Angewandte
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effect was not as obvious (Figure 2A,B). We next assessed the
performance of these probes for labeling complex cellular
proteomes. We used PKA and c-Src overexpressed bacterial
proteomes as model systems. In-gel fluorescence scanning
results of both labeled bacterial proteomes clearly showed
positive and highly specific labeling of PKA and c-Src by STS-
1/2 and DA-2/3, respectively (Figure 2C). DA-1 was previ-
ously shown to be less effective than DA-2 in such exper-
iments.[2b] Again, by direct comparison of the labeled kinases
under different labeling conditions, both STS-2 and DA-3
appeared to consistently give a stronger labeled-protein band
than STS-1 and DA-2. We previously showed small-molecule
probes such as STS-1 were suitable bioimaging probes to
detect endogenous kinase activities.[2a] Herein, we determined
whether STS-2, by virtue of its improved minimalist linker
design, might confer better cellular imaging activities than
STS-1. Live HepG2 cells were first treated with the probe
followed by UV irradiation to initiate photo-crosslinking.
Subsequently, cells were fixed, permeabilized, treated with
rhodamine-N3 under previously optimized click chemistry
conditions,[2] then imaged. Immunofluorescence (IF) was
performed on the same cells using anti-PKA antibodies. The
resulting images were then merged (Figure 2D); for cells
treated with STS-2, strong fluorescence signals were observed
throughout the whole cell excluding the nucleus, even at
0.4 mm probe concentration. On the other hand, fluorescence
was observed for cells treated with STS-1 at a higher probe
concentration (2 mm), but not at 0.4 mm. This indicated that
STS-2 either entered the cells more readily or bound to its
intended cellular targets more efficiently. The other 11 probes
were also shown to be effective bioimaging reagents as well
(Figure S7). Control imaging experiments with a negative
probe 13 (NP in Figure 1A) under similar conditions showed
it produced minimal background fluorescence compared to
labeled cells (Figure S7). Finally, we carried out endogenous
proteome labeling and PD/LC-MS/MS target identification
with STS-1/2 in both lysates and live HepG2 cells. NP was
used throughout the experiments to eliminate false hits from
non-specific photo-crosslinking. We optimized the number of
potential positive protein hits we could identify. Briefly,
probe-labeled proteomes (upon UV irradiation) were clicked
with biotin-N3, enriched using avidin-agarose beads, sepa-
rated on SDS-PAGE, followed by LC-MS/MS analysis, as
previously reported.[2b] Selected results are summarized in
Figure 2E, while the complete list of proteins are found in the
Supporting Information (Table S2, S3, and SI_3). Because
staurosporine (STS) is a pan-kinase inhibitor, we focused on
kinases that were positively identified from our experiments.
As shown in Figure 2E. STS-2 consistently identified more
kinases in both in vitro (cell lysates) and in situ (live cells)
than STS-1. A total of 65 kinases were identified by STS-2 (45
from in situ and 38 from in vitro experiments, with 18
overlapping targets). These numbers are significantly higher
than those from a previous study using a different photo-
crosslinking approach.[12] Notably, the in situ and in vitro
experiments yielded different sets of unique protein hits with
some degree of overlap. For example, 27 and 20 unique
kinases were identified in STS-2 experiments under in situ
and in vitro settings, respectively. Similar phenomenon had
been observed previously.[2] This underscores the importance
of our current affinity-based protein-profiling approach and
its unique capability of interrogating kinase–drug interactions
in living cells. To summarize, we attributed the improved
performance of our new probes (STS-2 and DA-3) to the
minimalist linkers, which could have minimized interference
with the genuine kinase–probe binding.
We next carried out endogenous proteome labeling
followed by PD/Western blotting (WB) to confirm the ability
of the 12 probes for labeling their known kinase targets
Figure 3. A,B) Proteome reactivity profiles in HepG2 cell lysate with
the 12 individual probes and NP (10 mm) in vitro (A) and in situ (B).
C) PD/WB results for target validation of 10 of the 12 probes in live
cells (in situ). See Figures S6,S8 for details.
(Figure 3). Both in vitro and in situ labeling were carried out,
followed by rhodamine-N3 click chemistry, and the resulting
proteome reactivity profiles were compared (Figure 3A,B).
Although there were some similarities in the labeling profiles,
obvious differences were evident, clearly indicating the
presence of different protein targets under the two exper-
imental settings. Within the same setting, however, the 12
probes also labeled both common and unique protein targets
as well, likely indicating their parental kinase inhibitors also
possess both common and unique cellular (on and off) targets.
Subsequently, the same probe-labeled proteomes were
clicked with biotin-N3, pulled-down, and immunoblotted
with the respective antibodies (Figure 3B; Figure S8); with
HepG2 cells alone, 10 out of the 12 probes were shown to
successfully label their known kinase targets both in vitro and
in situ.[13]
While each of these 12 kinase probes might be useful for
large-scale identification of the cellular targets (on and off) of
the parental inhibitor, we felt a “cocktail” approach contain-
ing a mixture of all 12 probes in equal concentrations might
offer better throughput and lower operational costs for testing
multiple mammalian cell lines. Previously, similar approaches
have been demonstrated for both activity- and affinity-based
probes.[14] We also thought the cocktail approach, when
coupled with quantitative mass spectrometry, could be used
for simultaneous measurement of the quantitative binding
Angew. Chem. Int. Ed. 2013, 52, 1 – 7
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!
Li, Zhengqiu
