogonal biotinylation reaction, resulting in the selective labeling of all prenylated proteins. Subsequent enrichment followed by quantitative proteomic analysis allows for the detection of many prenylated proteins. Work by Tate and coworkers reported the identification of 80 prenylated proteins from EA.hy926 cells using experiments performed with a combination of two different probes, one selective for farnesylation and one for geranylgeranylation [13]. Our group reported the identification of 78 prenylated proteins in COS-7 cells using a single probe, C15AlkOPP, a substrate for both the farnesylating and geranylgeranylating enzymes [17]. Those studies also showed that such experiments could simultaneously monitor changes in the prenylation of all observable prenylated proteins that occur upon treatment with various pharmacological agents. Recently, we reported the use of this metabolic labeling approach in vivo, in a mouse animal model for Alzheimer’s disease, to study the dysregulation of protein prenylation in that disease [18] and to study the impact of knock-down of a newly identified putative deprenylating enzyme [19].

In work reported by Li et al., it was shown that inhibitors of protein prenylation can serve as potent neurite-outgrowth-promoting agents [9]. The authors hypothesized that prenylation may limit axonal growth in normal and pathological situations, including early-onset forms of ALS. To unravel the molecular details involved in this process, we sought to determine whether it would be possible to characterize the prenylome in motor neurons. While chemical proteomic methods have been used to profile the prenylomes of immortalized cell lines in numerous cases, there is only one example of global profiling in primary cells [17]. Here, we first describe efforts to improve the synthesis of C15AlkOPP. Such improvements were necessary to obtain sufficient amounts of chemical probes in a single batch required to implement global profiling of prenylated proteins in a wide variety of cell cultures and animal studies. Next, the optimization of probe incorporation in metabolic labeling experiments is described, followed by an analysis of the prenylomes of embryonic stem cells (ESCs), and motor neurons and astrocytes derived from direct differentiation of ESCs. A quantitative analysis of prenylomic enrichment data obtained via metabolic labeling was then performed to examine the molecular origins of functional differences between the developmentally related cell-types.