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Supplementary Data
Photoactivatable protein labeling by singlet oxygen mediated reactions
Tsz-Leung Toa,b,§ , Katalin F. Medzihradszkya, Alma L. Burlingamea, William F. DeGradoa,b,
Hyunil Joa,b,* and Xiaokun Shua,b,*
aDepartment of Pharmaceutical Chemistry, University of California – San Francisco, San
Francisco, CA 94158, USA
bCardiovascular Research Institute, University of California – San Francisco, San Francisco, CA
94158, USA
§ Present Address: Broad Institute 415 Main St, Cambridge, MA 02142*To whom correspondence should be addressed: [email protected] or [email protected]
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Methods
Gene construction and protein purification
The plasmid constructs in this study were created by standard molecular biology techniques and
confirmed by sequencing the cloned fragments. MiniSOG-APC and APC were expressed with
C-terminal polyhistidine-tag on a pBAD expression vector (Invitrogen). A 5 aa SGGGS linker
was inserted between miniSOG and APC to create the miniSOG-APC direct fusion. For
phycocyanobilin and biliverdin production in E. coli, the PCB:ferredoxin oxidoreductase (PcyA)
and heme oxygenase-1 (HO1) genes codon-optimized for E. coli were co-expressed on the same
vector. The fusion protein was purified with the Ni-NTA purification system (Qiagen). The
protein concentration was quantified based on the absorbance of miniSOG and its published
extinction coefficient1 . To create the Skp2-miniSOG for mammalian expression, Skp2 (Addgene
plasmid #19947) and miniSOG were PCR amplified, fused by overlap extension PCR, and
subcloned into pcDNA3.1 vector (Life Technologies). A 9 aa SGGGSGGGS flexible linker was
inserted between Skp2 and miniSOG.
Synthesis and purification of the biotin-thiol labeling molecule
A biotin-conjugated thiol-containing molecule was synthesized by esterification reaction using a
slight modification of the literature method 2, followed by reduction with TCEP. Briefly, 2-
hydroxyethyldisulfide (476 mg, 3 mmol) and triethylamine (5 mL) were added to a solution of
(+)-biotin (1.00g, 4 mmol), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride
(EDC•HCl, 942 mg, 4.9 mmol), and 1-hydroxybenzotriazole (HOBt, 664 mg, 4.9 mmol) in N,N-
dimethylformamide (DMF, 15 mL). The reaction mixture was stirred at room temperature
overnight and diluted with dichloromethane. The reaction mixture was washed with 1M
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hydrochloric acid solution and the aqueous layer was extracted with dichloromethane twice. The
combine organic layer was washed with brine, dried over sodium sulfate and concentrated. The
crude mixture was filtered through a short pad of silica (chloroform:methanol 9:1) to yield a
white solid (1g). A mixture of the above disulfide (200 mg), Tris(2-caroxyethyl)phosphine
hydrochloride (TCEP•HCl, 174 mg) in 30% acetonitrile in H2O (6 mL) was stirred for 2 h at
room temperature and lyophilized. The solid was purified by RP-HPLC (Atlantis T3 OBD
column, 19.5x150 mm, solvent A: 0.1% TFA in H2O, solvent B: 0.1% TFA in i-
PrOH:CH3CN:H2O (6:3:1), gradient: 5% solvent B to 100% solvent B over 31 min after 5 min
5% solvent B, flow rate 15 mL/min). The biotin-ester thiol has retention time 18.8 min, 1H NMR
(CDCl3, 300 MHz) δ ppm 5.59 (s, 1H), 5.16 (s, 1H), 4.49 (s, 1H), 4.32 (s,1H), 4.21-4.17 (m,2H),
3.15 (s, 1H), 2.94-2.81 (m, 1H), 2.78-2.71 (m, 3H), 2.39-2.34 (m, 2H), 1.72-1.47 (m, 6H) ESI-
MS: calcd. m/z = 305.1 (MH+), found 305.6. yield: 130 mg (overall 53%)
Photo-activatable protein labeling with purified proteins
Various concentrations of the biotin-thiol substrate were added to 1 M of miniSOG-APC in a
reaction volume of 100 L in phosphate-buffered saline (PBS) buffer at pH 7.4. To photo-
generate 1O2, the reaction mixture was illuminated with a 450 nm blue LED (Innovations in
Optics, Woburn MA) at an intensity of ~ 200 mW/cm2 at room temperature. After the reaction,
the mixture was purified with an Amicon centrifugal filter of 3-kDa cutoff (EMD Millipore), in
order to remove the free biotin-thiol molecules. The streptavidin-biotin purification of labeled
protein was performed with 100 L of Strep-Tactin Superflow resin (IBA). Elution was done by
10 mM of desthiobiotin (Sigma-Aldrich). The purified labeled protein was then assayed by an
LDS-PAGE using NuPAGE® Novex® 4-12% Bis-Tris protein gel (Life Technologies). The
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protein gel was imaged with white light in an AlphaImager 3300 Imaging System (Alpha
Innotech). To prepare samples for mass spectrometric analysis, in-solution tryptic digestion of
the purified labeled protein was performed in 50 mM ammonium bicarbonate (pH = 8). Side-
chain-protected porcine trypsin (Promega) was added to the protein at 1:50 enzyme:protein ratio
(w/w). The mixture was incubated at 37 °C for 3 hours and desalted on a SepPak C18 cartridge
(Waters Corp.). Briefly, the cartridge was equilibrated sequentially with acetonitrile, methanol
and 1% formic acid. Peptide mixture was centrifuged at 13,200g for 10 min and the sample
supernatant was loaded into the cartridge. Cartridge was washed and 1% formic acid and
peptides were eluted using 90% acetonitrile, 10% water and 0.1% formic acid. After desalting,
the sample was dried in a vacuum concentrator and resuspended in 100 µL of 0.1% formic acid
in water for LC/MS/MS analysis.
Mammalian cell cultures.
HeLa cells (ATCC CCL-2) were obtained from ATCC. Cells were passaged in Dulbecco’s
Modified Eagle medium (DMEM) supplemented with 10% Foetal Bovine Serum (FBS), non-
essential amino acids, penicillin (100 units/mL) and streptomycin (100 g/mL). All culture
supplies were obtained from the UCSF Cell Culture Facility. Cells were seeded on a 150-mm
culture dish. At ~50% confluent, cells were transiently transfected with the Skp2-miniSOG
construct using the calcium phosphate method. For each transfection in a 150-mm culture dish,
77 g of plasmid DNA was mixed with 1.3 mL of 1X Hanks’ Balanced Salts buffer (HBS) and
77 of 2.5M CaCl2. SPRAY experiment was performed 24 hours after the transient transfection, at
which cells are at ~90% confluent. The total number of cells per dish was estimated to be 20
millions.
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Photo-activatable protein labeling in cultured mammalian cells
HeLa cells transfected as above were harvested by trypsinization, washed and resuspended
in 1 mL of PBS, and transferred to a 35-mm dish. The cells were incubated with 100 M of μ
biotin-thiol substrate for one hour. The 450 nm blue LED described above was used to
irradiate cells at a light intensity of 200 mW/cm2 for 20 min. The cells were then lysed with
RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1%
Triton X-100) supplemented with protease cocktail (Sigma-Aldrich). The lysed cells were further
illuminated for 10 min, giving a total illumination time of 30 min. The lysed cells were then
centrifuged for 10 min at 14,000 g and the supernatant was saved. The unreacted biotin-thiol
substrate in the lysate was removed with an Amicon column (3 kDa cutoff). The lysate (~1 mL)
was added to 200 L of magnetic strep-tactin bead slurry (Qiagen) and incubated at 4 °C for 1
hour with gentle rotation. The supernatant was removed from the beads with pipetting and the
beads were washed 3 times with RIPA lysis buffer. On-bead trypsinization was performed by
adding side-chain-protected porcine trypsin (Promega) to the beads in 50 mM ammonium
bicarbonate (pH = 8) and the mixture was incubated at 37 °C for 3 hours. To remove the
unbound peptides, the beads were washed 3 times with PBS. To elute the bound peptides from
the beads, 1M of hydroxylamine hydrochloride (Sigma-Aldrich) in PBS (pH adjusted to 8.5) was
added and the beads were incubated for 6 hours at 37 °C. The eluted peptides were desalted on a
SepPak C18 cartridge (Waters Corp.). Desalting of samples was performed using ZipTip
according to manufacturer’s protocol (EMD Millipore). After desalting, the sample was dried in
a vacuum concentrator and resuspended in 100 µL of 0.1% formic acid in water for LC/MS/MS
analysis.
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Tandem mass spectrometry
Data-dependent LC/MS/MS experiments were performed using a nanoACQUITY HPLC system
(Waters) directly linked to LTQ-Orbitrap XL mass spectrometer (Thermo). The precursor ions
were measured in the Orbitrap, the CID experiments were conducted in the linear ion trap.
Peaklist were generated using PAVA 3. Database searches were performed using the search
engine Protein Prospector. The mass accuracy required was 10 ppm and 0.6 Da for the
precursors and for the fragment ions, respectively. Only tryptic cleavages were considered, two
missed cleavages were permitted, and on top of the default variable modifications the appropriate
modifications of Cys-residues were considered.
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Supplementary Figure 1. Hydroxylamine cleavage of ester site on the spacer arm of the
biotin-thiol substrate. The labeled interacting partner(X) can be released from biotin-
streptavidin by hydroxylamine cleavage. The labeled peptide features the ‘leftover’ beta-
mercapto-ethanol modification (Cys-S)-S-CH2-CH2-OH, which can serve as a signature for mass
spectrometric identification.
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Supplementary Figure 2. Full LDS-PAGE of data shown in Figures 2c. Experimental details
are described in the Method section under “Photo-activatable protein labeling with purified
proteins”.
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Band Intensity Normalized
Lysate 12239 1.005min / 1μM 635 0.055min / 10μM 668 0.055min / 100μM 747 0.065min / 1mM 1362 0.1130min / 1μM 1089 0.0930min / 10μM 4271 0.3530min / 100μM 8237 0.6730min / 1mM 7353 0.60
Supplementary Figure 3. Recovery of miniSOG-APC fusing protein by SPRAY under
various concentrations of the biotin-thiol and illumination times. The amount of miniSOG-
APC recovered is compared to the amount in E. coli lysate prior to SPRAY (first lane from the
left). To quantify the recovery, we generated densitometric data of LDS-PAGE image with
ImageJ (NIH). The grayscale image was inverted such that the dark areas (protein bands) became
light. Background intensity was then subtracted from the image. A rectangular box was defined
manually to enclose a single band completely, and the integrated density within the box was
recorded. Under the best condition tested (30 min illumination, 100 μM substrate), the recovery
was estimated to be 67%.
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Supplementary Figure 4. Oxidative modification of singlet oxygen sensitive residues by
miniSOG. (a) Purified IFP1.4-miniSOG fusion protein was illuminated with a 450 nm blue LED
for 30 min at an intensity of ~ 200 mW/cm2 at room temperature. The fusion protein was
trypsinized and subjected to LC-MS/MS analysis. (b) Five tryptic peptides were detected from
CID data. The residues known to be singlet oxygen sensitive (Cys, Met, Trp, Tyr, His) are in
bold and the detected singlet oxygen specific modifications from CID data are underscored. (c)
The five identified tryptic peptides are highlighted in the primary sequence of IFP1.4. Analysis
was performed on LTQ Orbitrap XL. Database search was performed with PP against the Swiss-
Prot, without species specification, but with User-Protein sequence added when necessary. Strict
database search was performed, after which the modification searches were performed. For the
oxidative modifications, Met and Trp mono- and di-oxidation as well as Tyr oxidation were
allowed. For His, 1‐16 Da modification was selected as variable modifications.
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References
1. Shu, X.; Lev-Ram, V.; Deerinck, T. J.; Qi, Y.; Ramko, E. B.; Davidson, M. W.; Jin, Y.; Ellisman, M. H.; Tsien, R. Y., A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 2011, 9 (4), e1001041.2. Wulff, J. E.; Herzon, S. B.; Siegrist, R.; Myers, A. G., Evidence for the rapid conversion of stephacidin B into the electrophilic monomer avrainvillamide in cell culture. J Am Chem Soc 2007, 129 (16), 4898-9.3. Guan, S. H.; Price, J. C.; Prusiner, S. B.; Ghaemmaghami, S.; Burlingame, A. L., A Data Processing Pipeline for Mammalian Proteome Dynamics Studies Using Stable Isotope Metabolic Labeling. Molecular & Cellular Proteomics 2011, 10 (12).