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that undergoes farnesylation, as detected using mass spectrometry (Fig. 4.10b). Fol-
lowing this, experiments were performed in vivo by following the farnesylation of
Ras and Hdj-2. In this case, lovastatin was used as an inhibitor of farnesyl pyrophos-
phate biosynthesis to block natural farnesylation. As a result, lovastatin addition
resulted in a shift of the gel bands associated with these proteins, which was reversed
by the introduction of
39
, indicating successful lipidation with this azido-tagged
analog. Subsequently, global analysis of protein farnesylation was pursued using a
bioorthogonal labeling strategy. For these studies, whole cell lysates were subjected
to
38
, followed by biotinylation of the resulting labeled proteins using the Staudinger
ligation, which was analyzed by in-gel labeling and mass spectrometry. By using the
latter technique for sequencing, 21 proteins were identified, 17 of which contained
the CAAX consensus sequence for farnesylation, and in addition, three endogenously
biotinylated proteins and Annexin A2.
Probes bearing clickable tags have now been applied for extensive studies involv-
ing global analysis of protein prenylation [89-96] and fatty acylation [97-102] using
probes exemplified by those that introduce azido-geranylgeranyl (
40
), azido- and
alkynyl-myristate (
41a-b
), and azido- and alkynyl-palmitate (
42a-b
) groups onto
target proteins. In addition, studies have focused on particular systems to eluci-
date aspects of lipidation, including the myristoylation of proteins associated with
apoptosis [103], palmitoylation of mitochondrial proteins [104], palmitoylation in
T-cell activation [105], palmitoylation of histone H3 variants [106], the dependence
of IFITM3 antiviral activity on S-palmitoylation [107], palmitoylation of neuronal
proteins [108], and dynamic imaging of palmitoylation [109]. Furthermore, similar
approaches have proven effective for characterizing protein cholesterylation [110] as
well as bacterial lipoproteins [111]. Otherwise, covalent lipidation has been adapt-
able in order to label a broader range of proteins [112, 113]. For example, Ting
and coworkers identified a minimalist 22-amino acid sequence of the enzyme lipoic
acid ligase that can be added to the N- or C-terminus of various proteins to intro-
duce an azidoacyl chain [114]. Finally, the fact that covalent lipidation introduces a
bioorthogonal tag onto target proteins at a specific location has been exploited for
covalent modification of proteins, such as through immobilization onto surfaces in a
geometrically defined fashion [115-118].
While prior studies focused on the covalent modification of proteins via physiolog-
ical pathways, there has also been interest in using similar approaches to characterize
aberrant covalent modification events. Marnett and coworkers used a bioorthogonal
labeling strategy to characterize covalent adducts of 4-hydroxynonenal (HNE), an
electrophilic species that results from oxidative stress [119, 120]. Towards this end,
synthetic azido- and alkynyl-HNE analogs
43a-b
were prepared and studied for the
covalent labeling of proteins, which was initially analyzed using three short peptide
sequences with different known adduction sites (His, Lys, or Cys). While most of
the probe reactions provided the same results as natural HNE, the exception was
the labeling of Cys with azido-HNE
43a
, which yielded 50% less adduct. Probes
43a-b
were also shown to have similar toxicity profiles compared to natural HNE.
Next, the conditions for post-labeling via CuAAC were optimized, and sodium ascor-
bate was reported to be a significantly better reducing agent for promoting triazole
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