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twice with 0.05% Tween/PBS, then incubated with streptavidin–
horseradish peroxidase (HRP) conjugate (1:5000, BioLegend
#405210) in 0.05% Tween/PBS for 1 h. For anti-b-tubulin experi-
ments, membrane was blocked with 5% BSA in 0.1% Tween-20/
TBS for 1 h and then incubated with HRP-conjugated b-tubulin an-
tibody (1:1000, Cell Signaling Technology #5346) in 0.1% Tween-
20/TBS containing 5% BSA for overnight. After three washes with
0.05% Tween/PBS, the membrane was incubated in SuperSignal
West Pico chemiluminescent substrate mix (Thermo Scientific
#34080) for 8 min with gentle shaking. The autoradiology film
(GeneMate #F-9023-5X7) was then exposed to the membrane for
a suitable period (typically within 2 min) and was developed in an
automatic film developer (Konica Minolta SRX-101A).
study is critical for future rational optimization and translation-
al development of chalcone-based compounds as potential an-
ticancer agents.
Experimental Section
Cell line and culture conditions: Human non-small-cell lung ade-
nocarcinoma A549 cells, from ATCC, were cultured in Dulbecco’s
modified Eagle medium (DMEM) with 10% fetal bovine serum
(FBS), penicillin (100 UmLÀ1), and streptomycin (100 mgmLÀ1) at
378C with 5% CO2. Cells, upon reaching 80% confluency, needed
to be passaged by the standard trypsin method.
Intact cell-based photoaffinity labeling with PAL probes: A549
cells were seeded in six-well plates (2ꢁ105 cells per well in 2 mL
medium) or 100 mm cell culture dishes (2ꢁ106 cells per plate in
10 mL medium) and incubated for 20–24 h for attachment. The old
medium was removed and the new medium, containing the corre-
sponding probe at the specified concentrations, was added. After
1.5 h incubation, plates or dishes were exposed to UV light by
a UV illuminator (312 nm) for 8 min to activate the azide for
probe–target covalent labeling. Cells were then collected by stan-
dard trypsin treatment, pelleted, and lysed with EDTA-free radioim-
munoprecipitation assay (RIPA) buffer. The protein concentration of
the lysates was determined by the standard BCA assay. The lysates
were diluted to a final concentration of 1 mgmLÀ1 with phosphate-
buffered saline (PBS) and were stored at À808C until use.
Enrichment of biotinylated PAL-probe-labeled proteins with
streptavidin-coated beads: A streptavidin T1 beads slurry (67 mL,
Invitrogen #65601) was washed thoroughly with PBS in a microcen-
trifuge tube. The beads were then incubated with the cytoplasmic
fraction lysate (200 mL) with gentle rotation by using a rotary
shaker (48C, overnight). The beads were washed with 0.05%
Tween-20/PBS (400 mLꢁ2), 1m NaCl in 0.1% Tween-20/PBS
(400 mLꢁ2), and 0.5% SDS/PBS (100 mL). The beads were then
mixed with 0.5% SDS/PBS (14 mL) and heated (958C, 5 min) to re-
lease the proteins from the beads. The protein sample was then
subjected to SDS-PAGE analysis. Typically, 5% of such protein
sample was used for western blot analysis, whereas the remaining
sample was used for Coomassie blue staining, in-gel digestion, and
MS analysis.
Introduction of biotin to the PAL probe in the cell lysates by
click chemistry: To the lysate sample (30 mL) was sequentially
added azido-functionalized biotin (0.35 mm in DMSO, 3 mL), tris(2-
carboxyethyl)phosphine (14 mm in H2O, 3 mL), tris(benzyltriazolyl-
methyl)amine (2.8 mm in 3:1 tert-butyl alcohol/DMSO, 3 mL), and
copper(II) sulfate (16.8 mm in H2O, 3 mL) to reach a final concentra-
tion of 25 mm, 1 mm, 200 mm, and 1.2 mm, respectively. For the
click reaction with the use of subcellular fraction lysates, which
contained EDTA (1 mm), the concentration of copper(II) sulfate was
increased to 2 mm to achieve optimal reaction efficiency. The mix-
ture was incubated at room temperature for 1 h in the dark. For
larger scale sample preparation, up to 500 mL of lysate was used
and all reagents were proportionally adjusted.
In-gel digestion: The SDS-PAGE gel was stained with Imperial Coo-
massie staining (Thermo Scientific #24615) to visualize the target
proteins. The gel region corresponding to a protein molecular
weight of ~52 kDa was cut out and diced into 1 mm cubes. To the
diced gel samples was added ammonium bicarbonate (25 mm,
100 mL) and dithiothreitol (300 mm, 10 mL). The mixture was incu-
bated for 15 min at 508C. Then, saturated iodoacetamide (10 mL)
was added, and the mixture was incubated for 15 min at room
temperature. The supernatant was removed, and the gel pieces
were washed with a mixture of 1:1 v/v acetonitrile/25 mm ammoni-
um bicarbonate (100 mLꢁ2) and acetonitrile (100 mL). The residual
solvent was removed by speed-vac, and the gel pieces were resus-
pended in ammonium bicarbonate (25 mm, 75 mL); trypsin
(0.1 mgmLÀ1, 25 mL) was added to the gel pieces, and the mixture
was incubated at 378C overnight. The supernatant was collected.
The gel pieces were washed with 0.1% formic acid in 60% acetoni-
trile/25 mm ammonium bicarbonate (100 mLꢁ2). The washes were
combined with the supernatants and dried by speed-vac. The
dried samples were reconstituted in 0.1% formic acid (15 mL) and
then desalted with ZipTip C18 column (Millipore #ZTC18S096) by
following the manufacturer’s instruction.
Subcellular fractionation: A549 cells, cultured in 100 mm cell cul-
ture dishes, were treated with the PAL probes and exposed to UV
light the same way as that described above. Cells were then col-
lected by trypsin treatment and were fractionated by using a cellu-
lar fractionation kit (Thermo Scientific #78840) following the manu-
facturer’s protocol. Briefly, the cell pellet (~107 cells) was sequen-
tially extracted with cytoplasmic extraction buffer (400 mL, 10 min
at 48C, centrifuged at 500 g for 5 min), membrane extraction
buffer (200 mL, 10 min at 48C, centrifuged at 3000 g for 5 min), nu-
clear extraction buffer (75 mL, 30 min at 48C, centrifuged at 5000 g
for 5 min), RNase-containing nuclear extraction buffer (50 mL, 5 min
at 378C, centrifuged at 17000 g for 5 min), and pellet extraction
buffer (50 mL, 10 min at room temperature, centrifuged at 17000 g
for 5 min). Protein concentrations in each fraction were determined
following the BCA method and were then diluted to 1 mgmLÀ1 ac-
cordingly with PBS. Protein lysates were stored at À808C until use.
Mass spectrometry analysis and hit identification: The final de-
salted samples were analyzed by LC–MS2 by using a 75 mmꢁ11 cm,
15 mm orifice, Luna C18 (Phenomenex), 5 mm, 80 ꢂ LC column and
a
Thermo Scientific Orbitrap Velos (Rounds 1–4) or Fusion
(Round 5) MS system. The MS1 spectra were collected with an orbi-
trap detector (at a resolution of 30000), and the MS2 spectra were
collected with either higher-energy collisional dissociation (HCD)
(for rounds 1–3 and 5 at a normalized collision energy of 40.0) or
collision-induced dissociation (CID) (for round 4 at a normalized
collision energy of 40.0) MS2 fragmentation with an orbitrap detec-
tor (at a resolution of 7500). The eluent was acetonitrile/H2O (0.1%
formic acid) with a gradient from 2 to 35% within 1 h at a flow
rate of 300 mLminÀ1. The raw peptide data thus collected was
searched by Proteome Discoverer with the Sequest algorithm
Western blot analysis: Protein samples were resolved with the
Novex NuPAGE gel electrophoresis system following the manufac-
turer’s protocol by using commercial 4–12% SDS-PAGE gels (Invi-
trogen #NP0322). The proteins in the gel was transferred onto a cel-
lulose membrane and blocked with 1% bovine serum albumin
(BSA) in 0.05% Tween-20/PBS for 1 h. The membrane was washed
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ChemMedChem 2016, 11, 1 – 11
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