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Prenylomic analysis in AML-3 using tellurium-functionalized isoprenoid probes and OSTAC reaction

ABSTRACT: This experiment was designed to identify prenylated proteins in AML-3 cells using tellurium-functionalized isoprenoid probes in combination with the oxidation-controlled tellurophene–alkyne cycloaddition (OSTAC) reaction. AML-3 cells were treated with the tellurium probe, which can be metabolically incorporated into prenylated proteins by endogenous prenyltransferases. After labeling, lysates were subjected to the OSTAC reaction using BCN-biotin sulfone, allowing selective biotinylation of probe-modified proteins. These biotinylated proteins were subsequently captured on streptavidin beads and digested on-bead. The resulting peptides were fractionated and analyzed by LC–MS/MS/MS. This workflow enabled the selective enrichment and identification of prenylated proteins from complex AML-3 lysates.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Cell Suspension Culture, Leukocyte, Blood

DISEASE(S): Acute Leukemia

SUBMITTER: Yuanzhe Chen

LAB HEAD: Mark Distefano

PROVIDER: PXD068330 | Pride | 2026-02-12

REPOSITORIES: Pride

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			Action	DRS
	CYZBN2040.xml	Xml
	CYZ_BN2_040_20250310_AML3_OSTAC_MS3_Fusion_1.raw	Raw
	CYZ_BN2_040_20250310_AML3_OSTAC_MS3_Fusion_2.raw	Raw
	CYZ_BN2_040_20250310_AML3_OSTAC_MS3_Fusion_3.raw	Raw
	CYZ_BN2_040_20250310_AML3_OSTAC_MS3_Fusion_4.raw	Raw

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Project description:Prenylation consists of the modification of proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) at a cysteine near the C-terminus of target proteins to generate thioether-linked lipidated proteins. Given the widespread involvement of prenylation in protein localization, membrane trafficking, and cellular signaling, dysregulation of prenylation and prenylated proteins have been implicated in the pathogenesis of numerous diseases. In recent work, metabolic labeling with alkyne-containing isoprenoid analogues including C15AlkOPP has been used to identify prenylated proteins and track their levels in different diseases. Here, a systematic study of the impact of isoprenoid length on proteins labeled with these probes was performed. First docking experiments were used to examine how lipid length might impact prenyltransferase selectivity. Next, chemical synthesis was used to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing the total number of compounds to four used in this study. Enzyme kinetics were used to evaluate these molecules as substrates for prenyltransferases in vitro. In cellulo metabolic labeling, enrichment and proteomic analysis were performed, resulting in the labeling of 8 proteins for C10AlkOPP (lipid length = 12 atoms), 70 proteins for C15AlkOPP (lipid length = 16 atoms), 41 proteins for C15hAlkOPP (lipid length = 17 atoms) and 7 proteins for C20AlkOPP (lipid length = 20 atoms). While C10AlkOPP was the most selective for farnesylated proteins and C20AlkOPP was most selective for geranylgeranylated proteins, the number of proteins identified using those probes was relatively small. In contrast, C15AlkOPP labeled the most proteins including representatives from all classes of prenylated proteins. Functional analysis of these analogues demonstrated that C15AlkOPP was particularly well suited for biological studies since it was efficiently incorporated in cellulo, was able to confer correct plasma membrane localization of H-Ras protein and complement the effects of GGPP depletion in macrophages to yield correct cell polarization and filopodia. Collectively, these results indicate that C15AlkOPP is a biologically functional, universal probe for metabolic labeling experiments that has minimal effects on cellular physiology.

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Prenylomic analysis in AML-3 using tellurium-functionalized isoprenoid probes and OSTAC reaction

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