Chemical proteomic analysis of palmostatin beta-lactone analogs that affect N-Ras palmitoylation

Suciu, Radu M.; Luvaga, Irungu K.; Hazeen, Akram; Weerasooriya, Chulangani; Richardson, Stewart K.; Firestone, Ari J.; Shannon, Kevin; Howell, Amy R.; Cravatt, Benjamin F.

doi:10.1016/j.bmcl.2021.128414

Cited by 4 publications

(2 citation statements)

References 20 publications

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“…Examples include membrane trafficking of proto-oncogene Ras 5,6 and Src 7,8 isoforms, b-adrenergic receptor signaling [9][10][11] , vesicle/vacuole fusion 12,13 and proper assembly of respiratory viruses [14][15][16] . Large scale computational analyses suggest that aberrant S-palmitoylation may play a role in cancer and neurologic disorders 17 and pharmacological studies targeting the S-palmitoylation machinery show promise for the future development of antiviral 18,19 and antineoplastic 20,21 therapies.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap)

Forrester,

Egol,

Tata

et al. 2024

Preprint

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Protein cysteine thiols undergo reversibleS-acylation via a thioester linkage in vivo.S-palmitoylation, modification by C16:0 fatty acid, is a commonS-acylation that mediates protein-membrane and protein-protein interactions critical to an array of biological processes, from homeostatic lung surfactant function to cellular transformation. The most widely usedS-acylation assays, including acyl-biotin exchange (ABE) and acyl resin-assisted capture (Acyl-RAC), utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. ABE and Acyl-RAC have enabled both the proteome-wide identification ofS-palmitoylation sites and basic biochemical studies. Yet, these assays generally utilize hundreds of micrograms to milligrams of input material and require numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome this, we devisedAcyl-Trap, a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediatedS-acyl enrichment from 20-50 micrograms of input protein. The method is compatible with protein-level detection ofS-acylated proteins as well asS-acyl site-based identification and quantification using on-quartz isobaric (tandem mass tag) labeling and LC-MS/MS. Also described are conditions for long-term hydroxylamine storage, which further expedites the assay and minimizes waste. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional intact protein detection and chemoproteomic workflows.

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Section: Introductionmentioning

confidence: 99%

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap)

Forrester,

Egol,

Tata

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…Protein fatty acylation is a ubiquitous post-translational modification that involves esterification of a Cys thiol with a saturated lipid typically 16C in length ( S -palmitoylation), imparting significant hydrophobicity onto the modified protein. Starting with the discovery of viral S -palmitoylation in 1979 by radiolabeling and autoradiography, decades of methodological advancement have established roles of S -palmitoylation in a panoply of biological processes. − Examples include membrane trafficking of proto-oncogene Ras , and Src , isoforms, β-adrenergic receptor signaling, − vesicle/vacuole fusion , and proper assembly of respiratory viruses. − Large scale computational analyses suggest that aberrant S -palmitoylation may play a role in cancer and neurologic disorders and pharmacological studies targeting the S -palmitoylation machinery show promise for the future development of antiviral , and antineoplastic , therapies.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap)

Forrester,

Egol,

Tata

et al. 2024

J. Proteome Res.

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Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein−membrane and protein−protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylaminedependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 μg of protein input material per sample and numerous reagent removal and washing steps, making them laborious and illsuited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 μg of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamline the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

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Fatty Acyl Sulfonyl Fluoride as an Activity‐Based Probe for Profiling Fatty Acid‐Associated Proteins in Living Cells

Zhang

Chen

et al. 2021

ChemBioChem

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Fatty acids play fundamental structural, metabolic, functional, and signaling roles in all biological systems. Altered fatty acid levels and metabolism have been associated with many pathological conditions. Chemical probes have greatly facilitated biological studies on fatty acids. Herein, we report the development and characterization of an alkynyl‐functionalized long‐chain fatty acid‐based sulfonyl fluoride probe for covalent labelling, enrichment, and identification of fatty acid‐associated proteins in living cells. Our quantitative chemical proteomics show that this sulfonyl fluoride probe targets diverse classes of fatty acid‐associated proteins including many metabolic serine hydrolases that are known to be involved in fatty acid metabolism and modification. We further validate that the probe covalently modifies the catalytically or functionally essential serine or tyrosine residues of its target proteins and enables evaluation of their inhibitors. The sulfonyl fluoride‐based chemical probe thus represents a new tool for profiling the expression and activity of fatty acid‐associated proteins in living cells.

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Chemical proteomic analysis of palmostatin beta-lactone analogs that affect N-Ras palmitoylation

Cited by 4 publications

References 20 publications

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap)

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap)

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap)

Fatty Acyl Sulfonyl Fluoride as an Activity‐Based Probe for Profiling Fatty Acid‐Associated Proteins in Living Cells

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