An Alkyne-Containing Isoprenoid Analogue Based on a Farnesyl Diphosphate Scaffold Is a Biologically Functional Universal Probe for Proteomic Analysis

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. 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. Chemical synthesis was used
to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing
the total number of compounds to eight used in this study. Enzyme
kinetics performed in vitro combined with metabolic
labeling in cellulo, resulted in the identification
of 8 proteins for C10AlkOPP, 70 proteins for C15AlkOPP, 41 proteins
for C15hAlkOPP, and 7 proteins for C20AlkOPP. 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.