science.sciencemag.

org/content/367/6482/1091/suppl/DC1

Supplementary Material for

Microenvironment mapping via Dexter energy transfer on immune cells

Jacob B. Geri, James V. Oakley, Tamara Reyes-Robles, Tao Wang, Stefan J. McCarver,
Cory H. White, Frances P. Rodriguez-Rivera, Dann L. Parker Jr., Erik C. Hett,
Olugbeminiyi O. Fadeyi*, Rob C. Oslund*, David W. C. MacMillan*
*Corresponding author. Email: olugbeminiyi.fadeyi@merck.com (O.O.F); rob.oslund@merck.com
(R.C.O.); dmacmill@princeton.edu (D.W.C.M.)

Published 6 March 2020, Science 367, 1091 (2020)

DOI: 10.1126/science.aay4106

This PDF file includes:

Materials and Methods

Figs. S1 to S51
Table S1
References

Other Supplementary Material for this manuscript includes the following:

(available at science.sciencemag.org/content/367/6482/1091/suppl/DC1)

Data File S1
Table of Contents

SUPPLEMENTARY FIGURES ............................................................................................................... 3

GENERAL CONSIDERATIONS ........................................................................................................... 18
Synthetic methods ............................................................................................................................................ 18
General materials.............................................................................................................................................. 18
General Western blot protocol ......................................................................................................................... 19
General cell culture protocol ............................................................................................................................ 20
PHOTOCATALYST SCREENING ........................................................................................................ 21
SYNTHESIS .......................................................................................................................................... 22
Preparation of clickable photocatalyst 3........................................................................................................... 22
Synthesis of biotin-diazirine 4.......................................................................................................................... 44
Synthesis of EMARS-hexyl-Biotin .................................................................................................................. 47
Synthesis of water insertion product ................................................................................................................ 51
BIOTINYLATION: CATALYTIC DIAZIRINE SENSITIZATION AND BSA LABELING ....................... 54
Catalyst-dependent biotinylation of bovine serum albumin ............................................................................. 54
Photonic control over BSA biotinylation ......................................................................................................... 56
PREPARATION OF PHOTOCATALYST-ANTIBODY CONJUGATES ............................................... 58
PROXIMITY-SELECTIVE LABELING ON PROTEIN-FUNCTIONALIZED AGAROSE BEADS ........ 59
Targeting of VEGFR2 or EGFR2 with photocatalyst-conjugated secondary antibodies (µMap) .................... 59
Targeting of VEGFR2 and EGFR with peroxidase-conjugated secondary antibodies ..................................... 62
Irradiation time vs. selectivity for VEGFR2 targeting with µMap ................................................................... 65
Reaction time vs selectivity for VEGFR2 targeted peroxidase-based labeling ................................................ 68
Protein loading vs. selectivity with VEGFR2-targeted µMap .......................................................................... 71
Bead loading vs selectivity for VEGFR2 targeted peroxidase-based labeling ................................................. 74
Photocatalyst/antibody ratio vs VEGFR2 targeted labeling selectivity ............................................................ 77
Secondary antibody loading vs. selectivity of VEGFR2 targeted µMap.......................................................... 80
Impact of the site of photocatalyst-antibody conjugation and selectivity......................................................... 83
PROXIMITY-SELECTIVE LABELING ON CELLS ............................................................................... 86
Targeted labeling of CD45 on Jurkat cells for Western blot analysis .............................................................. 86
Confocal Microscopy Imaging of CD45 Targeting µMapping on Jurkat Cells ............................................... 87
Photolabeling on live Jurkat or JY cells for quantitative LC-MS/MS analysis ................................................ 88
Photolabeling on live A549 cells for quantitative LC-MS/MS analysis .......................................................... 94
Peroxidase labeling (1 min) on live cells for quantitative LC-MS/MS analysis .............................................. 97
Selective proteomic proximity labeling assay using tyramide (SPPLAT) method on live cells for quantitative
LC-MS/MS analysis ......................................................................................................................................... 98
Enzyme-mediated activation of radical sources (EMARS) method on live cells for quantitative LC-MS/MS
analysis ............................................................................................................................................................. 99
Protein extraction and digestion for LC-MS/MS analysis .............................................................................. 101
LC-MS/MS-based proteomic analysis of labeled cell experiments................................................................ 102
Bioinformatic analysis of mass spectrometry data ......................................................................................... 102
Correlation Analysis ....................................................................................................................................... 103
Linear modeling and fold change generation ................................................................................................. 103
Volcano plot generation ................................................................................................................................. 104
Cell receptor surface density determination ................................................................................................... 105
Analysis of IL-2 Production in Jurkat-JY Two cell system ........................................................................... 106
Flow Cytometry Analysis of CD45 on Jurkat and JY cells ............................................................................ 106
Flow Cytometry Analysis of µMapping in two-cell system ........................................................................... 107
Confocal microscopy imaging of µMapped cells ........................................................................................... 112
Data and Code availability ............................................................................................................................. 116
REFERENCES .................................................................................................................................... 117

S2
Supplementary Figures

Fig. S1. Diazirine photosensitization substrate scope.

Standard procedure for diazirine screen

Diazirine (100 µM), 4-trifluoromethylbenzoic acid (100 µM), and [Ir(dFCF3ppy)2(dtbbpy)]PF6
(2, 10 µM) were combined in 1.0 mL 1:1 DMSO:H2O and irradiated in a PennOC Photoreactor
M1 for 15 minutes using a 450 nm light source at 100% intensity. 100 µL D2O was then added
and samples removed for analysis by 19F-NMR spectroscopy. The trifluoromethyl peak in the
starting material was used for quantitation of remaining diazirine starting material against in
internal standard (4-trifluoromethyl benzoic acid). Experiments were performed in triplicate,
then averaged to provide the reported consumption percentages.

S3
Fig. S2. Absorption spectra of 1 and 2 vs. emission spectra of biophotoreactor.

A lack of overlap between the photoreactor emission and the absorption spectrum of 1,
combined with overlap between photoreactor emission and the absorption spectrum of
photocatalyst 2, explain the low background signal from direct photolysis of 1 by blue light
during µMap experiments.

Emission spectrum of PennOC Photoreactor M1 (450 nm LED):

1
0.9
0.8
Normalized Radience

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
350 400 450 500 550 600 650 700
Wavelength (nm)

S4
Wavelength spectrum of Efficiency Aggregators Biophotoreactor BPR200 (454 nm LED):
1
0.9

Normalized Radience
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
350 400 450 500 550 600 650 700
Wavelength (nm)

Photo of biophotoreactor (Efficiency Aggregators (Richland, Tx, BPR200)):

S5
Fig. S3. Absorption spectra of 1 vs. emission spectra of 2.

No overlap was observed between the absorption spectrum of 1 and the emission spectrum of
2, even at concentrations 1000 times higher than the highest used in protein labeling
experiments (100 mM). This overlap is required for Förster energy transfer, and its absence
here firmly rules out this mechanism of energy transfer. The sole alternative mechanism, Dexter
energy transfer, does not require any overlap as it operates through simultaneous two-electron
transfer with a substrate. By process of elimination we have therefore assigned the mechanism
of energy transfer between 1 and 2 as Dexter energy transfer.

S6
Table. S1. Emission quenching of 2 by 1.

[1] (mM) Abs (410 nm) Abs (480 nm) F (480 nm) F (abs. corr.) F0/F
0.0 0.048 0.004 9.7(3) × 102 1.03(2) × 103 1(0)
10.0 0.049 0.004 3.67(8) × 102 3.84(5) × 102 2.69(2)
20.0 0.050 0.001 2.14(4) × 102 2.29(3) × 102 4.5(2)
30.0 0.054 0.001 1.501(2) × 102 1.599(2) × 102 6.5(1)
40.0 0.059 0.001 1.19(3) × 102 1.25(2) × 102 8.2516(7)
50.0 0.063 0.003 9.2(3) × 101 1.02(4) × 102 10.1(6)

Solutions containing 2 (10.0 µM) and diazirine 1 (0–50 mM) were prepared in 1:1 DMSO:H2O
in 1.0 cm path-length quartz fluorescence cuvettes equipped with septum screwcaps. The
solutions were sparged for 15 minutes with N2. Emission spectra were recorded with excitation
at 410 nm, and emission intensity at the peak maximum was recorded. Experiments were
performed in duplicate and averaged. Error (as the standard deviation) in the trailing digit is
notated in parenthesis. The fluorescence quenching rate constant was calculated through
absorption-corrected Stern-Volmer analysis (using the reported lifetime of 2, 2.3 µs (44), and
correcting for the inner filter effect imposed by sample absorption at the excitation and emission
wavelengths (410 and 480 nm, respectively) (45). The calculated rate: kq(obs) = 7.9(5) × 107 M-
1 -1
s .

Formula for calculation of absorption-corrected emission intensity:

. . =

S7
Fig. S4. Stern-Volmer plot.
12

y = 181.38x + 1
10 R² = 0.9995

8
I/I0

0
0 0.01 0.02 0.03 0.04 0.05 0.06
Concentration of 1 (M)
Results: Data plotted from Table S1.

S8
Fig. S5. Example emission spectra of 10 µM 2 with changing [1].

1000
900
0 mM 10 mM
800 20 mM 30 mM
40 mM 50 mM
Emission Intensity (I)

700
600
500
400
300

200
100
0
420 470 520 570
Wavelength (nm)

Results: Increasing the concentration of 1 quenches emission from 2 in 1:1 DMSO, reflecting
an increase in nonradiative decay of the excited state of 2 likely caused by energy transfer to
1.

S9
Fig. S6. PEG-biotin-functionalized BSA mass spectrum (a), deconvoluted spectrum (b).

Results: The mass difference (619) is approximately equal to one carbene derived from N2
elimination from diazirine 4 (Exact mass: 616.2542), validating chemical tagging of BSA
through photocatalytic diazirine sensitization.

Panels: Intact Protein Analysis of BSA Biotinylation. 10 µL sample from sample 7 (see page
S54) was subjected to intact protein analysis using an Orbitrap Q-Exactive (Thermo Fisher). In
addition to the starting material observed at 66,571 (BSA theoretical MW: 66,432), a +619 Da
species appeared at 67,190 Da representing 19% of the total signal.

S10
Fig. S7. µMap labeling vs. peroxidase labeling on beads.

Results: Primary antibody targeted labeling of bead-bound VEGFR2 or EGFR with HRP-
secondary antibody conjugates was unselective. Error bars represent standard deviation (n = 3).

S11
Fig. S8. Confocal microscopy imaging of CD45-targeted µMap on Jurkat cells.

Confocal microscopy images of (top row) isotype-targeted Jurkat cells (10 minutes light
irradiation) or (bottom row) CD45-targeted Jurkat cells (10 minutes light irradiation) using
µMap. Prior to visible light irradiation, cells were maintained at 4 °C during antibody coating
steps to reduce protein aggregation on the membrane surface. Cells were imaged for
biotinylation (green) and nuclei (blue). Scale bar: 5 µm. Five replicate images are shown for
each condition.

S12
Fig. S9. Peroxidase-based targeted labeling of CD45 (1min) on Jurkat cells.

Volcano plot of LC-MS/MS analysis of proteins enriched from targeted labeling of CD45 using
peroxidase for 1 min at 4 °C. The relative protein fold change from CD45-targeted samples vs
isotype samples are plotted on the x axis as averaged log2 ratios across 3 replicates. On the y
axis are plotted the corresponding negative log10 transformed p values. In purple are proteins
with > 2.5-fold enrichment and p < 0.05 (Benjamini-Hochberg FDR-corrected moderated t
statistic). CD45 (PTPRC), CD47, and CD29 (ITGB1) are colored in green. This result shows
that performing peroxidase-based proximity labeling of CD45 (PTPRC) at 4 °C for 1 minute
does not significantly enrich CD45 (PTPRC) from CD47 and CD29 (ITGB1).

S13
Fig. S10. Flow cytometry analysis of JY-PD-L1 and Jurkat-PD-1 cells for CD45, CD45RA,
and CD45RO expression.

JY-PD-L1

Jurkat PD1

Flow cytometry analysis of CD45 surface expression on Jurkat-PD-1 and JY-PD-L1 cells used
for two-cell labeling. Histograms show Isotype (gray), CD45 (blue), CD45RA (purple), and
CD45RO (green) surface expression levels. CD45RO surface expression (right panels) was
selectively detected on Jurkat cells over JY cells.

S14
Fig. S11. Effect of staphylococcal enterotoxin D on cell activation and transient or stable
synaptic biotinylation.

a) JY-PD-L1 and Jurkat-PD-1 cells were co-cultured in the presence or absence of

staphylococcal enterotoxin D (SED) for 24 hours followed by analysis of IL-2 production to
test for the both the requirement of SED to induce T cell activation and the ability of the PD-
1/PD-L1 interaction to disrupt this effect. Bar plots of IL-2 production (as measured by an
increase in OD450) confirm that only the combined use of SED and PD-1/PD-L1 disruption
(through α-PD-L1 antibody-mediated blockade) results in increased IL-2 production. Error bars
represent standard deviation of n = 3 experiments. b) Flow cytometry analysis of PD-L1-
targeted µMap in the JY-PD-L1 and Jurkat-PD-1 co-culture system in the presence of SED with
2.5 hour incubation at 37 °C (+SED) or in the absence of SED (-SED) without 2.5 hour
incubation (10 min light irradiation) shows similar transcellular biotinylation on Jurkat-PD-1
(CD3+) cells (top panels) in both +SED and –SED conditions (Note that Jurkat and JY cells
were co-mixed by pelleting prior incubation). c) Bar plots of replicate analysis of Jurkat
transcellular biotinylation measured by flow cytometry in panel b for Jurkat-PD-1 cells in the
presence (blue bars) or absence (orange bars) of SED. Error bars represent standard deviation
of n = 3 experiments. Confocal microscopy images of PD-L1-targeted µMap of Jurkat-JY two
cell system in the d) presence of SED (+SED) or e) absence of SED (-SED). Cells were imaged
for CD3 surface expression (magenta), biotinylation (green), and nuclei (blue). Scale bar: 5 μm.
Three replicate images are shown for each condition.

S15
Fig. S12. µMap vs peroxidase: two cell cis/trans labeling flow cytometry quantitation.

a) Bar plots of replicate analysis of cell biotinylation measured by flow cytometry in Figures
4b and 4c. Error bars represent standard deviation of n = 4 experiments. These data reflect the
ability of µMap to selectively trans-label only when targeted to a protein present in the
intercellular synapse. b) Pseudocolor flow cytometry plots of Figure 4b. c) Pseudocolor flow
cytometry plots of Figure 4c.

S16
Fig. S13. PD-1-targeted µMap in Jurkat-JY two cell system.

a) Flow cytometry analysis of µMap of PD-1 on Jurkat-PD-1 cells in the Jurkat-JY two cell
system using 10 min light irradiation shows cis-labeling on Jurkat-PD-1 (CD3+) cells and
trans-cellular labeling on JY-PD-L1 (CD19+) cells. b) Bar plots of replicate analysis of cell
biotinylation measured by flow cytometry in panel a for Jurkat-PD-1 cells (orange bars) and
JY-PD-L1 cells (blue bars). Error bars represent standard deviation of n = 3 experiments. Two
cell system confocal microscopy images of c) isotype-targeted (10 minutes light irradiation) or
d) PD-1-targeted (10 minutes light irradiation) using μMap shows both cis- and synaptic-cell
biotinylation. Cells were imaged for biotinylation (green), CD3 surface expression (magenta),
and nuclei (blue). Scale bar: 5 μm. For each condition, two replicate microscopy images (top
and bottom) are shown.

S17
General Considerations
Synthetic methods
Organic solvents were purified according to the method of Grubbs (46). Water was purified
using a Millipore Milli-Q Integral Water Purification System. Organic solutions were
concentrated under reduced pressure on a Büchi rotary evaporator using a water bath. 1H NMR
spectra were recorded on a Bruker UltraShield Plus Avance III 500 MHz unless otherwise noted
and are internally referenced to residual solvent signals. Data for 1H NMR are reported as
follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
p = quintet, m = multiplet, dd = doublet of doublets, dt = doublet of triplets…etc, br = broad),
coupling constant (Hz) and integration. Irradiation of samples was performed in either a
PennOC Photoreactor for small-molecule sensitization experiments (PennOC, Pennsburg, PA,
Model M1) or a Biophotoreactor (Efficiency Aggregators (Richland, Tx, Fisher, NC1558343
BPR200). Spectral analysis of light sources was performed using a UPRtek MK350 handheld
spectrometer. Chromatographic purification was carried out using a Biotage Isolera Prime flash
chromatography system with SilaSep flash cartridges (60 mesh) and UV detection. 13C NMR
spectra were recorded on a Bruker UltraShield Plus Avance III 500 MHz (125 MHz) and data
are reported relative to the solvent employed. IR spectra were recorded on a Perkin Elmer
Spectrum 100 FTIR spectrometer and are reported in wavenumbers (cm-1). High resolution
mass spectra and intact protein mass spectra were obtained from the Princeton University Mass
Spectral Facility and Princeton Proteomics & Mass Spectrometry Core.

General materials
All buffers and synthetic starting materials were used as received from commercial sources.
Ascorbic acid (BP321-500) and Ethanol (BP2818100) were purchased from Fisher Scientific
(Pittsburgh, PA). Bovine Serum Albumin (BSA) (A7906), Eppendorf Protein LoBind tubes
(Z666505), goat α-human IgG agarose beads (A3316) and Trolox (238813) were purchased
from Sigma-Aldrich (St. Louis, MO). Sodium Azide (14314) was purchased from Alfa Aesar
(Haverhill, MA). Biotin phenol (LS-3500.1000) was purchased from Iris Biotech GMBH.
RIPA Buffer (89900), Click-iT Protein Reaction Buffer Kit (C10276), 1X DPBS (14190144),
Pierce BCA Protein Assay Kit (23227), Mouse α-human EGFR (MA5-13070), and iBright
Prestained Protein ladder (LC5615) were purchased from Thermo Scientific (Rockford, IL).
TBST (IBB-581X) was purchased from Boston BioProducts (Ashland, MA). 5M Sodium
Chloride (S24600-500.0) was purchased from Research Products International (Mt. Prospect,

S18
IL). 12% Criterion TGX precast gels (5671044) and 4x Laemmli sample buffer (161-0747)
were purchased from Bio-Rad (Hercules, CA). Goat α-Mouse secondary antibody (AP124) and
peroxidase conjugated goat α-Mouse secondary antibody (AP124P) were purchased from
Millipore (Billerica, MA). 20% SDS solution (351-066-721) was purchased from Quality
Biological (Gaithersburg, MD). rhVEGFR2/Fc (357-KD) was purchased from Frontier
Scientific. rhEGFR/Fc (344-ER) was purchased from R&D Systems. Mouse α-human
VEGFR2 (359902) was purchased from BioLegend. Mouse IgG1 κ isotype (556648) was
purchased from BD Biosciences.
Jurkat NF-κB GFP cells (TR850A-1) were purchased from System Biosciences (Palo Alto,
CA). JY-wt and JY-PD-L1 cells (47) were a gift from Rene De Waal Malefyt and Sabine Le
Saux (MRL, Merck & Co., Inc., Palo Alto, CA, USA). Jurkat PD-1 cells (48) were a gift from
Aaron Willingham and Bhagyashree Bhagwat (Merck & Co., Inc., Palo Alto, CA, USA). A549
cells (CCL-185) were purchased from ATCC.

General Western blot protocol

Gel electrophoresis was performed using a Bio-Rad Criterion Vertical Electrophoresis Cell
tank, Bio-Rad PowerPac Basic Power Supply, and Criterion TGX tris-glycine polyacrylamide
gel cassettes (SDS/Tris). After electrophoresis, gels were transferred from precast cassettes to
nitrocellulose or PVDF membranes using an iBlot 2 gel transfer device (Thermo Fisher,
IB21001, IB23001), and washed with water. The membranes were then immersed in REVERT
total protein stain (Li-Cor, 926-11011) for 5 minutes. Excess stain was decanted, membranes
washed with 6.7:30:63.3 AcOH:MeOH:H2O and imaged using a Li-Cor Odyssey CLx scanner
in the 700 nm channel. The membranes were washed with water, then immersed in Odyssey
Blocking Buffer (Li-Cor, 927-50000) and incubated for 1 hour. The blocking solution was then
decanted, and 35 mL of fresh blocking buffer containing 70 µL of Tween 20 was added. This
mixture was rocked for 5 minutes. Afterwards, 1.5 µL of IRDye 800CW streptavidin (Li-Cor,
926-32230) was added and the mixture incubated for 1 hour. The blocking buffer was then
decanted, and the membranes were washed with 1X TBST (3 x 5 min) and water before imaging
via Li-Cor Odyssey CLx scanner in the 800 nm channel. Pixel densitometry was performed
using Image Studio Lite V. 5.2 (Li-Cor). The streptavidin 800 channel pixel density was then
divided by the total protein stain 700 channel pixel density to provide a normalized biotinylation
signal for each protein band.

S19
General cell culture protocol
Jurkat NF-κB GFP cells were grown in Roswell Park Memorial Institute (RPMI) 1640 1X
medium containing L-Glutamine (Corning, 10-040-CV), 10% fetal bovine serum (FBS)
(HyClone, SH30910.03) and 100 IU Penicillin/100µg/mL Streptomycin (1X from a 100X
stock, Corning, 30-002-CI).

JY wildtype (wt) and JY PD-L1 cells were grown in RPMI 1640 1X with L-glutamine
(Corning, 10-040-CV), 10% fetal bovine serum (FBS) (HyClone, SH30910.03), 100 IU
Penicillin/100 µg/mL Streptomycin (1X from a 100X stock, Corning, 30-002-CI), 2 mM L-
Glutamine (Lonza, 17-605E), 1X MEM Non-Essential Amino Acid Solution (MEM NEAA,
Sigma, M7145-100ML), and 1 mM Sodium Pyruvate (Cellgro, 25-000-CI).

Jurkat PD-1 cells were grown in RPMI 1640 1X with L-glutamine (Corning, 10-040-CV),
10% fetal bovine serum (FBS) (HyClone, SH30910.03), 1X MEM Non-Essential Amino
Acid Solution (MEM NEAA, Sigma, M7145-100ML), 1 mM Sodium Pyruvate (Cellgro, 25-
000-CI), 10mM HEPES Buffer (Fisher, BP299-100), 500 μg/mL Geneticin (Thermo Fisher
Scientific, 10131-035) and 20 ng/mL Puromycin (Thermo Fisher Scientific, A11138-03).

A549 cells were grown in DMEM (Gibco, 10569-010), 10% fetal bovine serum (FBS)
(HyClone, SH30910.03), 100 IU Penicillin/100 µg/mL Streptomycin (1X from a 100X stock,
Corning, 30-002-CI).

After preparation, all cell culture media was sterilized using 0.2 µm Nalgene™ Rapid-Flow™
Sterile Disposable Filter Units with PES Membrane, 1,000 mL capacity (Thermo Scientific,
567-0020) or 500 mL capacity (Thermo Scientific, 569-0020) as needed and stored at 4 °C until
ready to use.

All cells were grown at 37 °C with 5% CO2 in 25cm2 (Corning: 430639), 75cm2 (Corning:
430641U) or 150cm2 (Corning: 430825) canted neck, vented cap sterile cell culture flasks (for
Jurkat NF-κB , Jurkat PD-1, JY PD-L1, and JY wt), or 10-cm (Falcon: 353003), 150-cm
(Falcon: 168381) or 6-well plates (Falcon: 353046) for A549 cells as needed. For passaging of
adherent cells, cells were detached from the plate using TrypLE Express (Gibco, 12604-021)
as indicated.

S20
Photocatalyst Screening
N N H OH
Photocatalyst (10 mol %)
CF3 CF3
1:1 DMSO:H2O
ClH3N 450 nm LEDs, 15 min ClH3N

1 2

Tabulated properties of screened photocatalysts:

Catalyst Conversion ET E1/2(ox)*(V) E1/2(red) (V) τ (ns)

(kcal)
[Ir(dF(CF3)ppy)2(dtbbpy)]PF6 (2) 100% 60.1 1.21 -1.37 2300
[Ir(dF(CF3)ppy)2(bpy)]PF6 100% 60.4 0.97 -1.23 2280
Ir(4’-F-ppy)3 0% 58.6 0.68 -2.18 2040
Ir(ppy)3 0% 55.2 0.31 -2.20 1900
[Ru(bpz)3](PF6)2 0% 48.4 1.45 -0.80 740
[Ru(bpy)3](PF6)2 0% 46.5 0.77 -1.33 1100
[Ru(4,4’-dm-bpy)3](PF6)2 0% 45.3 0.22 -1.43 875
(3) 100% - - - -

Physical data regarding photocatalysts adapted from Teegardin, et. al. Org. Proc. Res. Dev. 20,
1156-1163 (2016) (42)

Control experiments
Catalyst Deviation from standard conditions Conversion
none none 0%
[Ir(dF(CF3)ppy)2(dtbbpy)]PF6 0% light intensity 0%
(3) 0% light intensity 0%

Standard procedure for photocatalyst screen:

A solution containing diazirine 1 (100 µM), 4-trifluoromethylbenzoic acid internal standard
(100 µM), and photocatalyst (10 µM) was prepared in 1 mL 1:1 DMSO:H2O in an 8-mL
scintillation vial equipped with a septum screw-cap. After sparging with N2 for 15 minutes, the
mixture was irradiated in a PennOC Photoreactor M1 for 15 minutes using a 450 nm light
source at 100% intensity. Following irradiation, 100 µL of D2O was added to each vial and
samples were removed for 19F-NMR spectroscopy; conversion of diazirine 1 (δ -64.61 (3F, S))
was measured against internal standard (δ -61.35 (3F, S)). Experiments were performed in
triplicate and averaged to provide the reported consumption percentages; the presence of
carbinol product was validated through comparison with an authentic sample.

S21
Synthesis
Preparation of clickable photocatalyst 3

2,2'-([2,2'-bipyridine]-4,4'-diyl)bis(propan-2-ol) (5)

(Step 1) Bipyridine dicarboxylic acid dimethyl ester (11.98 g, 44 mmol) was dissolved in 500
mL anhydrous THF under nitrogen in a 1 L round bottom flask, then cooled to -78 °C.
Methylmagnesium bromide (90.3 mL, 271 mmol) was rapidly added to the flask with vigorous
stirring (5 cm stir bar). After continuing to stir the solution for 2 hours at room temperature, the
suspension was quenched with saturated aq. NH4Cl (ca. 100 mL), evaporated to dryness,
extracted into ethyl acetate (500 mL from 500 mL H2O), washed with brine (ca. 100 mL), dried
over MgSO4, the solvent evaporated, and the residual solid recrystallized from boiling ethyl
acetate / hexanes (75 mL EtOAc, 400 mL hexanes) to afford an off-white crystalline solid (9.0
g, 75%), which possessed spectral properties matching the reported values (49).

S22
2-(4'-(2-methoxypropan-2-yl)-[2,2'-bipyridin]-4-yl)propan-2-ol (6)

5 6

(Step 2) 5 (4.62 g, 17 mmol) was dissolved in THF (17 mL) in a septum-equipped 40 mL vial.
NaH (60% mineral oil dispersion, 350.4 mg NaH basis, 15.3 mmol) was carefully added with
stirring, then the mixture stirred for 1 hour at 25 °C while vented to a bubbler through a needle
puncturing the septum. Methyl iodide (0.84 mL, 13.6 mmol) was then added, the septum
replaced with a cap, and the reaction mixture heated to 80 °C for 16 hours. The mixture was
then quenched with saturated aq. NH4Cl (10 mL), diluted with water (100 mL), the product
extracted from the mixture with DCM (4 x 50 mL), the organic phase dried with MgSO4, and
solvent evaporated. The residue was purified by silica chromatography (40% ethyl acetate to
100% ethyl acetate / hexanes over 9 CV, 330 g SiO2, 180 mL/min) to afford 6 as a tan crystalline
solid (1.74 g, 35%).

1H-NMR (500 MHz, CDCl3): δ 8.69 (d, 1H, J = 0.9 Hz), 8.69 (d, 1H, J = 1.0 Hz), 8.50 (d, 1H,
J = 1.3 Hz), 8.42 (d, 1H, J = 1.2 Hz), 7.49 (dd, 1H, J = 5.1, 1.8 Hz), 7.43 (dd, 1H, J = 5.1, 1.8
Hz), 3.17 (s, 3H), 1.97 (s, 1H), 1.66 (s, 6H), 1.61 (s, 6H).

13C-NMR (126 MHz, CDCl3): δ 158.9, 156.5, 156.4, 156.4, 149.4, 149.3, 121.0, 119.7, 118.4,
117.0, 76.5, 72.3, 51.0, 31.3, 27.4.

IR (film): νmax 3393, 2967, 1587, 1548, 1461, 1367, 1353, 1287, 1251, 1229, 1169, 1149, 1109,
1096, 1058, 992, 962, 931, 909.

HRMS (ESI-TOF): m/z calcd. for C17H23N2O2 ([M+H]+) 287.1760, found 287.1727.

S23
6: 1H-NMR

6: 13C-NMR

S24
13,13,14,14-Tetramethyl-3,6,9-trioxa-13-silapentadec-11-yn-1-yl Tosylate (7)

Compound 7 was provided as a gift from Merck & Co., Inc., Kenilworth, NJ, USA and
was used as received.

1H-NMR (500 MHz, CDCl3): δ 7.81 (d, 2H, J = 8.4 Hz), 7.35 (d, 2H, J = 8.1 Hz), 4.20 (s, 2H),
4.16 (m, 2H), 3.68 (m, 6H) 3.59 (s, 4H), 2.45 (s, 3H), 0.93 (s, 9H), 0.10 (s, 6H)

13C-NMR (126 MHz, CDCl3): δ 144.8, 133.0, 129.8, 128.0, 102.0, 89.7, 70.7, 70.5, 70.4, 69.2,
68.8, 68.7, 59.1, 26.0, 21.6, 16.4, -4.6.

IR (film): νmax 2952, 2929, 2858, 2173, 1598, 1462, 1355, 1292, 1250, 1189, 1176, 1096, 1009,
989, 918.

HRMS (ESI-TOF): m/z calcd. for ([M+Na]+) C22H36NaO7SSi 479.1894, found 479.1887.

S25
7: 1H-NMR

7: 13C-NMR

S26
4-(2-Methoxypropan-2-yl)-4'-(2,16,16,17,17-pentamethyl-3,6,9,12-tetraoxa-16-silaoctadec-
14-yn-2-yl)-2,2'-bipyridine (8)

(Step 3) To a flame-dried 40 mL vial under nitrogen gas equipped with a magnetic stir bar was
added 5 (286 mg, 1 mmol) and anhydrous THF (10 mL). NaH (46 mg, 2 mmol) was then added,
and the reaction mixture stirred at room temperature for 2 hours. Then, 7 (456 mg, 1 mmol)
was added via syringe, and the reaction mixture stirred at 60 °C for 16 h. The reaction solution
was then evaporated onto silica and purified via column chromatography (120 g SiO2, 10 to
100 % EtOAc / Hexanes over 12 CV, 50 mL/min) to afford the product 8 as a colorless oil (230
mg, 40%).

1H-NMR (500 MHz, CDCl3): δ 8.65 (ddd, 2H, J = 4.9, 3.9, 0.8 Hz), 8.39 (s, 2H), 7.46 (dd, 1H,
J = 5.1, 1.8 Hz), 7.40 (dd, 1H, J = 5.1, 1.8 Hz), 4.21 (s, 2H), 3.69 (s, 4H), 3.67 (s, 4H), 3.65 (t,
2H, J = 5.2 Hz), 3.39 (t, 2H, J = 5.2 Hz), 3.14 (s, 3H), 1.60 (s, 6H), 1.59 (s, 6H), 0.92 (s, 9H),
0.10 (s, 6H).

13C-NMR (126 MHz, CDCl3): 156.6, 156.5, 156.5, 156.3, 149.3, 149.3, 121.1, 120.9, 118.4,
118.3, 102.0, 86.6, 76.5, 70.8, 70.7, 70.6, 70.4, 68.9, 62.6, 59.1, 51.0, 27.8, 27.4, 26.0, 16.5, -
4.7.

IR (film): νmax 2978, 2929, 2858, 2172, 1588, 1549, 1461, 1381, 1364, 1280, 1250, 1170, 1097,
1072, 1033, 1008, 991, 938.

HRMS (ESI-TOF): m/z calcd. for C32H51N2O5Si ([M+H]+) 571.3567, found 571.3537.

S27
8: 1H-NMR

8: 13C-NMR

S28
2-(2,4-difluorophenyl)-5-(trifluoromethyl)isonicotinic acid (9)

(Step 4) To a 500 mL RB flask equipped with a magnetic stir bar was added 2-chloro-5-
(trifluoromethyl)isonicotinic acid (11.28 g, 50 mmol), (2,4-difluorophenyl)boronic acid (9.47
g, 60 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane
complex (817 mg, 1 mmol) and dioxane (150 mL). Cesium carbonate (57 g, 175 mmol) was
added as an aqueous solution (50 mL total volume) at room temperature with stirring. The
mixture was degassed via bubbling with N2 for 20 min and refluxed for 12 hours. After cooling
to room temperature, the reaction mixture was concentrated to remove the dioxane and
partitioned between EtOAc and brine. The aqueous phase was extracted 3 times with EtOAc.
The combined organic phase was concentrated and purified by column chromatography
(hexanes:EtOAc:MeOH = 50:50:1) to give 2-(2,4-difluorophenyl)-5-
(trifluoromethyl)isonicotinic acid (9, 12.13 g, 40 mmol, 80% yield) as a white solid.

1H-NMR (500 MHz, CD3OD): δ 9.10 (1H, s), 8.18 (m, 2H), 7.19 (m, 2H).

13C-NMR (126 MHz, CD3OD): δ 166.2, 164.2 (dd, J = 252.1, 12.4 Hz), 161.2 (dd, J = 265.7,
12.4 Hz), 156.6, 147.4 (q, J = 5.8 Hz), 140.7, 132.4 (dd, J = 10.1, 3.8 Hz), 123.1 (q, J = 272.7
Hz), 122.9 (d, J = 10.8 Hz), 121.7 (dd, J = 11.1, 3.8 Hz), 121.3 (q, J = 34.0 Hz), 111.9 (dd, J =
21.7, 3.7 Hz), 104.2 (t, J = 26.6 Hz).

19F-NMR (478 MHz, CD3OD): δ -60.8 (s, 3F), -108.5 (m, 1F), -113.3 (m, 1F).

IR (film): νmax 2477, 1888, 1726, 1609, 1557, 1515, 1482, 14344, 1315 ,1284, 1245, 1169,
1145, 1109, 1061, 1038, 968, 951, 918.

HRMS (ESI-TOF): m/z calcd. for C13H7F5NO2 ([M+H]+) 304.0397, found 304.0364.

S29
9: 1H-NMR

9: 13C-NMR

S30
9: 19F-NMR

S31
Compound 10

(Step 5) IrCl3•H2O (2.98 g, 10 mmol) and 9 (6.66 g, 22 mmol) were combined in a 500 mL, 3
necked round bottom flask equipped with a large stirbar, an N2 inlet port, a septum-capped
reflux condenser, and a septum. A bubbler was connected to the top of the reflux condenser
through a needle, and the solution as heavily sparged for 30 minutes. After sparging, the N2
manifold was connected to the top of the reflux condenser through a needle and a constant flow
through the bubbler maintained. The reaction mixture was then heated to 135 °C for 12 hours.
The reaction mixture was then allowed to cool to room temperature, and poured into 1 L H2O.
The suspension was heated to 90 °C for 10 minutes, then filtered hot. The filter cake was then
dried, suspended in 1 liter of boiling ethyl acetate, and filtered to afford a red homogeneous
solution. This solution was then concentrated through distillation at atmospheric pressure to a
volume of 150 mL, cooled to -20 °C for 1 hour, the precipitated solid recovered by filtration,
and then the solid dried under high vacuum for 48 hours to afford the product 10•(1/3 EtOAc)
as a crystalline red solid (2.08 g, 25%).

1H-NMR (500 MHz, CD3OD): δ 10.35 (1H, s), 9.23 (1H, s), 8.67 (1H, s), 8.66 (1H, s), 6.67
(t, 1H, J = 10.8 Hz), 6.57 (t, 1H, J = 10.2 Hz), 5.75 (d, 1H, J = 8.4 Hz), 5.56 (d, 1H, J = 8.7
Hz).

13C-NMR (126 MHz, CD3OD): δ 169.3, 167.8, 165.1, 165.0, 164.8 (dd, J = 260.5, 13.2 Hz),
163.7 (dd, J = 258.0, 12.7 Hz), 162.3, (dd, J = 261.2, 13.9 Hz), 161.8 (dd, J = 259.4, 12.9 Hz),
153.5 (d, J = 7.4 Hz), 149.5 (q, J = 6.2 Hz), 146.3 (m), 144.9 (d, J = 8.8 Hz), 142.3, 141.4,
126.7, 126.3, 122.2 (q, J = 279.3 Hz), 122.0-121.2 (m), 114.1 (d, J = 21.1 Hz), 98.3 (t, J = 27.3
Hz), 97.5 (t, J = 26.8 Hz).

S32
19F-NMR (376 MHz, CD3OD): δ -60.8 (s, 3F), -60.8 (s, 3F), -105.0 (m, 1F), -205.8 (m, 1F, -
109.1 (m, 1F), -110.2 (m, 1F).

IR (film): νmax 2955, 88, 1724, 1602, 1575, 1540, 1494, 1452, 1420, 1376, 1298, 1249, 1158,
1112, 1065, 1041, 994, 948, 912.

HRMS (ESI-TOF): m/z calcd. for IrC30H16F10N4O4 ([M/2 – Cl + 2 MeCN]+) 879.0630, found
879.0698.

S33
10: 1H-NMR

10: 13C-NMR

S34
10: 19F-NMR

S35
Compound 11

(Step 6) 10 (0.998 g, 0.6 mmol) was combined with AgOTf (316 mg, 1.2 mmol) in 15 mL
acetonitrile in a 40 mL vial. This mixture was stirred at room temperature overnight in the dark.
The resulting suspension was then filtered through celite and concentrated. The residue was
redissolved in 1:1 DCM:MeOH (5 mL), filtered through celite, and concentrated to yield the
product 11 as a yellow powder that was used without further purification (1.21 g, 98%).

19F-NMR (376 MHz, CD3OD) stereoisomeric mixture: δ -61.0 (m, 6F; stereoisomers at -60.8
and -61.5), -80.1 (s, 3F), -103.5 (m, 2F; stereoisomers at -103.4 and -103.9), -108.0 (m, 2F;
stereoisomers at -108.0 and -108.6).
IR (film): νmax 2938, 1737, 1604, 1576, 1538, 1494, 1421, 1299, 1219, 1142, 1110, 1064, 1027,
991, 924.
HRMS (ESI-TOF): m/z calcd. for IrC30H16F10N4O4 ([M]+) 879.0630, found 879.0682.

S36
11 19F-NMR

S37
Compound 12

(Step 7) To an 8 mL vial was added 8 (57 mg, 0.1 mmol) and 11 (77.2 mg, 0.075 mmol). The
mixture was then stirred at room temperature for 16 hours, the solution evaporated onto silica
gel (4 g) and purified by chromatography (1 to 25% MeOH/DCM over 12 CV, 120 g SiO2, 50
mL/min) to afford the product 12 as a yellow powder (90.2 mg, 66%).

1H-NMR (500 MHz, CD3OD): 8.89 (s, 1H), 8.83 (s, 1H), 8.39 (s, 2H), 8.10 (d, 1H, J = 2.6
Hz), 8.09 (d, 1H, J = 2.6 Hz), 7.93 (dd, 1H, J = 5.8, 1.8 Hz), 7.82 (dd, 1H, J = 5.8, 1.8 Hz), 7.50
(s, 2H), 6.80 (ddd, 2H, J = 12.0, 9.1, 2.3 Hz), 5.88 (dt, 2H, J = 8.5, 2.7 Hz), 4.13 (s, 2H), 3.63
(m, 10H), 3.50 (t, 2H, J = 4.4 Hz), 3.25 (s, 3H), 1.68 (s, 3H), 1.67 (s, 3H), 1.66 (s, 3H), 1.65 (s,
3H), 0.96 (s, 9H), 0.11 (s, 6H).

13C-NMR (126 MHz, CD3OD): 169.3, 167.5, 164.7 (dd, J = 259.1, 12.6), 162.5 (dd, J = 253.7,
11.9 Hz), 161.9, 161.8, 156.0, 155.7, 154.9 (t, J = 7.1 Hz), 151.1, 150.7, 145.5, 126.6, 126.4
(m), 126.1, 125.2, 123.0, 122.3 (d, J = 21.7 Hz), 122.3 (d, J = 21.7 Hz), 121.9 (q, J = 263.9
Hz), 121.2–120.7 (m), 113.7 (d, J = 18.6 Hz), 99.1 (t, J = 27.8 Hz), 88.7, 76.5, 76.4, 70.3, 70.2,
70.1, 69.9, 68.5, 62.7, 58.2, 49.9, 28.1, 26.5, 26.2, 25.7, 25.0, 15.8, -5.9.

19F-NMR (376 MHz, CD3OD): δ -61.9 (s, 3F), -62.0 (s, 3F), -104.7 (dt, 2F, J = 20.0, 9.8

IR (film): νmax 2978, 2929, 2858, 2172, 1588, 1549, 1461, 1381, 1364, 1280, 1250, 1170, 1097,
1072, 1033, 1008, 991, 938.

HRMS (ESI-TOF): m/z calcd. for IrC58H60F10N4O9Si ([M+H]+) 1367.3599, found 1367.3581.

S38
12: 1H-NMR

12: 13C-NMR

S39
12: 19F-NMR

S40
Compound 3

12 (41 mg, 0.03 mmol) was suspended in 3 mL anhydrous acetonitrile in an 8 mL vial and
sonicated for one minute. To this mixture was added TBAF (60 µL, 1.0 M in THF) at room
temperature. The reaction mixture was stirred for 16 hours at 30 °C. Mass spectroscopy showed
full conversion of the starting material. The reaction mixture was then concentrated and purified
with normal phase column chromatography (DCM/MeOH, 2–40% over 15 CV, 80 g SiO2, 0.5
CV/min) to afford 3 (36.4 mg, 97% yield) as a bright yellow solid.

1H-NMR (500 MHz, CD3OD): δ 8.89 (1H, d, J = 1.8 Hz), 8.84 (1H, d, J = 1.9 Hz), 8.39 (2H,
s), 8.10 (2H, dd, J = 5.8, 4.3 Hz), 7.96 (1H, dd, J = 5.8, 1.8 Hz), 7.82 (1H, dd, J = 5.8, 1.8 Hz),
7.51 (2H, s), 6.80 (2H, ddt, J = 11.7, 9.0, 2.4 Hz), 5.86 (2H, dt, J = 8.2, 2.7 Hz), 4.06 (2H, d, J
= 2.4 Hz), 3.69 (4H, m), 3.62 (2H, m), 3.57 (4H, m), 3.49 (2H, m), 3.25 (3H, s), 2.71 (1H, t, J
= 2.4 Hz), 1.69 (3H, s), 1.67 (3H, s), 1.66 (3H, s), 1.66 (3H, s).

13C-NMR (126 MHz, CD3OD): δ 169.4, 167.5, 164.8 (ddd, J = 259.6, 12.5, 3.7 Hz), 162.5 (dd,
J = 263.3, 13.2 Hz), 161.8, 156.0, 155.7, 155.0, 151.1, 150.8, 150.7, 145.5 (m), 126.8, 126.5
(m), 126.1, 122.4 (d, J = 21.5 Hz), 121.9 (q, J = 278.3 Hz), 121.1, 121.0 (d, J = 20.6 Hz), 120.9
(d, J = 22.2 Hz), 120.8 (q, J = 33.3 Hz), 113.7 (d, J = 18.0 Hz), 99.1 (t, J = 27.1 Hz), 79.1, 76.5,
76.4, 74.4, 70.2, 70.2, 70.1, 69.9, 68.7, 62.8, 57.5, 54.6, 49.8, 28.1, 26.7, 26.1, 25.7, 25.7.

19F-NMR (376 MHz, CD3OD): δ -60.5 (3F, s), -60.6 (3F, s), -103.4 (q, J = 9.8 Hz), -106.9 (dt,
J = 23.5, 12.3 Hz).

IR (film): νmax 2935, 1601, 1574, 1535, 1486, 1411, 1354, 1308, 1242, 1165, 1141, 1108, 1064,
990, 892, 829, 789, 722, 700.

HRMS (ESI-TOF): m/z calcd. for IrC52H46F10N4O9 ([M+H]+) 1253.2734, found 1253.2775.

S41
3: 1H-NMR

3: 13C-NMR

S42
3: 19F-NMR

S43
Synthesis of biotin-diazirine 4

To a solution of diazirine amine (100 mg, 0.397 mmol) in DMF (2 mL) was added Biotin-
PEG3-NHS ester (216 mg, 0.397 mmol) and diisopropylethylamine (0.138 mL, 0.795 mmol)
in an 8 mL vial. The mixture was stirred at room temperature overnight. The mixture was dried
with a stream of nitrogen and purified by silica chromatography using DCM: MeOH (0 to 50%)
to afford biotin diazirine 4 as a white solid (150 mg, 62%).

1H-NMR (500 MHz, DMSO-d6): 8.44 (t, 1H, J = 6.0 Hz), 7.84 (t, 1H, J = 5.7 Hz), 7.39 (d,
2H, J = 8.3 Hz), 7.25 (d, 2H, J = 8.0 Hz), 6.42 (s, 1H), 6.36 (s, 1H), 4.30 (m, 3H), 4.12 (m, 1H),
3.63 (t, 2H, J = 6.3 Hz), 3.50 (m, 8H), 3.39 (t, 2H J = 6.0 Hz), 3.18 (m, 2H), 3.09 (ddd, 1H, J
= 8.5, 6.2, 4.3 Hz), 2.81 (dd, 1H, J = 12.4, 5.1 Hz), 2.57 (d, 1H, J = 12.5 Hz), 2.06 (t, 2H, J =
7.5 Hz), 1.60 (ddt, 1H, J = 12.3, 9.4, 6.1 Hz), 1.49 (m, 3H), 1.29 (m, 4H).

13C-NMR (126 MHz, DMSO-d6): δ 173.2, 172.5, 170.7, 163.1, 142.7, 130.1, 128.4, 127.2,
126.9, 126.3, 122.4 (q, J = 274.8 Hz), 70.1, 70.1, 69.6, 67.2, 61.4, 59.6, 55.8, 42.0, 38.8, 36.5,
35.5, 28.6, 28.5, 25.7, 25.7.

19F-NMR (376 MHz, DMSO-d6): δ -64.7 (s, 3F).

IR (film): νmax 3285, 2923, 1704, 1641, 1550, 1461, 1344, 1231, 1183, 1139, 1052, 938, 857,
805.

HRMS (ESI-TOF): m/z calcd. for C28H40F3N6O6S ([M+H]+) 645.2682, found 645.2693.

S44
4: 1H-NMR

4: 13C-NMR

S45
4: 19F-NMR

S46
Synthesis of EMARS-hexyl-Biotin

EMARS-hexyl-NBoc ((6-(4-azido-2-hydroxybenzamido)hexyl)carbamate)

In an 8 mL vial was added 6.0 mL of DCM, azido-salicylic acid (53. 0 mg, 0.30 mmol), and
triethylamine (206 μL, 1.47 mmol). Then, N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide
hydrochloride (85.0 mg, 0.44 mmol) and hydroxybenzotriazole hydrate (59.9 mg, 0.44 mmol)
were added and the mixture was let to stir at room temperature for 20 minutes before the
addition of tert-butyl (6-aminohexyl)carbamate (76.8 mg, 0.35 mmol). This mixture was stirred
for 12 hours, and the mixture was purified via normal phase column chromatography
(DCM/MeOH, 0–5%) to give tert-butyl (6-(4-azido-2-hydroxybenzamido)hexyl)carbamate
(30.4 mg, 37% yield) as a white solid.
1H NMR (500 MHz, CD3OD): δ 12.90 (s, 1H), 7.56 (d, 1H, J = 8.5 Hz), 6.96 (s, 1H), 6.65 (d,
1H, J = 2.3 Hz), 6.51 (d, 1H, J = 8.7 Hz), 4.61 (s, 1H), 3.44 (d, 2H, J = 6.4 Hz), 3.18 (d, 2H, J
= 6.6 Hz), 1.54–1.37 (m, 17 H).

13C NMR (125 MHz, CD3OD): δ 169.4, 163.1, 156.4, 145.5, 127.3, 111.4, 109.7, 108.0, 79.3,
39.3, 38.5, 30.1, 28.9, 28.4, 25.1, 24.8.

IR (film) vma 3356, 2939, 2344, 2256, 2133, 2034, 1955, 1683, 1640, 1591, 1525, 1345, 1278,
1209, 1172, 866, 773, 730.

HRMS (ESI-TOF) m/z calcd. for C18H27N5NaO4+ ([M + H]+) 400.1955, found 400.1930.

S47
EMARS-hexyl-NBoc: 1H-NMR

EMARS-hexyl-NBoc: 13C-NMR

S48
EMARS-hexyl-biotin (4-azido-2-hydroxy-N-(6-(6-((3aS,4S,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl)hexanamido)hexyl)benzamide)

In an 8-mL vial was added (6-(4-azido-2-hydroxybenzamido)hexyl)carbamate (15.7 mg, 0.04

mmol) and 1.0 mL of 4M HCl in dioxane. The solution was let to stir for 1 hour and then
concentrated under reduced pressure. The resultant residue was taken up in 1 mL of DCM and
cooled to 0 °C before the addition of triethylamine (2.0 mg, 0.20 mmol) and NHS-biotin (13.7
mg, 0.04 mmol). The reaction was then stirred for 12 hours, concentrated under reduced
pressure, and purified via reverse phase chromatography (0–73% H2O/MeCN) to yield the
product as a white solid (5.5 mg, 26% yield).
1H NMR (500 MHz, CD3OD): δ 7.80 (d, 1H, J = 8.6 Hz), 6.59-6.54 (m, 2H), 4.49 (dd, 1H, J
= 7.9, 4.9 Hz), 4.31 (dd, 1H, J = 7.9, 4.4 Hz), 3.39 (t, 2H, J = 7.1 Hz), 3.24–3.15 (m, 3H), 2.93
(dd, 1H, J = 12.7, 5.0 Hz), 2.71 (d, 1H, J = 12.7 Hz), 2.21 (t, 2H, J = 7.3 Hz), 1.79-1.58 (m,
6H), 1.58–1.51 (m, 2H), 1.49–1.37 (m, 7H).

13C NMR (125 MHz, CD3OD): δ 174.6, 168.9, 164.7, 162.4, 145.0, 129.2, 113.0, 108.9, 107.1,
61.9, 60.2, 55.6, 39.6, 38.9, 38.8, 35.4, 29.0, 28.9, 28.4, 28.1, 26.3, 26.2, 25.5.

IR (film) vma 3294, 2931, 2411, 2114, 2036, 1687, 1625, 1586, 1453, 1368, 1271, 1228, 848,
760, 683.

HRMS (ESI-TOF) m/z calcd. for C23H34N7O4S+ ([M + H]+) 504.2387, found 504.2368.

S49
EMARS-hexyl-biotin: 1H-NMR

EMARS-hexyl-biotin: 13C-NMR

S50
Synthesis of water insertion product

1 (56 mg, 0.22 mmol) was dissolved in 2.0 mL H2O in an 8 mL scintillation vial and irradiated
with UV light (365 nm) for 1 hour with stirring at room temperature. The resulting solution was
evaporated to afford the corresponding water insertion product as a white, crystalline solid (53
mg, 98%).

1H-NMR (500 MHz, D2O): δ 7.50 (2H, d, J = 8.0 Hz), 7.43 (2H, d, J = 8.4 Hz), 5.18 (1H, q, J
= 7.0 Hz), 4.11 (2H, s).

13C-NMR (126 MHz, D2O): δ 135.1, 133.8, 129.0, 128.3, 124.3 (q, J = 281.7 Hz), 71.0 (q, J =
31.7 Hz), 42.6.

19F-NMR (478 MHz, D2O): δ -77.8 (3F, d, J = 7.0 Hz)

IR (film): νmax 3307, 2988, 2600, 2361, 1604, 1501, 1459, 1420, 1387, 1346, 1251, 1217, 1159,
1120, 1094, 1074, 1020, 969, 956, 888, 862, 819, 772, 741, 692.

HRMS (ESI-TOF): m/z calcd. for C9H11F3NO ([M-Cl]+) 206.0793, found 206.0733.

S51
1
H-NMR

13
C-NMR

S52
19
F-NMR

S53
Biotinylation: Catalytic Diazirine Sensitization and BSA Labeling
Catalyst-dependent biotinylation of bovine serum albumin

N N Ir (3, 1 µM )
+ BSA Biotin BSA
F3C Biotin DPBS, 10 min
4, 100 µM 450 or 375 nm LED (Western Blot)
10 µM

Photocatalyst 3 was combined with diazirine 4 and BSA in DPBS to afford reaction mixtures
with 100 µL total solution volume and desired component concentrations (see table below).
These samples were then either placed in the dark, irradiated with UV (375 nm) light, or
irradiated with visible (450 nm) light in a biophotoreactor for 10 minutes at 100% intensity. 30
µL samples were then removed, combined with 10 µL of 4x reducing Laemmli sample buffer
(5% ß-mercaptoethanol), vortexed, and heated at 95 °C for 10 minutes. 10 µL of each sample
was then analyzed by Western blot according to the Western Blot General Protocol described
above.

Sample conditions
Lane [4] (µM) [3] (µM) [BSA] (µM) Light
1 100 0.0 10.0 None
2 100 0.0 10.0 375 nm
3 100 0.0 10.0 450 nm
4 100 2.5 10.0 450 nm
5 100 5.0 10.0 450 nm
6 100 7.5 10.0 450 nm
7 100 10.0 10.0 450 nm

Fig. S14. Representative Western blot (n = 3)

(kDa)
250

80

55

800 channel (streptavidin) 700 channel (total protein)

S54
Fig. S15. Normalized intensity (streptavidin 800 channel).

1.2

0.9
Normalized Intensity

0.6

0.3

0
1 2 3 4 5 6 7
Lane

Results: The dependence of biotinylation on catalyst concentration in the illuminated lanes

reflects the catalyst-dependent nature of diazirine sensitization in µMap.

Panel: The normalized intensity values were calculated by densitometry analysis of Western
blot lanes using Image Studio Lite and dividing the 800 channel (streptavidin) band intensity
by the 700 channel (total protein) band intensity. Error bars represent standard deviation, n = 3.

S55
Photonic control over BSA biotinylation

Photocatalyst 3 was combined with diazirine 4 and bovine serum albumin (BSA) in DPBS to
afford reaction mixtures with 1,000 µL total solution volume to give 10 µM 3, 10 µM BSA,
and 100 µM 4. 20 µL aliquots were removed every 2 minutes. The solution was kept in the
dark, with irradiation lasting two minutes occurring at the 8, 16, and 24-minute time points.
The removed aliquots were combined with 6.6 µL 4x reducing Laemmli sample buffer (5% ß-
mercaptoethanol), vortexed, and heated at 95 °C for 10 minutes. 10 µL of each sample was then
imaged according to the Western Blot General Protocol described above. The experiment was
repeated three times. Lanes: 2-4, 0-8 minutes; 5-8, 10-16 minutes; 9-12: 18-24 minutes; 13-16:
26-32 minutes.

Fig. S16. Representative Western blot: photonically controlled BSA labeling (n = 3).

800 channel (streptavidin)

(kDa)
250

55

700 channel (total protein)

S56
Fig. S17. Time-dependent, light-controlled biotinylation.

1.2

0.9
Normalized Intensity

0.6

0.3

0
0 10 20 30
Time (min)

Results: That biotinylation proceeds only under irradiation, and not in the periods of time that
the reaction was left in the dark, reflects the temporal control over reaction progress afforded
by sensitized energy transfer mechanisms.

Panel: The normalized intensity values were calculated by densitometry analysis of Western
blot lanes using Image Studio Lite and dividing the 800 channel (streptavidin) band intensity
by the 700 channel (total protein) band intensity. Error bars represent standard deviation, n = 3.

S57
Preparation of Photocatalyst-Antibody Conjugates
The following operations were carried out at room temperature. 150 µL of a 2.0 mg/mL stock
of goat α-mouse polyclonal antibody (Millipore, AP124) was combined with 15 µL of 1.0 M
NaHCO3 and 10 µL of a 10 mM stock of azidobutyric acid NHS ester (Enamine, EN300-
265680) in DMSO. The mixture was incubated for 1.5 hours. Alternatively, a PBS solution of
azidogalactose-functionalized antibody was prepared using the SiteClick kit according to
manufacturer’s instructions (Thermo Fisher Scientific, S10467). After this time, the reaction
mixture was passed through a ZEBA 40 kDa 2 mL desalting column which had been
equilibrated with Tris-HCl (pH 8.0, 50 mM). The Tris-HCl solution (ca. 200 µL) was then
treated with 8 µL of a 5.0 mM solution of 3 in DMSO, then the components of the Click-It kit
(Thermo Fisher Scientific, C10276) were added: 50 µL reaction buffer A, 20 µL Cu(OAc)2,
and 20 µL additive 1. The mixture was incubated for 5 minutes, then 30 µL additive 2 was
added and the mixture allowed to incubate in the dark for 30 minutes. The reaction mixture was
then passed through a DPBS-equilibrated desalting column to afford ca. 250 µL of Ir-antibody
stock. The light-yellow solution of photocatalyst-antibody conjugate was analyzed by BCA
protein assay kit (Thermo Fisher) for total protein concentration and A350 for Ir concentration
against serial dilutions of BSA and 3 as authentic standards, respectively, indicating a final
concentration of 1.0 mg/mL and an Ir/primary Ab ratio of 6. Different ratios were obtained by
using either more or less azidobutyric acid NHS ester. Using azidogalactose functionalized
antibody, a 1:1 Ir/antibody ratio was obtained. Note: Antibody photocatalyst conjugate was
stored at 4 °C in the dark and should be used within a 7 day window after preparation for best
results.

S58
Proximity-selective Labeling on Protein-Functionalized Agarose Beads
Targeting of VEGFR2 or EGFR2 with photocatalyst-conjugated secondary
antibodies (µMap)
The following procedures were conducted at room temperature. To a 0.5 mL centrifuge tube
was added 12.5 µL of a suspension of goat α-human IgG agarose beads (Sigma A3316) and
333 µL of TBST. Then, 1.2 µg rhVEGFR2/Fc (Frontier Scientific 357-KD) and 1.0 µg of
rhEGFR/Fc (R&D Systems, 344-ER) were added as 0.5 mg/mL solutions in DPBS, and the
solution was incubated for 30 minutes with continuous inversion. Following incubation, the
beads were washed through pelleting (4000g, 2 min), removal of supernatant, resuspension in
1.0 mL TBST, and incubation with continuous inversion for 30 minutes. The beads were
washed in this manner one additional time. Next, the beads were then suspended in 333 µL of
TBST containing 0.5 µg of α-primary antibody (mouse α-human VEGFR2 (BioLegend,
359902) for samples 1 & 3; mouse α-human EGFR (Thermo Fisher, MA5-13070) for samples
2 & 4; mouse IgG1 κ isotype (BD Biosciences, 556648) for sample 5, added as 0.5, 0.2, and
0.5 mg/mL solutions in PBS, respectively, and incubated for 30 minutes with continuous
inversion. The beads were then washed three times with TBST as described above. The beads
were then suspended in 333 µL of TBST containing 0.4 µg of photocatalyst-3-conjugated goat
α-mouse secondary IgG (5.8 photocatalysts/antibody, added as a 1.0 mg/mL in DPBS (prepared
according to the NHS/Click-ligation protocol described on page S58)) and incubated for 30
minutes with continuous inversion. The beads were then washed twice with TBST, once with
DPBS, and then resuspended in 333 µL of a 187.5 µM solution of diazirine 4 in DPBS. These
samples were then irradiated under visible light in the biophotoreactor for 10 minutes at 100%
intensity. For sample 6, the following deviations to the protocol were made: antibodies were
not added, and after the final washing step, the beads were taken up in 333 µL of a 187.5 µM
solution of diazirine 4 and 4 µM of photocatalyst 3 in DPBS. After irradiation, the beads were
pelleted (4000g, 2 minutes) and the supernatant removed. Then, 8.5 µL of 4x Laemmli sample
buffer w/ ß-mercaptoethanol was added to each centrifuge tube, the samples denatured at 95 °C
for 10 minutes, and the supernatant analyzed according to the Western Blot General Protocol
described above using a 4–15% SDS-PAGE gel.

S59
Conditions

lane primary antibody secondary antibody additive light

1 α-VEGFR2 α-Mouse-IgG-Ir-conj. None None
2 α-EGFR α-Mouse- IgG-Ir-conj. None None
3 α-VEGFR2 α-Mouse- IgG-Ir-conj. None 10 min
4 α-EGFR α-Mouse- IgG-Ir-conj. None 10 min
5 Isotype α-Mouse- IgG-Ir-conj. None 10 min
6 None None 4 µM 3 10 min

Fig. S18. Representative Western blot for photocatalyst-conjugated antibody targeted

labeling on agarose beads (n = 4)

Lane
(kDa) 250 1 2 3 4 5 6
VEGFR2

130 EGFR

90

55

30

800 channel (streptavidin)

S60
 700 channel (total protein)

Results: µMap is capable of selectively labeling either VEGFR2 or EGFR

coimmunoprecipitated on agarose beads using α-VEGFR2 or α-EGFR targeting, respectively.

S61
Targeting of VEGFR2 and EGFR with peroxidase-conjugated secondary
antibodies

The following procedures were conducted at room temperature. To a 0.5 mL centrifuge tube
was added 12.5 µL of a suspension of goat α-human IgG agarose beads (Sigma A3316) and
333 µL of TBST. Then, 1.2 µg rhVEGFR2/Fc (Frontier Scientific 357-KD) and 1.0 µg of
rhEGFR/Fc (R&D Systems, 344-ER) were added as 0.5 mg/mL solutions in DPBS, and the
solution was incubated for 30 minutes with continuous inversion. Following incubation, the
beads were washed through pelleting (4000g, 2 min), removal of supernatant, resuspension in
1.0 mL TBST, and incubation with continuous inversion for 30 minutes. The beads were
washed in this manner one additional time. Next, the beads were then suspended in 333 µL of
TBST containing 0.5 µg of α-primary antibody (mouse α-human VEGFR2 (BioLegend,
359902) for samples 1 & 3; mouse α-human EGFR (Thermo Fisher, MA5-13070) for samples
2 & 4; mouse IgG1 κ isotype (BD Biosciences, 556648) for sample 5, added as 0.5, 0.2, and
0.5 mg/mL solutions in PBS, respectively) and incubated for 30 minutes with continuous
inversion. The beads were then washed three times with TBST as described above. The beads
were then suspended in 333 µL of TBST containing 0.4 µg of peroxidase-conjugated goat α-
mouse secondary IgG (Millipore Sigma, AP124P, added as a 1.0 mg/mL solution in PBS) and
incubated for 30 minutes with continuous inversion. The beads were then washed twice with
TBST, once with DPBS, and then resuspended in 333 µL of a 187.5 µM solution of biotinyl
tyramide in DPBS either without H2O2 (Samples 1 and 2) or containing 1.0 mM H2O2 (Samples
3, 4 and 5). These samples were then incubated for 5 minutes with continuous inversion before
the addition of 333 µL of quench solution (10 mM NaN3, 5 mM Trolox, 10 mM ascorbic acid
in DPBS). After quenching, the beads were pelleted (4000g, 2 min) and the supernatant
removed. Then, 8.5 µL of 4x Laemmli sample buffer w/ ß-mercaptoethanol was added to each
sample, the samples denatured at 95 °C for 10 minutes, and the supernatant analyzed according
to the Western Blot General Protocol described above using a 4–15% SDS-PAGE gel.
Conditions

Lane Primary AB Secondary antibody Reaction time

1 α-VEGFR2 α-Mouse-IgG-peroxidase-conj. 5 min (no H2O2)
2 α-EGFR α-Mouse- IgG-peroxidase-conj. 5 min (no H2O2)
3 α-VEGFR2 α-Mouse- IgG-peroxidase-conj. 5 min
4 α-EGFR α-Mouse- IgG-peroxidase-conj. 5 min
5 Isotype α-Mouse- IgG-peroxidase-conj. 5 min

S62
 Fig. S19. Representative Western blot for peroxidase targeted labeling of VEGFR2 and
EGFR on agarose beads (n = 3)

VEGFR2
VEGFR2
EGFR
EGFR

S63
Fig. S 20. Streptavidin stain intensity (normalized to total protein stain) of targeted
peroxidase labeling of VEGFR2 and EGFR (n = 3)

3
Normalized intensity ( n = 3) a b
2.5
VEGFR
VEGFR2
2
EGFR
EGFR
1.5

1 c

0.5

0
1 2 3 4 5
Lane

The normalized intensity values were calculated by densitometry analysis of Western blot lanes
using Image Studio Lite and dividing the 800 channel (streptavidin) band intensity by the 700
channel (total protein) band intensity. Error bars represent standard deviation, n = 3. p-values:
a) 0.017, b) 0.025, c) 0.000015.

Results: Peroxidase based labeling is incapable of selectively labeling either VEGFR2 or

EGFR coimmunoprecipitated on agarose beads using α-VEGFR2 or α-EGFR targeting,
respectively.

S64
Irradiation time vs. selectivity for VEGFR2 targeting with µMap

The following procedures were conducted at room temperature. To a 0.5 mL centrifuge tube
was added 12.5 µL of a suspension of goat α-human IgG agarose beads (Sigma A3316) and
333 µL of TBST. Then, 1.2 µg rhVEGFR2/Fc (Frontier Scientific 357-KD) and 1.0 µg of
rhEGFR/Fc (R&D Systems, 344-ER) were added as 0.5 mg/mL solutions in DPBS, and the
solution was incubated for 30 minutes with continuous inversion. Following incubation, the
beads were washed through pelleting (4000g, 2 min), removal of supernatant, resuspension in
1.0 mL TBST, and incubation with continuous inversion for 30 minutes. The beads were
washed in this manner one additional time. Next, the beads were then suspended in 333 µL of
TBST containing 0.5 µg of α-primary antibody (mouse α-human VEGFR2 (BioLegend,
359902) added as a 0.5 mg/mL solution in PBS) and incubated for 30 minutes with continuous
inversion. The beads were then washed three times with TBST as described above. The beads
were then suspended in 333 µL of TBST containing 0.4 µg of photocatalyst-3-conjugated goat
α-mouse secondary IgG (5.8 photocatalysts/antibody, added as a 1.0 mg/mL in DPBS (prepared
according to the NHS/Click-ligation protocol described on page S58)) and incubated for 30
minutes with continuous inversion. The beads were then washed twice with TBST, once with
DPBS, and then resuspended in 333 µL of a 187.5 µM solution of diazirine 4 in DPBS. These
samples were then irradiated under visible light in the biophotoreactor for 1, 2, 5, or 10 minutes
at 100% intensity (samples 1, 2, 3, and 4, respectively). After irradiation, the beads were
pelleted (4000g, 2 minutes) and the supernatant removed. Then, 8.5 µL of 4x Laemmli sample
buffer w/ ß-mercaptoethanol was added to each centrifuge tube, the samples denatured at 95 °C
for 10 minutes, and the supernatant analyzed according to the Western Blot General Protocol
described above using a 4–15% SDS-PAGE gel.

Conditions
Lane Reaction time
1 1 min
2 2 min
3 5 min
4 10 min

S65
 Fig. S21. Representative Western blot for VEGFR2 targeted µMapping vs. reaction time
on agarose beads (n = 3)

VEGFR2
VEGFR2
EGFR
EGFR

800 channel (streptavidin) 700 channel (total protein)

Fig. S22. Streptavidin stain intensity (normalized to total protein stain) of VEGFR2 and
EGFR vs. reaction time for VEGFR2-targeted µMap (n = 3)

1.20

VEGFR
VEGFR2
1.00
Normalized intensity (n = 3)

d
EGFR
EGFR
0.80 c

0.60 b
a
0.40

0.20

0.00
1 2 3 4
Lane

The normalized intensity values were calculated by densitometry analysis of Western blot lanes
using Image Studio Lite and dividing the 800 channel (streptavidin) band intensity by the 700
channel (total protein) band intensity. Error bars represent standard deviation, n = 3. p-values:
a) 0.005, b) 0.00000023, c.) 0.0012 , d) 0.019.

S66
Fig. S23. Ratio of VEGFR2:EGFR normalized intensity vs reaction time for VEGFR2-
targeted µMap (n = 3)

VEGFR2:EGFR (n = 3) 5

0
1 2 3 4
Lane

Results: No change in selectivity for VEGFR2 is observed over a 10 minute time range using
α-VEGFR2-targeted µMap labeling at 1, 2, 5, and 10 minute time points.

S67
Reaction time vs selectivity for VEGFR2 targeted peroxidase-based labeling
The following procedures were conducted at room temperature. To a 0.5 mL centrifuge tube
was added 12.5 µL of a suspension of goat α-human IgG agarose beads (Sigma A3316) and
333 µL of TBST. Then, 1.2 µg rhVEGFR2/Fc (Frontier Scientific 357-KD) and 1.0 µg of
rhEGFR/Fc (R&D Systems, 344-ER) were added as 0.5 mg/mL solutions in DPBS, and the
solution was incubated for 30 minutes with continuous inversion. Following incubation, the
beads were washed through pelleting (4000g, 2 min), removal of supernatant, resuspension in
1.0 mL TBST, and incubation with continuous inversion for 30 minutes. The beads were
washed in this manner one additional time. Next, the beads were then suspended in 333 µL of
TBST containing 0.5 µg of α-primary antibody (mouse α-human VEGFR2 (BioLegend,
359902), added as a 0.5 mg/mL solution in PBS) and incubated for 30 minutes with continuous
inversion. The beads were then washed three times with TBST as described above. The beads
were then suspended in 333 µL of TBST containing 0.4 µg of peroxidase-conjugated goat α-
mouse secondary IgG (Millipore Sigma, AP124P, added as a 1.0 mg/mL solution in PBS) and
incubated for 30 minutes with continuous inversion. The beads were then washed twice with
TBST, once with DPBS, and then resuspended in 333 µL of a 187.5 µM solution of biotinyl
tyramide in DPBS containing 1.0 mM H2O2. The samples were then incubated for 1, 2, 5, or 10
minutes (samples 1, 2, 3, and 4, respectively) with continuous inversion before the addition of
333 µL of quench solution (10 mM NaN3, 5 mM Trolox, 10 mM ascorbic acid in DPBS). After
quenching, the beads were pelleted (4000g, 2 min) and the supernatant removed. Then, 8.5 µL
of 4x Laemmli sample buffer w/ ß-mercaptoethanol was added to each centrifuge tube, the
samples denatured at 95 °C for 10 minutes, and the supernatant analyzed according to the
Western Blot General Protocol described above using a 4–15% SDS-PAGE gel.

Conditions
Lane Reaction time
1 1 min
2 2 min
3 5 min
4 10 min

S68
Fig. S 24. Representative Western blot for VEGFR2 targeted peroxidase labeling vs.
reaction time on agarose beads (n = 3)

VEGFR2 VEGFR2
EGFR EGFR

Fig. S25. Streptavidin stain intensity (normalized to total protein stain) of VEGFR2 and
EGFR vs. reaction time for peroxidase labeling (n = 3) targeting VEGFR2

0.35
VEGFR
VEGFR2 d
c
Normalized Intensity (n = 3)

0.30 EGFR
EGFR b
a
0.25

0.20

0.15

0.10

0.05

0.00
1 2 3 4
Lane

The normalized intensity values were calculated by densitometry analysis of Western blot lanes
using Image Studio Lite and dividing the 800 channel (streptavidin) band intensity by the 700
channel (total protein) band intensity. Error bars represent standard deviation, n = 3. p-values:
a) 0.00027, b) 0.00085, c) 0.0042, d) 0.0095.

S69
Fig. S26. Ratio of VEGFR2:EGFR normalized intensity vs reaction time for peroxidase
labeling targeting VEGFR2

0.8
VEGFR2:EGFR (n = 3) 0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1 2 3 4
Lane
Results: No selectivity for VEGFR2 is observed over a 10 minute time range using α-VEGFR2-
targeted peroxidase-based labeling at 1, 2, 5, and 10 minute time points.

S70
Protein loading vs. selectivity with VEGFR2-targeted µMap
The following procedures were conducted at room temperature. To a 0.5 mL centrifuge tube
was added 12.5 µL of a suspension of goat α-human IgG agarose beads (Sigma A3316) and
333 µL of TBST. Then, 3.5 and 3.0, 2.25 and 1.95, 1.5 and 1.3, 0.75 and 0.65, or 0.5 and 0.43
µg of rhVEGFR2/Fc (Frontier Scientific, 357-KD) and rhEGFR/Fc (R&D Systems, 344-ER)
were added as 0.5 mg/mL solutions in DPBS to samples 1, 2, 3, 4, and 5, respectively, and the
solution was incubated for 30 minutes with continuous inversion. Following incubation, the
beads were washed through pelleting (4000g, 2 min), removal of supernatant, resuspension in
1.0 mL TBST, and incubation with continuous inversion for 30 minutes. The beads were
washed in this manner one additional time. Next, the beads were then suspended in 333 µL of
TBST containing 0.5 µg of α-primary antibody (mouse α-human VEGFR2 (BioLegend,
359902) added as a 0.5 mg/mL solution in PBS) and incubated for 30 minutes with continuous
inversion. The beads were then washed three times with TBST as described above. The beads
were then suspended in 333 µL of TBST containing 0.4 µg of photocatalyst-3-conjugated goat
α-mouse secondary IgG (5.8 photocatalysts/antibody, added as a 1.0 mg/mL in DPBS (prepared
according to the NHS/Click-ligation protocol described on page S58)) and incubated for 30
minutes with continuous inversion. The beads were then washed twice with TBST, once with
DPBS, and then resuspended in 333 µL of a 187.5 µM solution of diazirine 4 in DPBS. These
samples were then irradiated under visible light in the biophotoreactor for 10 minutes at 100%
intensity. After irradiation, the beads were pelleted (4000g, 2 minutes) and the supernatant
removed. Then, 8.5 µL of 4x Laemmli sample buffer w/ ß-mercaptoethanol was added to each
centrifuge tube, the samples denatured at 95 °C for 10 minutes, and the supernatant analyzed
according to the Western Blot General Protocol described above using a 4–15% SDS-PAGE
gel.
Conditions

Lane VEGFR2 loading (µg) EGFR loading (µg)

1 3.5 3.0
2 2.25 1.95
3 1.5 1.3
4 0.75 0.65
5 0.50 0.43

S71
 Fig. S27. Representative Western blot for VEGFR2 targeted µMapping vs. bead loading
on agarose beads (n = 2) targeting VEGFR2

VEGFR2
EGFR

800 channel (streptavidin) 700 channel (total protein)

Fig. S28. Streptavidin stain intensity (normalized to total protein stain) of VEGFR2 and
EGFR vs. VEGFR2 loading for µMapping (n = 2) targeting VEGFR2
Streptavidin 800 intensity normalized to total

1.2
VEGFR2
1 EGFR

0.8
protein (n = 2)

0.6

0.4 a
0.2

0
1 2 3 4 5

Lane

The normalized intensity values were calculated by densitometry analysis of Western blot lanes
using Image Studio Lite and dividing the 800 channel (streptavidin) band intensity by the 700
channel (total protein) band intensity. Error bars represent standard deviation, n = 2.

S72
Fig. S 29. Ratio of VEGFR2:EGFR normalized intensity vs VEGFR2 loading for
µMapping (n = 2) targeting VEGFR2

9
8
Normalized intensity ratio
(VEGFR2:EGFR) (n = 2)

7
6
5
4
3
2
1
0
1 2 3 4 5

Lane

Results: Decreasing total protein loading on the agarose bead surface results in higher observed
targeting selectivity. We believe this observed increase in targeting selectivity results from an
increase in inter-protein distance on the surface of the beads.

S73
Bead loading vs selectivity for VEGFR2 targeted peroxidase-based labeling
The following procedures were conducted at room temperature. To a 0.5 mL centrifuge tube
was added 12.5, 16.5, 25, 37.5, or 50 µL (for samples 1, 2, 3, 4, and 5, respectively) of a
suspension of goat α-human IgG agarose beads (Sigma A3316) and 333 µL of TBST. Then,
1.2 µg rhVEGFR2/Fc (Frontier Scientific 357-KD) and 1.0 µg of rhEGFR/Fc (R&D Systems,
344-ER) were added as 0.5 mg/mL solutions in DPBS, and the solution was incubated for 30
minutes with continuous inversion. Following incubation, the beads were washed through
pelleting (4000g, 2 min), removal of supernatant, resuspension in 1.0 mL TBST, and incubation
with continuous inversion for 30 minutes. The beads were washed in this manner one additional
time. Next, the beads were then suspended in 333 µL of TBST containing 0.5 µg of α-primary
antibody (mouse α-human VEGFR2 (BioLegend, 359902), added as a 0.5 mg/mL solution in
PBS) and incubated for 30 minutes with continuous inversion. The beads were then washed
three times with TBST as described above. The beads were then suspended in 333 µL of TBST
containing 0.4 µg of peroxidase-conjugated goat α-mouse secondary IgG (Millipore Sigma,
AP124P, added as a 1.0 mg/mL solution in PBS) and incubated for 30 minutes with continuous
inversion. The beads were then washed twice with TBST, once with DPBS, and then
resuspended in 333 µL of a 187.5 µM solution of biotinyl tyramide in DPBS containing 1.0
mM H2O2. The samples were then incubated for 5 minutes with continuous inversion before
the addition of 333 µL of quench solution (10 mM NaN3, 5 mM Trolox, 10 mM ascorbic acid
in DPBS). After quenching, the beads were pelleted (4000g, 2 min) and the supernatant
removed. Then, 15.0 µL of 4x Laemmli sample buffer w/ ß-mercaptoethanol was added to each
centrifuge tube, the samples denatured at 95 °C for 10 minutes, and the supernatant analyzed
according to the Western Blot General Protocol described above using a 4–15% SDS-PAGE
gel.

Conditions

Lane VEGFR2 loading (µg) EGFR loading (µg) Bead loading (µL)
1 1.2 1.0 12.5
2 1.2 1.0 16.5
3 1.2 1.0 25
4 1.2 1.0 37.5
5 1.2 1.0 50

S74
 Fig. S30. Representative Western blot for VEGFR2 targeted peroxidase labeling vs. bead
loading on agarose beads (n = 3)

VEGFR2 VEGFR2

EGFR EGFR

700 channel (total protein) 800 channel (streptavidin)

Fig. S31. Streptavidin stain intensity (normalized to total protein stain) of VEGFR2 and
EGFR vs. bead loading for VEGFR2 targeted peroxidase labeling (n = 3)

VEGFR2
VEGFR
Normalized Intensity (N = 3)

5
EGFR
EGFR

0
1 2 3 4 5
Lane

The normalized intensity values were calculated by densitometry analysis of Western blot lanes
using Image Studio Lite and dividing the 800 channel (streptavidin) band intensity by the 700
channel (total protein) band intensity. Error bars represent standard deviation, n = 3.

S75
Fig. S32. Ratio of VEGFR2:EGFR normalized intensity vs reaction time for VEGFR2
targeted peroxidase labeling (n = 3)

0.7
VEGFR2:EGFR (normalized 0.6

0.5
intensity, N =3)

0.4

0.3

0.2

0.1

0
1 2 3 4 5
Lane

Results: Using peroxidase-based labeling, no increase in targeting selectivity is observed as the

total protein loading is decreased. This contrast the observed targeting selectivity trend with
µMap, where a decrease in total protein loading results in an increase in targeting selectivity.

S76
Photocatalyst/antibody ratio vs VEGFR2 targeted labeling selectivity
The following procedures were conducted at room temperature. To a 0.5 mL centrifuge tube
was added 12.5 µL of a suspension of goat α-human IgG agarose beads (Sigma A3316) and
333 µL of TBST. Then, 1.2 µg rhVEGFR2/Fc (Frontier Scientific 357-KD) and 1.0 µg of
rhEGFR/Fc (R&D Systems, 344-ER) were added as 0.5 mg/mL solutions in DPBS, and the
solution was incubated for 30 minutes with continuous inversion. Following incubation, the
beads were washed through pelleting (4000g, 2 min), removal of supernatant, resuspension in
1.0 mL TBST, and incubation with continuous inversion for 30 minutes. The beads were
washed in this manner one additional time. Next, the beads were then suspended in 333 µL of
TBST containing 0.5 µg of α-primary antibody (mouse α-human VEGFR2 (BioLegend,
359902) added as a 0.5 mg/mL solution in PBS) and incubated for 30 minutes with continuous
inversion. The beads were then washed three times with TBST as described above. The beads
were then suspended in 333 µL of TBST containing 0.4 µg of photocatalyst-3-conjugated goat
α-mouse secondary IgG (0.88, 1.5, 2.4, 3.1, 5.8, 8.3 photocatalysts/antibody in samples 1, 2, 3,
4, 5, 6, respectively), added as 1.0 mg/mL stock solutions in DPBS (prepared according to the
NHS/Click-ligation protocol described on page S58)) and incubated for 30 minutes with
continuous inversion. The beads were then washed twice with TBST, once with DPBS, and
then resuspended in 333 µL of a 187.5 µM solution of diazirine 4 in DPBS. These samples were
then irradiated under visible light in the biophotoreactor for 10 minutes. After irradiation, the
beads were pelleted (4000g, 2 minutes) and the supernatant removed. Then, 8.5 µL of 4x
Laemmli sample buffer w/ ß-mercaptoethanol was added to each centrifuge tube, the samples
denatured at 95 °C for 10 minutes, and the supernatant analyzed according to the Western Blot
General Protocol described above using a 4–15% SDS-PAGE gel.

Conditions

Lane Ir/2o antibody

1 0.88
2 1.5
3 2.4
4 3.1
5 5.8
6 8.3

S77
 Fig. S33. Representative Western blot for targeted µMapping of VEGFR2 vs. amount of
photocatalysts/secondary antibody (n = 3)

VEGFR2 VEGFR2
EGFR EGFR

Fig. S34. Streptavidin stain intensity (normalized to total protein stain) for targeted
µMapping of VEGFR2 vs. amount of photocatalysts/secondary antibody (n = 3)

0.14

0.12
Normalized intensity (N =3)

VEGFR
VEGFR2
0.1 EGFR
EGFR

0.08

0.06

0.04

0.02

0
1 2 3 4 5 6
Lane

The normalized intensity values were calculated by densitometry analysis of Western blot lanes
using Image Studio Lite and dividing the 800 channel (streptavidin) band intensity by the 700
channel (total protein) band intensity. Error bars represent standard deviation, n = 3.

S78
Fig. S 35. Ratio of VEGFR2:EGFR normalized intensity for targeted µMapping of
VEGFR2 vs. amount of photocatalysts/secondary antibody (n = 3)

7
(normalized intensity, N = 3)
6
VEGFR2:EGFR

0
1 2 3 4 5 6
Lane
Error bars represent standard deviation, n = 3.

Results: Increasing the number of conjugated photocatalysts per secondary antibody results in
increased intensity and selectivity over a range of 0.88 to 8.3 phoatacalyst/secondary antibody.

S79
Secondary antibody loading vs. selectivity of VEGFR2 targeted µMap
The following procedures were conducted at room temperature. To a 0.5 mL centrifuge tube
was added 12.5 µL of a suspension of goat α-human IgG agarose beads (Sigma A3316) and
333 µL of TBST. Then, 1.2 µg rhVEGFR2/Fc (Frontier Scientific 357-KD) and 1.0 µg of
rhEGFR/Fc (R&D Systems, 344-ER) were added as 0.5 mg/mL solutions in DPBS, and the
solution was incubated for 30 minutes with continuous inversion. Following incubation, the
beads were washed through pelleting (4000g, 2 min), removal of supernatant, resuspension in
1.0 mL TBST, and incubation with continuous inversion for 30 minutes. The beads were
washed in this manner one additional time. Next, the beads were then suspended in 333 µL of
TBST containing 0.5 µg of α-primary antibody (mouse α-human VEGFR2 (BioLegend,
359902) added as a 0.5 mg/mL solution in PBS) and incubated for 30 minutes with continuous
inversion. The beads were then washed three times with TBST as described above. The beads
were then suspended in 333 µL of TBST containing 0.1, 0.25, 0.5, 1.0, or 1.5 µg of
photocatalyst-3-conjugated goat α-mouse secondary IgG in samples 1, 2, 3, 4, and 5,
respectively (5.8 photocatalysts/antibody, added as a 1.0 mg/mL stock solution in DPBS
(prepared according to the NHS/Click-ligation protocol described on page S58)) and incubated
for 30 minutes with continuous inversion. The beads were then washed twice with TBST, once
with DPBS, and then resuspended in 333 µL of a 187.5 µM solution of diazirine 4 in DPBS.
These samples were then irradiated under visible light in the biophotoreactor for 10 minutes.
After irradiation, the beads were pelleted (4000g, 2 minutes) and the supernatant removed.
Then, 8.5 µL of 4x Laemmli sample buffer w/ ß-mercaptoethanol was added to each centrifuge
tube, the samples denatured at 95 °C for 10 minutes, and the supernatant analyzed according to
the Western Blot General Protocol described above using a 4–15% SDS-PAGE gel.

Conditions
Lane Primary antibody loading (µg) Secondary antibody loading (µg)
1 0.5 0.1
2 0.5 0.25
3 0.5 0.5
4 0.5 1.0
5 0.5 1.5

S80
Fig. S36. Western blot for targeted µMapping of VEGFR2 vs. loading of secondary
antibody

VEGFR2
EGFR

VEGFR2
EGFR

S81
Fig. S37. Streptavidin stain intensity (normalized to total protein stain) for targeted
µMapping of VEGFR2 vs. loading of secondary antibody (n = 3)

0.35
VEGFR
VEGFR2
0.3
EGFR
EGFR
Normalized Intensity (N = 3)

0.25

0.2

0.15

0.1

0.05

0
1 2 3 4 5
Lane

The normalized intensity values were calculated by densitometry analysis of Western blot lanes
using Image Studio Lite and dividing the 800 channel (streptavidin) band intensity by the 700
channel (total protein) band intensity. Error bars represent standard deviation, n = 3.

Fig. S 38. Ratio of VEGFR2:EGFR normalized intensity for targeted µMapping of

VEGFR2 vs. loading of secondary antibody (n = 3)

3.5
Normalized Intensity (N = 3)

2.5

1.5

0.5

0
1 2 3 4 5
Lane
Error bars represent standard deviation, n = 3.
Results: The targeted labeling selectivity of µMap increased slightly as loading of secondary
antibody loading was increased over a range of 0.1–1.5 µg.
S82
Impact of the site of photocatalyst-antibody conjugation and selectivity
The following procedures were conducted at room temperature. To a 0.5 mL centrifuge tube
was added 12.5 µL of a suspension of goat α-human IgG agarose beads (Sigma A3316) and
333 µL of TBST. Then, 1.2 µg rhVEGFR2/Fc (Frontier Scientific 357-KD) and 1.0 µg of
rhEGFR/Fc (R&D Systems, 344-ER) were added as 0.5 mg/mL solutions in DPBS, and the
solution was incubated for 30 minutes with continuous inversion. Following incubation, the
beads were washed through pelleting (4000g, 2 min), removal of supernatant, resuspension in
1.0 mL TBST, and incubation with continuous inversion for 30 minutes. The beads were
washed in this manner one additional time. Next, the beads were then suspended in 333 µL of
TBST containing 0.5 µg of α-primary antibody (mouse α-human VEGFR2 (BioLegend,
359902) for samples 1 and 3, photocatalyst-3-conjugated mouse α-human VEGFR2 (1.9
photocatalysts/antibody, added as a 0.21 mg/mL solution in DPBS (prepared according to the
NHS/Click-ligation protocol described on page S58)) for sample 2, or photocatalyst-3-
conjugated mouse α-human VEGFR2 (0.7 photocatalysts/antibody, added as a 0.17 mg/mL
solution in DPBS (prepared according to the siteclick/click-ligation protocol described on page
S58)) for sample 4, and incubated for 30 minutes with continuous inversion. The beads were
then washed three times with TBST as described above. The beads were then suspended in 333
µL of TBST containing 0.4 µg of photocatalyst-3-conjugated goat α-mouse secondary IgG (1.5
photocatalysts/antibody, added as a 1.1 mg/mL solution in DPBS (prepared according to the
NHS/Click-ligation protocol described on page S58)), photocatalyst-3-conjugated goat α-
mouse secondary IgG (0.9 photocatalysts/antibody, added as a 0.21 mg/mL solution in DPBS
(prepared according to the siteclick/click-ligation protocol described on page S58)) (for
samples 1 and 3, respectively), or without secondary antibody (for samples 2 and 4), and
incubated for 30 minutes with continuous inversion. The beads were then washed twice with
TBST, once with DPBS, and then resuspended in 333 µL of a 187.5 µM solution of diazirine 4
in DPBS. These samples were then irradiated under visible light in the biophotoreactor for 10
minutes at 100% intensity. After irradiation, the beads were pelleted (4000g, 2 minutes) and
the supernatant removed. Then, 8.5 µL of 4x Laemmli sample buffer w/ ß-mercaptoethanol was
added to each centrifuge tube, the samples denatured at 95 °C for 10 minutes, and the
supernatant analyzed according to the Western Blot General Protocol described above using a
4–15% SDS-PAGE gel.

S83
 Conditions
Lane primary antibody secondary antibody Ir/antibody Light
1 Mouse α-VEGFR2 NHS α-mouse 1.5 10 min
2 NHS mouse α-VEGFR2 None 1.9 10 min
3 Mouse α-VEGFR2 Site-click α-mouse 0.9 10 min
4 Site-click α-VEGFR2 None 0.7 10 min

Fig. S39. Representative Western blot for targeted µMapping of VEGFR2 vs. site of
photocatalyst conjugation (n = 3)

VEGFR2
VEGFR2

EGFR
EGFR

S84
Fig. S40. Streptavidin stain intensity (normalized to total protein stain) for targeted
µMapping of VEGFR2 vs. site of photocatalyst conjugation (n = 3)

0.4

0.35
VEGFR
VEGFR2
0.3 EGFR
EGFR
Normalized intensity

0.25

0.2

0.15

0.1

0.05

0
1 2 3 4
Lane

The normalized intensity values were calculated by densitometry analysis of Western blot lanes
using Image Studio Lite and dividing the 800 channel (streptavidin) band intensity by the 700
channel (total protein) band intensity. Error bars represent standard deviation, n = 3.

Results: Site-selective conjugation of photocatalysts at the glycosidic side chains of primary

or secondary antibodies ablates signal intensity and targeted labeling selectivity. The highest
targeted labeling selectivity is observed when the primary targeting antibody is NHS decorated
with photocatalysts. These results may suggest that some sites of conjugation on targeting
antibodies do not position the catalysts in an orientation that allows for productive labeling of
the target proteins to occur.

S85
Proximity-selective Labeling on Cells
Targeted labeling of CD45 on Jurkat cells for Western blot analysis
Jurkat NF-κB GFP cells were used at 5 million cells for each reaction condition and, unless
otherwise noted, were pelleted at 1,000xg for 4 min at 4 °C. To initiate the labeling reaction,
cells were removed from culture, washed twice in cold DPBS and resuspended in cold DPBS
at 5 million cells/mL. Cell suspensions were transferred to Protein LoBind tubes in 1 mL
aliquots. The cells were pelleted, the supernatant removed, and then resuspended in 1 mL of
cold DPBS containing 5 µg Isotype control (BD Biosciences, Purified Mouse IgG1 κ, clone
MOPC-21, 556648) or 5 µg purified mouse α-human CD45 antibody (BD Biosciences, clone
HI30, 555480). The cells were incubated on a rotisserie for 30 min at 4 °C. After incubation,
the cells were pelleted to remove the supernatant and washed twice with 1 mL cold DPBS.
After the final wash, the cells were pelleted to remove the supernatant and resuspended in 1 mL
of cold DPBS containing 5 µg of photocatalyst (3)-conjugated goat α-mouse secondary IgG
(antibody photocatalyst ratio typically ranged from 1:6 to 1:10) and incubated on a rotisserie
for 30 min at 4 °C. After incubation, the cells were pelleted to remove the supernatant, washed
twice with 1 mL cold DPBS and resuspended in 1 mL of cold DPBS containing 250 µM
diazirine biotin 4. The samples were left on ice (0-min treatment) or placed in the
biophotoreactor for 2 min or 10 min and irradiated at full light intensity. After irradiation, the
cells were then pelleted to remove the supernatant. The cell pellets were then lysed in 150 µL
4x Laemmli sample buffer (Bio-Rad: 161-0747) that was pre-diluted 1:1 in RIPA buffer (2x
final concentration). The samples were then sonicated for 5s at power level 6 using a Sonic
Dismembrator, Model 100 (Fisher Scientific) and then heated for 5 min at 95 °C. 15 µL of each
sample was then analyzed by Western blot. Gel electrophoresis was performed using a Bio-Rad
Criterion Vertical Electrophoresis Cell tank, Bio-Rad PowerPac Basic Power Supply, and
Criterion TGX tris-glycine polyacrylamide gel cassettes (SDS/Tris). After electrophoresis, gels
were transferred from precast cassettes to PVDF membranes using an iBlot 2 gel transfer device
(Thermo Fisher, IB21001, IB24001), and washed with water. The membrane was then blocked
for 60 min in TBST with 3% BSA (Sigma Aldrich, A2153). The membrane was then washed
3xs with TBST and incubated overnight with rabbit anti-CD45 (Cell Signaling Technologies,
13917S) and IRDye 800CW streptavidin (Li-Cor, 926-32230) in TBST with 3% BSA. The
membrane was then washed 3x with TBST and incubated for 1 hour with IRDye 680RD goat
anti-rabbit IgG (Li-Cor, 926-68071). The membrane was then washed 3xs with TBST and 1X

S86
with MQ-H2O. The membrane was then imaged using the Li-Cor Odyssey CLx laser scanner
in the 800 nm and 700 nm channels.

Confocal Microscopy Imaging of CD45 Targeting µMapping on Jurkat Cells

10 million Jurkat NF-κB cells were washed 1x with Dulbecco’s Phosphate Buffered Saline
(DPBS) (Gibco: 14190-144) by suspending cell in 5 mL cold DPBS and pelleting the cells by
centrifugation (800xg for 4 min at 4 °C was used for all washing steps of this experiment unless
otherwise noted). The cell pellet was then resuspended in 0.5 mL of cold DPBS containing 5
µg of Mouse α-Human CD45 (BD Biosciences, clone HI30: 555480) or Isotype control (BD
Biosciences, Purified Mouse IgG1 κ, clone MOPC-21: 556648). The cells were incubated at 4
°C on a nutator for 30 min and then washed 2x with cold DPBS. The cells were then
resuspended in 0.5 mL of DPBS containing 5 µg of photocatalyst (3)-conjugated goat α-mouse
secondary IgG (antibody photocatalyst ratio typically ranged from 1:6 to 1:10). The cells were
incubated at 4 °C on a nutator for 30 min and then washed 2x with cold DPBS. The cells were
resuspended in 0.5 mL cold DPBS containing 250 µM diazirine biotin probe 4 and immediately
irradiated for 10 min the bio-photoreactor. After irradiation, the cells were washed with 1X cold
DPBS.

For microscopy analysis, round-shaped glass microscope coverslips (Fisherbrand: 12-545-81)

were acid-etched by incubating in 1 N HCl (Fisher: SA56-1) for 30 min at 50 °C. The coverslips
were then washed in distilled water 3x and placed in 100% ethanol (Fisher: BP2818-500). Acid-
etched glass coverslips were placed into a 24-well plate (Thermo Fisher Scientific: 142485),
one coverslip per well. Coverslips were washed with 1 mL of 1X DPBS (Gibco: 14190-144)
2x to remove ethanol. 1ml of poly-L-lysine solution (Sigma: P4707-50ML) was added per well
and the plate was incubated for 30 min at 37 °C. Coverslips were washed 2x with 1 mL of 1X
DPBS. Approximately 2 million labeled cells in 500 μL of 1X DPBS were loaded per well on
the 24-well plate. The plate was then centrifuged at 400xg for 3 min with deceleration set at 3
to attach cells to the coverslips.

After centrifugation, 6% paraformaldehyde (PFA, Electron Microscopy Sciences, 15710) and

0.2% glutaraldehyde (Sigma-Aldrich, G5882-10X10ML) in 1X DPBS were gently added at a
1:1 ratio for a final concentration of 3% PFA and 0.1% glutaraldehyde per well and incubated
for 10 min at 4 °C. The fixative was removed, and the coverslips were washed 3x in Stain

S87
Buffer (BD Biosciences: 554656) and incubated overnight in 1 mL of Stain Buffer at 4 °C to
block. The following day, the monoculture samples were stained with 500 µL of streptavidin,
Alexa Fluor 488 streptavidin (BioLegend: 405235) at a 1:200 dilution in 500 µL of Stain Buffer.
The plate was sealed with parafilm, covered in foil, and incubated overnight at 4 °C. The next
day, the coverslips were washed 1x in 500 µL of Stain Buffer. Hoechst DNA dye (Cayman
Chemical Company: 600332) was added at a 1:10,000 dilution in 500 µL of Stain Buffer and
incubated for 10 min at ambient temperature, protected from light. The coverslips were washed
2x in Stain Buffer, followed by a final fixing step with 500 µL of 3% PFA and 0.1%
Glutaraldehyde solution in 1X DPBS for 5 min at room temperature. The coverslips were
washed 2x in Stain Buffer. One drop of ProLong Gold Anti-fade mountant (Invitrogen: P36934)
was added to each microscope slide (J. Melvin Freed Brand: 301MF, frosted, 3 x 1”) using
High Precision Straight Tapered Ultra Fine Point tweezers (Fisherbrand: 12-000-122). Each
coverslip was placed on top of the mountant on their respective slides and allowed to dry
overnight at room temperature, protected from light. The slides were imaged using a Zeiss
LSM800 inverted, confocal microscope using a 63X oil immersion objective and Airyscan
settings.

Photolabeling on live Jurkat or JY cells for quantitative LC-MS/MS analysis

Jurkat NF-κB GFP cells, JY-wt cells, or JY PD-L1 overexpression (JY-PD-L1) cells were used
at 50 million cells/labeling experiment and, unless otherwise noted, were pelleted at 1,000xg
for 4 min at 4 °C. To initiate the labeling reaction, cells were removed from culture, washed
twice in cold DPBS and resuspended in cold DPBS at 50 million cells/mL and transferred in 1
mL aliquots to Protein LoBind tubes. The cells were then pelleted to remove the supernatant
and resuspended in 1 mL of cold DPBS containing 25 µg of targeting antibody (see the
following list below of targeting antibodies used for µMap on Jurkat or JY cells).

Antibody Source Cell Tested

Isotype control Clone MOPC-21, BD Biosciences, 556648 Jurkat/JY
α-Human CD45 Clone HI30, BD Biosciences, 555480 Jurkat
α-Human CD45 Clone 2D1, BioLegend, 368502 Jurkat
α-Human CD45 Clone F10-89-4, Thermo Fisher, MA5-16669 Jurkat
α-Human CD47 Clone B6H12, Thermo Fisher, 16-0479-85 Jurkat
α-Human CD29 Clone TS2/16, BioLegend, 303002 Jurkat
α-Human PD-L1 Clone MIH1, Thermo Fisher Scientific, 14598382 JY
α-Human CD300a Clone MEM-260, Thermo Fisher, MA1-10190 JY
α-Human CD30 (TNFRSF8) Clone BerH8, BD Biosciences, 555827 JY
S88
The cells were then incubated on a rotisserie for 30 min at 4°C. After incubation, the cells were
pelleted to remove the supernatant, washed twice with 1 mL cold DPBS, and then resuspended
in 1 mL of cold DPBS containing 25 µg of photocatalyst (3)-conjugated goat α-mouse
secondary IgG (antibody photocatalyst ratio typically ranged from 1:6 to 1:10). The samples
were incubated on a rotisserie for 30 min at 4 °C and then pelleted to remove the supernatant.
The cells were washed twice with 1 mL of cold DPBS and then resuspended in 1 mL of cold
DPBS containing 250 µM diazirine biotin 4. The samples were placed in the biophotoreactor
for the indicated time points (0, 2, or 10 min) and irradiated at full intensity. After irradiation,
the cells were then pelleted to remove the supernatant and washed twice with 1 mL cold DPBS.
Each sample was resuspended in 1 mL of membrane permeabilization buffer (MEM-PER Plus
Membrane Fractionation Kit, Thermo Fisher Scientific, 89842) containing 1X protease
inhibitors (Sigma-Aldrich, 11873580001) and incubated for 20 min at 4 °C. The samples were
then spun at 16,000xg for 15 min at 4 °C. The supernatant enriched in cytosolic proteins was
removed, and the pellet was resuspended in 300 µL lysis buffer (RIPA buffer, Thermo Fisher
Scientific, 89900) containing 1% SDS and 1X protease inhibitors. The samples were sonicated
in the lysis buffer to break up the membrane pellet once for 5s at power level 6 using a Sonic
Dismembrator, Model 100 (Fisher Scientific) and then heated for 5 min at 95 °C. The samples
were then diluted to 1.3 mL with RIPA and sonicated (twice for 5s at power level 6). The protein
concentration was measured by BCA protein assay and the samples were stored at -80 °C until
streptavidin bead enrichment. For bead enrichment, 1-1.2 mg of cell lysate was added to a
Protein LoBind tube containing 250 µL of streptavidin magnetic beads (Thermo Fisher
Scientific, 88817) that were pre-washed twice with 1 mL RIPA buffer. The samples were
incubated for 3 hours at room temperature on a rotisserie and the beads were then pelleted on a
magnetic rack. The supernatant was removed, and the beads were washed 3x with 1 mL 1%
SDS in DPBS, 3x with 1 mL 1 M NaCl in DPBS, 3x with 1 mL 10% EtOH in DPBS, and once
with 1 mL RIPA. The beads were incubated in each of the washes for 5 min before pelleting to
remove the wash. After the final wash, the beads were resuspended in 25 µL of 4x Laemmli
sample buffer containing 20 mM DTT and 25 mM Biotin and heated to 95 °C for 10 min. The
samples were placed on the magnetic rack and the supernatant was collected and transferred to
a new Protein LoBind tube and stored at -80 °C until quantitative proteomic sample preparation
and analysis (performed at IQ Proteomics, Cambridge, MA). In the case of PD-L1 µMapping
at 2 min and 10 min (Fig. S44), 3 µl of the elution sample was used for Western blot analysis
for biotinylation and PD-L1 protein levels. Gel electrophoresis was performed using a Bio-Rad
S89
Criterion Vertical Electrophoresis Cell tank, Bio-Rad PowerPac Basic Power Supply, and
Criterion TGX tris-glycine polyacrylamide gel cassettes (SDS/Tris). After electrophoresis, gels
were transferred from precast cassettes to PVDF membranes using an iBlot 2 gel transfer device
(Thermo Fisher, IB21001, IB24001), and washed with water. The membrane was then blocked
for 60 min in TBST with 3% BSA (Sigma Aldrich, A2153). The membrane was then washed
3xs with TBST and incubated overnight with rabbit anti-PD-L1 (Cell Signaling Technologies,
13684S) and IRDye 800CW streptavidin (Li-Cor, 926-32230) in TBST with 3% BSA. The
membrane was then washed 3x with TBST and incubated for 1 hour with IRDye 680RD goat
anti-rabbit IgG (Li-Cor, 926-68071). The membrane was then washed 3x with TBST and 1x
with MQ-H2O. The membrane was then imaged using the Li-Cor Odyssey CLx laser scanner
in the 800 nm and 700 nm channels.

Fig. S41. Venn diagram analysis of significantly enriched proteins from CD45-targeted
µMap across four independent experiments: This result shows the ability of µMap to
consistently enrich the same proteins across multiple independent µMap experiments.

Proteins enriched from LC-MSMS analysis of targeted labeling of CD45 on Jurkat cells (>2.5-
fold enrichment, p < 0.05 (Benjamini-Hochberg FDR-corrected moderated t statistic) from 4
experiments containing 3 replicates each. Venn diagram overlap from all 4 independent
experiments shows consistent detection of CD45 (PTPRC), and two known CD45 associating
proteins (CD45 associated protein (PTPRCAP), and CD2), as well as additional surface
proteins.

S90
Fig. S42. Targeted labeling of CD45 using primary antibody clones that bind different
epitopes: This result shows that the use of α-CD45 antibodies known to bind similar or different
CD45 epitopes does not significantly influence the list of enriched proteins.

Left, Venn diagram analysis of enriched proteins (>2-fold enrichment, p-value < 0.05
(Benjamini-Hochberg FDR-corrected moderated t statistic) from 3 replicates) from LC-MSMS
analysis of targeted labeling of CD45 using antibody clones that bind similar CD45 epitopes
(clone HI30 and 2D1) (see BioLegend application notes for product 368502). Right, Venn
diagram analysis of enriched proteins ( > 2-fold enrichment, p-value < 0.05 (Benjamini-
Hochberg FDR-corrected moderated t statistic) from 3 replicates) from LC-MSMS analysis of
targeted labeling of CD45 using antibody clones that bind different CD45 epitopes (50) (clone
2D1 and F10894).

S91
Fig. S 43. PD-L1 targeted labeling on JY-wt and JY PD-L1 cells: This experiment
investigates PD-L1-targeted µMap on JY cells with or without PD-L1 surface expression and
highlights the selectivity of this technology by showing that PD-L1 expression is required to
achieve significant surface protein enrichment.

a) Schematic depicting labeling of JY-wt cells (no PD-L1 expression) and JY cells expressing
PD-L1 (JY-PD-L1 cells). Bar graph shows enrichment for PD-L1 with PD-L1 targeting over
isotype targeting. PD-L1 protein was not detected (denoted as n.d.) in JY-wt cells. Error bars
represent standard deviation of n = 3 experiments. Volcano plot of LC-MS/MS analysis of
proteins enriched from targeted labeling of PD-L1 in b) JY-wt cells or c) JY-PD-L1 cells. The
relative protein fold change from PD-L1 targeted samples vs isotype samples are plotted on the
x axis as averaged log2 ratios across 3 replicates. On the y axis are plotted the corresponding
negative log10 transformed p values. In purple are proteins with > 4-fold enrichment and p <
0.05 (Benjamini-Hochberg FDR-corrected moderated t statistic). Highly enriched PD-L1
(CD274) is labeled in green.

S92
Fig. S44. Analysis of time-dependent targeted PD-L1 µMapping on JY-PD-L1 Cells: This
result shows that increased light irradiation time during µMap leads to increased protein
biotinylation and enrichment, showcasing the ability for exquisite control over cellular protein
labeling with visible light irradiation.

a) JY-PD-L1 cells were labeled with isotype or PD-L1 primary antibody and secondary
antibody photocatalyst conjugate and irradiated with visible light for 2 minutes or 10 minutes
in the presence of diazirine biotin. Following streptavidin bead enrichment, the bound proteins
were eluted from the beads and analyzed by Western blot. Left panel shows the biotinylation
signal (green) and PD-L1 signal (red) in each of the fractions. Middle panel shows the
biotinylation signal only. Right panel shows the PD-L1 signal (~50 kDa band). As indicated in

S93
the Western blot, increased biotinylation and PD-L1 protein enrichment correlates with
increased exposure to visible light in the PD-L1 targeted samples, but not in the isotype. b)
Protein correlation analysis bar plot of PD-L1 targeted labeling at different time points. Bar
graphs of protein correlation analysis of targeted labeling of PD-L1 (green bars) and other
proteins with similar bar graph correlations (correlation value > 0.98). For each color bar graph
set, left bar is isotype targeting with 10 minutes photoactivation, middle bar is targeting with 2
minutes photoactivation, and right bar is targeting with 10 minutes photoactivation. Error bars
represent ± standard deviation of n = 3 experiments.

Photolabeling on live A549 cells for quantitative LC-MS/MS analysis

A549 cells were dissociated from the culture dish (15 min incubation with TrypLE Express
(Gibco, 12604-021) at 37 °C) and used in suspension at 20 million cells/labeling experiment
and, unless otherwise noted, were pelleted at 1,000xg for 4 min at 4 °C. To initiate the labeling
reaction, cells were removed from culture, washed twice in cold DPBS and resuspended in cold
DPBS at 20 million cells/mL and transferred in 1 mL aliquots to Protein LoBind tubes. The
cells were then pelleted to remove the supernatant and resuspended in 1 mL of cold DPBS
containing 25 µg of isotype control (BD Biosciences Purified Mouse IgG1 κ, clone MOPC-21,
556648), 25 µg of purified mouse α-human EGFR antibody (clone EGFR.1, BD Biosciences,
555996), or 25 µg of purified mouse α-human CD44 (clone G44-26, BD: 555476). The cells
were incubated on a rotisserie for 30 min at 4 °C. After incubation, the cells were pelleted to
remove the supernatant, washed twice with 1 mL cold DPBS, and then resuspended in 1 mL of
cold DPBS containing 25 µg of photocatalyst (3)-conjugated goat α-mouse secondary IgG
(antibody photocatalyst ratio typically ranged from 1:6 to 1:10). The samples were incubated
on a rotisserie for 30 min at 4 °C and then pelleted to remove the supernatant. The cells were
washed twice with 1 mL of cold DPBS and then resuspended in 1 mL of cold DPBS containing
250 µM diazirine biotin 4. The samples were placed in the biophotoreactor for 2 min and
irradiated at full intensity. After irradiation, the cells were then pelleted to remove the
supernatant and washed twice with 1 mL cold DPBS. Each sample was resuspended in 1 mL
of membrane permeabilization buffer (MEM-PER Plus Membrane Fractionation Kit, Thermo
Fisher Scientific, 89842) containing 1X protease inhibitors (Sigma-Aldrich, 11873580001) and
incubated for 20 min at 4 °C. The samples were then spun at 16,000xg for 15 min at 4 °C. The
supernatant enriched in cytosolic proteins was removed, and the pellet was resuspended in 300

S94
µL lysis buffer (RIPA buffer, Thermo Fisher Scientific, 89900) containing 1% SDS and 1X
protease inhibitors. The samples were sonicated in the lysis buffer to break up the membrane
pellet once for 5s at power level 6 using a Sonic Dismembrator, Model 100 (Fisher Scientific)
and then heated for 5 min at 95 °C. The samples were then diluted to 1.3 mL with RIPA and
sonicated (twice for 5 s at power level 6). The protein concentration was measured by BCA
protein assay and the samples were stored at -80 °C until streptavidin bead enrichment. For
bead enrichment, 2.5 mg of cell lysate was added to a Protein LoBind tube containing 250 µL
of streptavidin magnetic beads (Thermo Fisher Scientific, 88817) that were pre-washed twice
with 1 mL RIPA buffer. The samples were incubated for 3 hours at room temperature on a
rotisserie and the beads were then pelleted on a magnetic rack. The supernatant was removed,
and the beads were washed 3x with 1 mL 1% SDS in DPBS, 3x with 1 mL 1M NaCl in DPBS,
3x with 1 mL 10% EtOH in DPBS, and once with 1 mL RIPA. The beads were incubated in
each of the washes for 5 min before pelleting to remove the wash. After the final wash, the
beads were resuspended in 25 µL of 4X Laemmli sample buffer containing 20 mM DTT and
25 mM Biotin and heated to 95 °C for 10 min. The samples were placed on the magnetic rack
and the supernatant was collected and transferred to a new Protein LoBind tube and stored at -
80 °C until quantitative proteomic sample preparation and analysis (performed at IQ
Proteomics, Cambridge, MA).

S95
Fig. S45. Targeted labeling of endogenously expressed EGFR and CD44 on A549 cells:
this result shows that µMap can be used on non-immune cells such as A549 cells to profile
EGFR and CD44 microenvironments, showcasing the versatility of this method with different
cell types.

Left panel: Volcano plot of LC-MS/MS analysis of proteins enriched from targeted labeling
of EGFR (top) or CD44 (bottom) on A549 cells. The relative protein fold change from EGFR-
or CD44-targeted samples vs isotype samples are plotted on the x axis as averaged log2 ratios
across 3 replicates. On the y axis are plotted the corresponding negative log10 transformed p
values. In purple are proteins with > 2-fold (EGFR) or 3-fold enrichment (CD44) and p < 0.05
(Benjamini-Hochberg FDR-corrected moderated t statistic). EGFR and CD44 are colored in
green. Right panel: Venn diagram analysis of enriched membrane proteins (>2-fold enrichment
for EGFR and > 3-fold enrichment for CD44, p < 0.05 (Benjamini-Hochberg FDR-corrected
moderated t statistic).

S96
Peroxidase labeling (1 min) on live cells for quantitative LC-MS/MS analysis
Jurkat NF-κB cells were used at 50 million cells/labeling experiment and, unless otherwise
noted, were pelleted at 1,000xg for 4 min at 4 °C. To initiate the labeling reaction, cells were
removed from culture, washed twice with cold DPBS and resuspended in cold DPBS at 50
million cells/mL and transferred in 1 mL aliquots to Protein LoBind tubes. The cells were then
pelleted to remove the supernatant and resuspended in 1 mL of cold DPBS containing 25 µg of
isotype control (BD Biosciences, Purified Mouse IgG1 κ, clone MOPC-21, 556648) or 25 µg
of purified mouse α-human CD45 antibody (BD Biosciences, clone HI30, 555480). The cells
were incubated at 4 °C for 30 min on a rotisserie, followed by centrifugation to remove the
supernatant and then washed twice in cold DPBS. The cells were then resuspended in 1 mL
cold DPBS containing 25 µg goat α-Mouse IgG HRP (Millipore, AP124P) and incubated for
30 min at 4 °C on a rotisserie. The cells were then pelleted to remove the supernatant and
washed twice in cold DPBS. Each sample was resuspended in 1 mL of cold DPBS and added
directly to a 15 mL conical tube containing 4 mL of reaction buffer (250 µM biotin phenol and
1 mM H2O2 in cold DPBS) and incubated for 1 min at 4 °C. After 1 min, 5 mL quenching buffer
(DPBS containing 5 mM Trolox, 10 mM Sodium Ascorbate, 10 mM NaN3) was added to the
cells. The cells were then pelleted by centrifugation (4 min at 1,000xg, 4 °C) and washed once
with 10 mL of quenching buffer (DPBS containing 5 mM Trolox, 10 mM Sodium Ascorbic
Acid, 10 mM NaN3). The cells were resuspended in 1 mL of cold DPBS and transferred to
Protein LoBind tubes and washed twice in cold DPBS. After the final wash, the cells were
pelleted and each sample was resuspended in 1 mL of membrane permeabilization buffer
(MEM-PER Plus Membrane Fractionation Kit, Thermo Fisher Scientific, 89842) containing
1X protease inhibitors (Sigma-Aldrich, 11873580001) and incubated for 20 min at 4 °C. The
samples were then spun at 16,000xg for 15 min at 4 °C. The supernatant enriched in cytosolic
proteins was removed, and the pellet was resuspended in 300 µL lysis buffer (RIPA buffer,
Thermo Fisher Scientific, 89900) containing 1% SDS and 1X protease inhibitors. The samples
were sonicated in the lysis buffer to break up the membrane pellet once for 5s at power level 6
using a Sonic Dismembrator, Model 100 (Fisher Scientific) and then heated for 5 min at 95 °C.
The samples were then diluted to 1.3 mL with RIPA and sonicated twice for 5s at power level
6 using a Sonic Dismembrator, Model 100 (Fisher Scientific). The protein concentration was
measured by BCA and the samples were stored at -80 °C until streptavidin bead enrichment.
For bead enrichment, 1-1.2 mg of cell lysate was added to a Protein LoBind tube containing
250 µL of streptavidin magnetic beads (Thermo Fisher Scientific, 88817) that were pre-washed
twice with 1 mL RIPA buffer. The samples were incubated for 3 hours at room temperature on
S97
a rotisserie and the beads were then pelleted on a magnetic rack. The supernatant was removed,
and the beads were washed 3x with 1 mL 1% SDS in DPBS, 3x with 1 mL 1M NaCl in DPBS,
3x with 1 mL 10% EtOH in DPBS, and once with 1 mL RIPA. The beads were incubated in
each of the washes for 5 min before pelleting to remove the wash. After the final wash, the
beads were resuspended in 25 µL of 4x Laemmli sample buffer containing 20 mM DTT and 25
mM Biotin and heated to 95 °C for 10 min. The samples were placed on the magnetic rack and
the supernatant was collected and transferred to a new Protein LoBind tube and stored at -80
°C until quantitative proteomic sample preparation and analysis (performed at IQ Proteomics,
Cambridge, MA).

Selective proteomic proximity labeling assay using tyramide (SPPLAT)

method on live cells for quantitative LC-MS/MS analysis
Jurkat NF-κB cells were used at 50 million cells/labeling experiment and, unless otherwise
noted, were pelleted at 1,000xg for 4 min at 4 °C. To initiate the labeling reaction, cells were
removed from culture, washed twice with cold DPBS and resuspended in cold DPBS at 50
million cells/mL and transferred in 1 mL aliquots to Protein LoBind tubes. The cells were then
pelleted to remove the supernatant and resuspended in 1 mL of cold DPBS containing 20 µg of
isotype control (BD Biosciences, Purified Mouse IgG1 κ, clone MOPC-21, 556648) or 20 µg
of purified mouse α-human CD45 antibody (BD Biosciences, clone HI30, 555480). The cells
were incubated at 4 °C for 60 min on a rotisserie, followed by centrifugation to remove the
supernatant and then washed twice in cold DPBS. The cells were then resuspended in 1 mL
cold DPBS containing 20 µg goat α-Mouse IgG HRP (Millipore, AP124P) and incubated for
60 min at 4 °C on a rotisserie. The cells were then pelleted to remove the supernatant and
washed twice in cold DPBS. Each sample was resuspended in 1 mL of room temp 50 mM Tris
buffer pH 7.5 containing 220 µM biotin phenol and 0.03% H2O2 and incubated for 5 min at
room temperature. After the 5 min incubation, 100 units of Catalase (Sigma-Aldrich, C40) was
added to each sample and incubated for 5 min to stop the reaction. The wells were washed twice
with DPBS at room temperature. After the final wash, the cells were pelleted and each sample
was resuspended in 1 mL of membrane permeabilization buffer (MEM-PER Plus Membrane
Fractionation Kit, Thermo Fisher Scientific, 89842) containing 1X protease inhibitors (Sigma-
Aldrich, 11873580001) and incubated for 20 min at 4 °C. The samples were then spun at
16,000xg for 15 min at 4 °C. The supernatant enriched in cytosolic proteins was removed, and
the pellet was resuspended in 300 µL lysis buffer (RIPA buffer, Thermo Fisher Scientific,

S98
89900) containing 1% SDS and 1X protease inhibitors. The samples were sonicated in the lysis
buffer to break up the membrane pellet once for 5 s at power level 6 using a Sonic
Dismembrator, Model 100 (Fisher Scientific) and then heated for 5 min at 95 °C. The samples
were then diluted to 1.3 mL with RIPA and sonicated twice for 5s at power level 6 using a
Sonic Dismembrator, Model 100 (Fisher Scientific). The protein concentration was measured
by BCA and the samples were stored at -80 °C until streptavidin bead enrichment. For bead
enrichment, 1-1.2 mg of cell lysate was added to a Protein LoBind tube containing 250 µL of
streptavidin magnetic beads (Thermo Fisher Scientific, 88817) that were pre-washed twice with
1 mL RIPA buffer. The samples were incubated for 3 hours at room temperature on a rotisserie
and the beads were then pelleted on a magnetic rack. The supernatant was removed, and the
beads were washed 3x with 1 mL 1% SDS in DPBS, 3x with 1 mL 1 M NaCl in DPBS, 3x with
1 mL 10% EtOH in DPBS, and once with 1 mL RIPA. The beads were incubated in each of the
washes for 5 min before pelleting to remove the wash. After the final wash, the beads were
resuspended in 25 µL of 4X Laemmli sample buffer containing 20 mM DTT and 25 mM Biotin
and heated to 95 °C for 10 min. The samples were placed on the magnetic rack and the
supernatant was collected and transferred to a new Protein LoBind tube and stored at -80 °C
until quantitative proteomic sample preparation and analysis (performed at IQ Proteomics,
Cambridge, MA).

Enzyme-mediated activation of radical sources (EMARS) method on live

cells for quantitative LC-MS/MS analysis
Jurkat NF-κB cells were used at 50 million cells/labeling experiment and, unless otherwise
noted, were pelleted at 1,000xg for 4 min at room temperature. To initiate the labeling reaction,
cells were removed from culture, washed twice with cold DPBS and resuspended in cold DPBS
at 50 million cells/mL and transferred in 1 mL aliquots to Protein LoBind tubes. The cells were
then pelleted to remove the supernatant and resuspended in 1 mL of DPBS at room temperature
containing 5 µg of isotype control (BD Biosciences, Purified Mouse IgG1 κ, clone MOPC-21,
556648) or 5 µg of purified mouse α-human CD45 antibody (BD Biosciences, clone HI30,
555480). The cells were incubated at room temperature for 30 min on a rotisserie, followed by
centrifugation to remove the supernatant and then washed twice in DPBS at room temperature.
The cells were then resuspended in 1 mL DPBS containing 5 µg goat α-Mouse IgG HRP
(Millipore, AP124P) and incubated for 20 min at room temperature on a rotisserie. The cells
were then pelleted to remove the supernatant and washed twice in DPBS at room temperature.
The cells were resuspended in 0.5 mL of DPBS at room temperature containing 80 µM
S99
EMARS-hexyl-biotin and incubated for 15 min at room temperature. The wells were washed
3x with 1 mL DPBS at room temperature. After the final wash, the cells were pelleted and each
sample was resuspended in 1 mL of membrane permeabilization buffer (MEM-PER Plus
Membrane Fractionation Kit, Thermo Fisher Scientific, 89842) containing 1X protease
inhibitors (Sigma-Aldrich, 11873580001) and incubated for 20 min at 4 °C. The samples were
then spun at 16,000xg for 15 min at 4 °C. The supernatant enriched in cytosolic proteins was
removed, and the pellet was resuspended in 300 µL lysis buffer (RIPA buffer, Thermo Fisher
Scientific, 89900) containing 1% SDS and 1X protease inhibitors. The samples were sonicated
in the lysis buffer to break up the membrane pellet once for 5s at power level 6 using a Sonic
Dismembrator, Model 100 (Fisher Scientific) and then heated for 5 min at 95 °C. The samples
were then diluted to 1.3 mL with RIPA and sonicated twice for 5s at power level 6 using a
Sonic Dismembrator, Model 100 (Fisher Scientific). The protein concentration was measured
by BCA and the samples were stored at -80 °C until streptavidin bead enrichment. For bead
enrichment, 1-1.2 mg of cell lysate was added to a Protein LoBind tube containing 250 µL of
streptavidin magnetic beads (Thermo Fisher Scientific, 88817) that were pre-washed twice with
1 mL RIPA buffer. The samples were incubated for 3 hours at room temperature on a rotisserie
and the beads were then pelleted on a magnetic rack. The supernatant was removed, and the
beads were washed 3x with 1 mL 1% SDS in DPBS, 3x with 1 mL 1M NaCl in DPBS, 3x with
1 mL 10% EtOH in DPBS, and once with 1 mL RIPA. The beads were incubated in each of the
washes for 5 min before pelleting to remove the wash. After the final wash, the beads were
resuspended in 25 µL of 4x Laemmli sample buffer containing 20 mM DTT and 25 mM Biotin
and heated to 95 °C for 10 min. The samples were placed on the magnetic rack and the
supernatant was collected and transferred to a new Protein LoBind tube and stored at -80 °C
until quantitative proteomic sample preparation and analysis (performed at IQ Proteomics,
Cambridge, MA).

S100
Fig. S 46. Volcano plot showing proteins enriched through CD45-targeted EMARS
proximity labeling: this result shows that low levels of protein enrichment are observed using
the CD45-targeted EMARS method which is consistent with the low labeling signals described
in a previous literature report.(51)

Volcano plot of LC-MS/MS analysis of proteins enriched from targeted labeling of CD45 using
peroxidase following the EMARS method described above. The relative protein fold change
from CD45-targeted samples vs isotype samples are plotted on the x axis as averaged log2 ratios
across 3 replicates. On the y axis are plotted the corresponding negative log10 transformed p
values. In purple are proteins with >2-fold enrichment and p < 0.05 ((Benjamini-Hochberg
FDR-corrected moderated t statistic)). CD45 (PTPRC), CD47, and CD29 (ITGB1) are colored
in green.

Protein extraction and digestion for LC-MS/MS analysis

Prior to LC-MS/MS analysis, proteins were reduced with 20 mM dithiothreitol (DTT) for 1
hour at room temperature. Cysteine residues were alkylated with iodoacetamide (60 mM) for 1
hour in the dark and quenched with DTT (40 mM). Proteins were extracted by methanol-
chloroform precipitation and digested with 1 µg of trypsin (Promega) in 100 mM EPPS (pH
8.0) for 4 hours at 37 °C. Each of the tryptic peptide samples were labeled with 400 µg of
Tandem Mass Tag (TMT; Pierce) isobaric reagents for 2 hours at room temperature. A label
efficiency check was performed by pooling 2 µL from each sample within a single plex to
ensure at least 98% labeling of all N-termini and lysine residues. All samples were quenched
with hydroxylamine (0.5%), acidified with TFA (2%), pooled, and dried by speedvac
evaporation. Pooled TMT labeled peptides were fractionated using the high pH reverse-phase
S101
peptide fractionation kit (Pierce) into 3 fractions (20%, 25%, and 50% acetonitrile in 0.1%
triethylamine) and desalted with Empore-C18 (3M) in-house packed StageTips prior to analysis
by mass spectrometry.

LC-MS/MS-based proteomic analysis of labeled cell experiments

All mass spectra were acquired on an Orbitrap Fusion Lumos coupled to an EASY nanoLC-
1000 (or nanoLC-1200) (Thermo Fisher) liquid chromatography system. Approximately 2 µg
of peptides were loaded on a 75 µm capillary column packed in-house with Sepax GP-C18
resin (1.8 µm, 150 Å, Sepax) to a final length of 35 cm. Peptides were separated using a 110-
minute linear gradient from 8% to 28% acetonitrile in 0.1% formic acid. The mass spectrometer
was operated in a data dependent mode. The scan sequence began with FTMS1 spectra
(resolution = 120,000; mass range of 350–1400 m/z; max injection time of 50 ms; AGC target
of 1e6; dynamic exclusion for 60 seconds with a +/- 10 ppm window). The ten most intense
precursor ions were selected for MS2 analysis via collisional-induced dissociation (CID) in the
ion trap (normalized collision energy (NCE) = 35; max injection time = 100ms; isolation
window of 0.7 Da; AGC target of 1.5e4). Following MS2 acquisition, a synchronous-precursor-
selection (SPS) MS3 method was enabled to select eight MS2 product ions for high energy
collisional-induced dissociation (HCD) with analysis in the Orbitrap (NCE = 55; resolution =
50,000; max injection time = 86 ms; AGC target of 1.4e5; isolation window at 1.2 Da for +2
m/z, 1.0 Da for +3 m/z or 0.8 Da for +4 to +6 m/z). All mass spectra were converted to mzXML
using a modified version of ReAdW.exe. MS/MS spectra were searched against a concatenated
2018 human Uniprot protein database containing common contaminants (forward + reverse
sequences) using the SEQUEST algorithm (52). Database search criteria are as follows: fully
tryptic with two missed cleavages; a precursor mass tolerance of 50 ppm and a fragment ion
tolerance of 1 Da; oxidation of methionine (15.9949 Da) was set as differential modifications.
Static modifications were carboxyamidomethylation of cysteines (57.0214) and TMT on
lysines and N-termini of peptides (229.1629). Peptide-spectrum matches were filtered using
linear discriminant analysis (53) and adjusted to a 1% peptide false discovery rate (FDR) (54).

Bioinformatic analysis of mass spectrometry data

All bioinformatic analysis of LC-MS/MS data was performed in the R statistical computing
environment (55). Peptide level abudance data is used to identify the number of peptides
corresponding to a protein in the experiment. Any protein with a single peptide quantification
is removed to reduce the possibility that outliers will affect downstream proximal calls. Peptide
S102
level abundance data is then normalized to the summed total abundance for each sample
separately. These totals are then averaged, and each normalized protein abundance value is
multiplied by this average to rescale abundance data. Peptide level data is then merged to
protein level data by taking the median of all peptides corresponding to a protein. Proteins were
then filtered to remove any known contaminants identified from the database search. For
volcano plot generation, proteins were also filtered to remove known antibodies identified as
having IGKV or IGKL present in the gene symbol and Immunoglobulin present in the Uniprot
description as these result from antibody targeting (see Fig. S47 showing volcano plots that
showed enrichment of targeting antibodies). Finally, data are filtered to remove PRNP, a protein
which is a known false positive consistently detected across almost all experiments. See
extended supplementary tables (Data S1) for processed proteomic data as well as the GitHub
link below for raw peptide abundance data.

Correlation Analysis
For experiments in which isotype vs. antibody targeting are tested at two different time points
(Fig. S44), correlation was measured using the cor.test function in R with the pearson
correlation measure. Proteins were filtered before plotting. Proteins were first filtered by those
that were significantly differentially abundant when comparing the 10 minute timepoint against
the isotype control (FDR corrected p-value < 0.05). The proteins were further filtered to include
only those annotated in Uniprot to have an association with the plasma membrane. Finally,
protein abundances were correlated against the abundance measurements of the target (e.g. PD-
L1) and filtered to remove any proteins with correlation FDR corrected p-values below 0.05 as
given by the cor.test function and corrected by the p.adjust function within R. These significant
proteins were then ranked based upon this correlation measure. Bar plots were generated in R
with the ggplot library (56).

Linear modeling and fold change generation

For experiments in which isotype vs. antibody targeting are tested at a single time point, protein
abundances are log2 transformed and subjected to linear modeling analysis with Limma (57).
Limma utilizes an empirical Bayes approach that allows for a realistic distribution of biological
variance with small sample sizes per group. This program further utilizes the full dataset to
shrink the observed sample variances towards a pooled estimate. This borrowing of variance

S103
information across proteins allows for a more accurate estimate of true variance, and improved
power to detect real differences between groups. For each protein, abundance data is fit to a
linear model with the experimental group as the input variable using the lmFit function. The
log2FC values are estimated and p-values calculated for significance. P-values are then
corrected for multiple comparisons using the false discovery rate (FDR) method by Benjamini
and Hochberg (58).

Volcano plot generation

Volcano plots in the manuscript were generated using Microscoft Excel. Volcano plots in the
Supplementary Materials were generated in R with the ggplot2 library (56). Log2FC and p-
value estimates from Limma were subset to those reaching a specified log2FC cutoff. Proteins
were colored based on whether they fell above or below the log2-fold cutoff threshold and were
statistically significant (FDR corrected p-value of < 0.05).

S104
Fig. S 47. Volcano plots showing enrichment of targeting antibodies. Significantly
enriched (FDR-corrected p-value < 0.05) proteins are shown in purple, the targeted
protein and known associators are labeled green, and targeting antibodies are labeled in
maroon).

Cell receptor surface density determination

1 million Jurkat NF-κB cells were resuspended in 100 µL of PBS followed by addition of either
PE α-human CD45 (BD Biosciences, 555483), FITC α-human CD47 (BD Biosciences,
556045), or PE α-human CD29 (BioLegend, 303004) for cell surface staining. Antibody
amounts were added to achieve cell surface saturation (determined empirically for each
antibody). Stained cells were incubated for 30 min at 4 °C in the dark and then washed 2xs
with 500 µL of PBS. Cells were then resuspended in 500 µL PBS and the geometric mean
fluorescence intensity (MFI) of the cell surface antigens was measured using a BD
FACSCelesta (SN: H66034400170, Model No: 660344) with BD FACSDiva software

S105
(v8.0.1.1). Data was analyzed using FlowJo v10 (FlowJo, LLC). Geometric MFI values were
converted into antibody binding capacity values (which correlate to cell receptor surface density
at antibody saturation) using the Quantum Simply Cellular α-Mouse IgG microbead kit (Bangs
Laboratories, 815) according to manufacturer’s instructions using the flow cytometer and
software described above. Cell surface numbers were determined to be the following:

CD45: 665,000 +/- 2000 copies/cell

CD47: 790,000 +/-12,000 copies/cell
CD29: 360,000 +/- 2500 copies/cell

Analysis of IL-2 Production in Jurkat-JY Two cell system

45 µL of a 8e6 cells/mL stock of Jurkat PD-1 cells in assay media (RPMI 1640, Corning: 10-
040-CV + 10% dialyzed FBS, HyClone: SH30079.03) were mixed with 45 µL of mouse IgG1
κ isotype control antibody, clone MOPC-21 (BD Biosciences: 556648), or α-human PD-L1
antibody, clone MIH1 (Invitrogen: 14-5983-82) at a final concentration of 10 µg/mL in assay
medium and incubated for 30 min at room temperature in a 96-well U-bottom plate. JY-PD-L1
cells were suspended at 8•105 cells/mL in assay medium and incubated for 30 min at 37 °C +
5% CO2 with or without 120 ng/mL of Staphylococcal Enterotoxin D (SED) (Toxin
Technology: DT303).

After the 30 min incubation, 125 µL of SED-loaded JY-PD-L1 cells were aliquoted per well in
a separate 96-well U-bottom plate and mixed with 25 µL of antibody-loaded Jurkat PD-1 cells
(100,000 cells per cell line). The plate was incubated for 24 hours at 37 °C + 5% CO2. The
following day, the plate was centrifuged for 1 min at 1,100 RPM and room temperature. 50 µL
of culture supernatant per well were used to perform an ELISA using a Human IL-2 ELISA kit
(Thermo Fisher: EH2IL25) per manufacturer’s instructions to approximate IL-2 production.
Data was analyzed and graphed using MS Excel and GraphPad Prism software (8.1.1.330).

Flow Cytometry Analysis of CD45 on Jurkat and JY cells

Approximately 1 million Jurkat PD-1 cells and JY PD-L1 cells were centrifuged for 5 min at
500xg and 4 °C, washed 1x in Stain Buffer (BD Biosciences, 554656) and centrifuged again.
The cell pellets were resuspended in 100 µL of Stain Buffer containing Fc Block (BD
Biosciences, 564220) at a 1:100 dilution and incubated for 20 min on ice. The cells were
centrifuged for 5 min at 500xg and 4 °C, washed once in 200 µL of Stain Buffer and centrifuged

S106
again as above. Cells were stained in 100 µL of Stain Buffer using the following at a 1:20
dilution: BV605 Mouse IgG1, κ Isotype control, clone X40 at (BD Biosciences, 562652),
BV605 Mouse α-Human CD45, clone HI30 (BD Biosciences, 564047), APC Mouse IgG2b, κ
clone MPC-11 (BioLegend, 400322), APC α-Human CD45RA, clone HI100 (BioLegend,
304112), BV421 Mouse IgG2a, κ Isotype control, clone G155-178 (BD Biosciences, 562439),
and BV421 Mouse α-Human CD45RO, clone UCHL1 (BD Biosciences, 562641) and incubated
for 30 min on ice, protected from light. Cells were washed 1x in cold 1X DPBS (Gibco, 14190-
144) and resuspended in 100 µL of 1X DPBS containing a 1:500 dilution of Zombie Violet
Fixable Viability kit (BioLegend, 423113) and incubated for 15 min at room temperature,
protected from light. Cells were centrifuged as above, washed once in 1X DPBS, and the pellets
were resuspended in 200 μL of Stain Buffer and transferred to 5 mL FACS tubes (Fisherbrand,
14-956-3D). Samples were acquired on a BD FACSCelesta (SN: H66034400170, Model No:
660344) with BD FACSDiva software (v8.0.1.1). Data was analyzed using FlowJo v10
(FlowJo, LLC).

Flow Cytometry Analysis of µMapping in two-cell system

For transcellular labeling using µMapping, 10 million Jurkat PD-1 cells and 10 million JY PD-
L1 cells per sample were centrifuged for 4 min at 800xg, 4 °C in Protein LoBind tubes, and
each cell pellet was resuspended in 500 μL of their respective complete media containing 5 µg
of targeting antibody (see the following list below of targeting antibodies used for µMap on
Jurkat or JY cells).

Antibody Source Cell Tested

Isotype control clone MOPC-21, BD Biosciences, 556648 Jurkat/JY
α-Human CD45RO clone UCHL1, , BD Pharmingen, 562641 Jurkat
α-human PD-1 clone J116, Invitrogen, 14-9989-82 Jurkat
α-human PD-L1 clone MIH1, Thermo Fisher Scientific, 14598382 JY

Samples were incubated on a rotisserie at 4 °C for 30 min followed by 2 washes in 1 mL of

cold complete media (cells centrifuged at 800xg for 4 min at 4 °C to pellet cells). Cell pellets
were then resuspended in 500 μL of cold complete media, and 5 μg of photocatalyst (3)-
conjugated goat α-mouse secondary IgG (antibody photocatalyst ratio typically ranged from
1:6 to 1:10) was added to each sample. All cells were incubated at 4 °C for 30 min on the
rotisserie and then washed 2x with 1 mL of cold 1X DPBS (cells centrifuged at 800xg for 4
min at 4 °C to pellet cells).
S107
For transcellular labeling using peroxidase, 10 million cells per sample were resuspended in
500 μL of cold complete media for each cell line (see General Materials section for cell line
growth media) in Protein LoBind tubes containing 5 μg of Purified Mouse α-Human CD45RO
(BD Pharmingen, clone UCHL1, 562641), Purified Mouse α-Human PD-L1 (Thermo Fisher
Scientific, clone MIH1, 14598382), or Isotype control (BD Biosciences, Purified Mouse IgG1
κ, clone MOPC-21, 556648) and incubated for 30 min in a rotisserie at 4 °C. The cells were
pelleted at 800xg for 4 min at 4 °C, washed 2x in cold complete media and resuspended in 500
μL of complete media containing 5 μg of Goat α-Mouse IgG Horseradish Peroxidase (HRP)
and incubated for 30 min in a rotisserie at 4 °C. The cells were pelleted at 800xg for 4 min at 4
°C, washed 2x with cold 1X DPBS and the cell pellet resuspended in 500 μL of cold 1X DPBS.

JY-PD-L1 cells that were pre-coated with or without antibody were then incubated in the
presence of 120 ng/mL of Staphylococcal Enterotoxin D (SED) (Toxin Technology, DT303)
and incubated for 30 min on ice. 1 million JY-PD-L1 cells treated with SED were then
combined with 1 million Jurkat PD-1 cells and pelleted at 800xg for 4 min at 4 °C. The cell
pellet was resuspended in a final volume of 45 μL of 1X DPBS and incubated for 2.5 hours at
37 °C + 5% CO2 prior to the light irradiation step below. For no SED (-SED) experiments, JY
cells were not treated with SED and immediately mixed with Jurkat cells in 45 μL of 1X DPBS
just prior to the light irradiation step below. For PD-1 targeted µmap in Jurkat-Jurkat co culture,
Jurkat NF-κB cells were pre-treated with SED for 30 min on ice and then immediately
combined with Jurkat-PD-1 cells pre-coated with targeting antibody at a ratio of 1 million
Jurkat NF-κB cells to 1 million Jurkat-PD-1 cells in 45 μL of 1X DPBS just prior to the light
irradiation step below.

For iridium photocatalyst-based labeling, a 25 mM diazirine biotin probe 4 in DMSO was

diluted 1:10 in 1X DPBS and 5 μL added to the 45 μL cell suspension with gentle mixing to
give a final concentration of 250 μM. The cells were then irradiated for the indicated times at
100% light intensity in the biophotoreactor (BPR). For “No light” controls, the samples were
protected from light for the same amount of time as the maximal light irradiation samples. After
irradiation, cells were centrifuged for 4 min at 800xg, 4 °C and washed once with 1 mL of cold
1X DPBS.

For peroxidase labeled samples, reaction buffer (final concentration of 250 μM biotin phenol
and 1 mM H2O2 in cold 1X DPBS) was added to each sample for a final volume of 50 μL and
incubated for 1 min at 4 °C. After 1 min, 50 μL of quenching buffer (cold 1X DPBS containing
5 mM Trolox, 10 mM Sodium Ascorbic Acid, 10 mM NaN3) was added to the cells. The cells
S108
were then pelleted by centrifugation (4 min at 800xg, 4 °C) and washed 1x with 50 μL of
quenching buffer and centrifuged again.

The cell pellets from either iridium-photocatalyst or peroxidase labeling were then washed in 1
mL of cold 1X DPBS, centrifuged again, and resuspended in 100 μL of Stain Buffer (BD
Biosciences, 554656) containing a 1:100 dilution of Fc block (BD Biosciences, 564220) per
sample. The cells were incubated for 20 min on ice, then centrifuged for 4 min at 500xg, 4 °C,
washed once with 200 μL Stain Buffer and centrifuged again as above. Cells were resuspended
in 100 μL of Stain Buffer containing the following antibodies at the indicated dilutions: Alexa
Fluor 488 Mouse α-Human CD3, clone SP34-2 at a 1:40 dilution (BD Biosciences, 557705) or
FITC Mouse α-Human CD279 (PD-1), clone MIH4 at a 1:20 dilution (BD Biosciences,
557860), APC Mouse α-Human CD19, clone HIB19 at a 1:10 dilution (BD Biosciences,
555415), and Streptavidin PE at a 1:200 dilution (BD Biosciences, 554061). Compensation
controls were generated using UltraComp beads (BD Biosciences, 01-2222-42), BV421 Mouse
α-Human CD45RO, clone UCHL1 (BD Biosciences, 562641), PE Mouse α-Human CD3, clone
SK7 (BioLegend, 344806), Alexa Fluor 488 Mouse α-Human CD3, clone SP34-2, and APC
Mouse α-Human CD19, clone HIB19. The plate was incubated for 30 min on ice, the cells were
washed once with 200 μL of cold 1X DPBS then resuspended in 100 μL of a 1:500 dilution of
Zombie Violet Viability kit (BioLegend, 423113) and incubated for 15 min at room
temperature, protected from light. The cells were centrifuged as above and resuspended in 200
μL of cold 1X DPBS and centrifuged again. The final pellets were resuspended in 200 μL of
Stain Buffer and transferred to 5 mL FACS tubes (Fisherbrand, 14-956-3D) and acquired
samples on a BD FACSCelesta (SN: H66034400170, Model No: 660344) with BD FACSDiva
software (v8.0.1.1). Data was analyzed using FlowJo v10 (FlowJo, LLC). See Harvard
Dataverse link below for raw flow cytometry data.

S109
Fig. S48. Two cell system µMapping time course: This result shows that the degree of cis
and trans-cellular biotinylation induced by PD-L1 targeted µMap increases with longer light
irradiation time.

a) JY-PD-L1 cells (CD19+) were pre-labeled with α-PD-L1 or isotype control and
photocatalyst (3)-conjugated goat α-mouse secondary IgG. The cells were then mixed with
Jurkat PD-1 cells (CD3+) in the presence of staphylococcal enterotoxin D (SED) for 2.5 hours
followed by visible light irradiation (450 nm) in the presence of diazirine-biotin probe for the
indicated time points. Flow cytometry analysis shows both cis-biotinylation on the JY-PD-L1
cells and trans-biotinylation on Jurkat PD-1. b) Bar plots of replicate analysis of cell
biotinylation measured by flow cytometry in panel a. Error bars represent standard deviation of
n = 3 experiments.

S110
Fig. S49. Two cell system PD-1 µMapping of Jurkat-Jurkat co-culture: This result shows
that trans-cellular biotinylation is dependent on direct cellular interactions.

a) Flow cytometry analysis of PD-1 targeted µMap on Jurkat-PD-1 cells in co-culture with JY-
PD-L1 cells or Jurkat NF-κB using 10 min light irradiation. Cis-labeling on Jurkat-PD-1
(CD3+) cells is observed in both cases. Minimal to no trans-labeling was observed in the Jurkat-
Jurkat co-culture system compared to Jurkat-JY highlighting the dependence of direct cellular
interactions for trans-cellular biotinylation to occur. b) Bar plots of replicate analysis flow
cytometry data from panel a of trans-biotinylation on JY-PD-L1 cells (blue bars) or Jurkat NF-
κB cells (orange bars). Error bars represent standard deviation of n = 3 experiments; *P < 0.01.

Fig. S50. Representative gating strategy for fluorescence-activated cell sorting (FACS)
of Jurkat (CD3+) and JY (CD19+) two cell system: This result shows the gating strategy
used for monitoring cis and trans-cellular biotinylation.

S111
Confocal microscopy imaging of µMapped cells
For microscopy analysis, cells were biotinylated exactly as described in the above “Flow
Cytometry Analysis of µMapping in two-cell system” section. Round-shaped glass microscope
coverslips (Fisherbrand, 12-545-81) were acid-etched by incubating in 1 N HCl (Fisher, SA56-
1) for 30 min at 50 °C. The coverslips were then washed in distilled water 3x and stored in
100% ethanol (Fisher, BP2818-500) at room temperature until ready to use. Acid-etched glass
coverslips were placed into a 24-well plate (Thermo Fisher Scientific, 142485), one coverslip
per well and washed 2x with 1 mL of 1X DPBS (Gibco, 14190-144). 0.5 mL of poly-L-lysine
solution (Sigma: P4707-50ML) was added per well and the plate was incubated for 30 min at
37 °C. Coverslips were washed 2x with 1 mL of 1X DPBS. Approximately 2 million of labeled
cells were loaded in 500 μL of 1X DPBS per well on the 24-well plate. The plate was then
centrifuged at 400g for 3 min with deceleration set at 3 using a Sorvall Legend XTR table-top
centrifuge (Thermo Scientific).

After centrifugation, 6% paraformaldehyde (PFA, Electron Microscopy Sciences, 15710) and

0.2% glutaraldehyde (Sigma-Aldrich, G5882-10X10ML) in 1X DPBS were gently added at a
1:1 ratio for a final concentration of 3% PFA and 0.1% glutaraldehyde per well and incubated
for 10 min at 4 °C. The fixative was removed, and the coverslips were washed 3x in Stain
Buffer (BD Biosciences, 554656) and incubated overnight in 1 mL of Stain Buffer at 4 °C. The
following day, samples were stained with Alexa Fluor 488 streptavidin (BioLegend, 405235)
at a 1:200 dilution and BV650 Mouse α-human CD3 antibody (BD Biosciences, clone SK7,
563999) at a 1:40 dilution in 500 μL of Stain Buffer. The plate was sealed with parafilm and
incubated overnight at 4 °C protected from light. The next day, the coverslips were washed 1x
with 500 μL of Stain Buffer. Hoechst DNA dye (Cayman Chemical Company, 600332) was
added at a 1:10,000 dilution in 500 μL of Stain Buffer and incubated for 10 min at room
temperature, protected from light. The coverslips were washed 2x in Stain Buffer and fixed
with 3% PFA and 0.1% glutaraldehyde solution in 1X DPBS for 5 min at room temperature.
The coverslips were washed 2x in Stain Buffer and 1 drop of ProLong Gold Anti-fade mountant
(Invitrogen, P36934) was added to microscope slides (J. Melvin Freed Brand: 301MF, frosted,
3 x 1”) using High Precision Straight Tapered Ultra Fine Point tweezers (Fisherbrand, 12-000-
122). Coverslips were placed on top of the mountant on their respective slides and allowed to
dry overnight at room temperature, protected from light. The slides were imaged using a Zeiss
LSM800 inverted, confocal microscope using a 63X oil immersion objective and Airyscan
settings.
S112
Fig. S51. Split channel microscopy images of Figure 4d.

S113
S114
S115
Data and Code availability
Raw flow cytometry data are available through the Harvard Dataverse:
(doi:10.7910/DVN/O6O04O). Code for replicating proteomic data analysis, generation of
volcano plots, and raw peptide level abundance data for each of the experiments were deposited
in a GitHub repository (41).

S116
References and Notes
1. E. Lundberg, G. H. H. Borner, Spatial proteomics: A powerful discovery tool for cell biology.
Nat. Rev. Mol. Cell Biol. 20, 285–302 (2019). doi:10.1038/s41580-018-0094-y Medline
2. N. C. Bauer, P. W. Doetsch, A. H. Corbett, Mechanisms regulating protein localization.
Traffic 16, 1039–1061 (2015). doi:10.1111/tra.12310 Medline
3. B. Z. Stanton, E. J. Chory, G. R. Crabtree, Chemically induced proximity in biology and
medicine. Science 359, eaao5902 (2018). doi:10.1126/science.aao5902 Medline
4. R. R. Minter, A. M. Sandercock, S. J. Rust, Phenotypic screening-the fast track to novel
antibody discovery. Drug Discov. Today. Technol. 23, 83–90 (2017).
doi:10.1016/j.ddtec.2017.03.004 Medline
5. D. S. Johnson, A. Mortazavi, R. M. Myers, B. Wold, Genome-wide mapping of in vivo
protein-DNA interactions. Science 316, 1497–1502 (2007). doi:10.1126/science.1141319
Medline
6. M. Hanoun, M. Maryanovich, A. Arnal-Estapé, P. S. Frenette, Neural regulation of
hematopoiesis, inflammation, and cancer. Neuron 86, 360–373 (2015).
doi:10.1016/j.neuron.2015.01.026 Medline
7. S. S. Lam, J. D. Martell, K. J. Kamer, T. J. Deerinck, M. H. Ellisman, V. K. Mootha, A. Y.
Ting, Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat.
Methods 12, 51–54 (2015). doi:10.1038/nmeth.3179 Medline
8. J. S. Rees, X.-W. Li, S. Perrett, K. S. Lilley, A. P. Jackson, Protein neighbors and proximity
proteomics. Mol. Cell. Proteomics 14, 2848–2856 (2015). doi:10.1074/mcp.R115.052902
Medline
9. N. Hashimoto, K. Hamamura, N. Kotani, K. Furukawa, K. Kaneko, K. Honke, K. Furukawa,
Proteomic analysis of ganglioside-associated membrane molecules: Substantial basis for
molecular clustering. Proteomics 12, 3154–3163 (2012). doi:10.1002/pmic.201200279
Medline
10. J. S. Rees, X.-W. Li, S. Perrett, K. S. Lilley, A. P. Jackson, Selective proteomic proximity
labeling assay using tyramide (SPPLAT): A quantitative method for the proteomic
analysis of localized membrane-bound protein clusters. Curr. Protoc. Protein Sci. 80,
19.27.11–19.27, 18 (2015). doi:10.1002/0471140864.ps1927s80 Medline
11. E. Choi-Rhee, H. Schulman, J. E. Cronan, Promiscuous protein biotinylation by Escherichia
coli biotin protein ligase. Protein Sci. 13, 3043–3050 (2004).
12. L. K. Folkes, M. Trujillo, S. Bartesaghi, R. Radi, P. Wardman, Kinetics of reduction of
tyrosine phenoxyl radicals by glutathione. Arch. Biochem. Biophys. 506, 242–249 (2011).
doi:10.1016/j.abb.2010.12.006 Medline
13. H.-W. Rhee, P. Zou, N. D. Udeshi, J. D. Martell, V. K. Mootha, S. A. Carr, A. Y. Ting,
Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic
tagging. Science 339, 1328–1331 (2013). doi:10.1126/science.1230593 Medline
14. D. I. Kim, K. C. Birendra, W. Zhu, K. Motamedchaboki, V. Doye, K. J. Roux, Probing
nuclear pore complex architecture with proximity-dependent biotinylation. Proc. Natl.
Acad. Sci. U.S.A. 111, E2453–E2461 (2014). doi:10.1073/pnas.1406459111 Medline
15. K. Bersuker, C. W. H. Peterson, M. To, S. J. Sahl, V. Savikhin, E. A. Grossman, D. K.
Nomura, J. A. Olzmann, A proximity labeling strategy provides insights into the
composition and dynamics of lipid droplet proteomes. Dev. Cell 44, 97–112.e7 (2018).
doi:10.1016/j.devcel.2017.11.020 Medline
16. K. H. Loh, P. S. Stawski, A. S. Draycott, N. D. Udeshi, E. K. Lehrman, D. K. Wilton, T.
Svinkina, T. J. Deerinck, M. H. Ellisman, B. Stevens, S. A. Carr, A. Y. Ting, Proteomic
analysis of unbounded cellular compartments: Synaptic clefts. Cell 166, 1295–1307.e21
(2016). doi:10.1016/j.cell.2016.07.041 Medline
17. D. Z. Bar, K. Atkatsh, U. Tavarez, M. R. Erdos, Y. Gruenbaum, F. S. Collins, Biotinylation
by antibody recognition-a method for proximity labeling. Nat. Methods 15, 127–133
(2018). doi:10.1038/nmeth.4533 Medline
18. D. Z. Bar, K. Atkatsh, U. Tavarez, M. R. Erdos, Y. Gruenbaum, F. S. Collins, Addendum:
Biotinylation by antibody recognition-a method for proximity labeling. Nat. Methods 15,
749 (2018). doi:10.1038/s41592-018-0073-4 Medline
19. X.-W. Li, J. S. Rees, P. Xue, H. Zhang, S. W. Hamaia, B. Sanderson, P. E. Funk, R. W.
Farndale, K. S. Lilley, S. Perrett, A. P. Jackson, New insights into the DT40 B cell
receptor cluster using a proteomic proximity labeling assay. J. Biol. Chem. 289, 14434–
14447 (2014). doi:10.1074/jbc.M113.529578 Medline
20. N. Kotani, J. Gu, T. Isaji, K. Udaka, N. Taniguchi, K. Honke, Biochemical visualization of
cell surface molecular clustering in living cells. Proc. Natl. Acad. Sci. U.S.A. 105, 7405–
7409 (2008). doi:10.1073/pnas.0710346105 Medline
21. T. Hayashi, Y. Yasueda, T. Tamura, Y. Takaoka, I. Hamachi, Analysis of cell-surface
receptor dynamics through covalent labeling by catalyst-tethered antibody. J. Am. Chem.
Soc. 137, 5372–5380 (2015). doi:10.1021/jacs.5b02867 Medline
22. A. Admasu, A. D. Gudmundsdóttir, M. S. Platz, D. S. Watt, S. Kwiatkowski, P. J. Crocker, A
laser flash photolysis study of p-tolyl(trifluoromethyl)carbene. J. Chem. Soc., Perkin
Trans. 2 (5): 1093–1100 (1998). doi:10.1039/a707586c
23. H. C. Berg, Random Walks in Biology (Princeton Univ. Press, 1993).
24. S.-S. Ge, B. Chen, Y.-Y. Wu, Q.-S. Long, Y.-L. Zhao, P.-Y. Wang, S. Yang, Current
advances of carbene-mediated photoaffinity labeling in medicinal chemistry. RSC Adv. 8,
29428 (2018).
25. J. Brunner, H. Senn, F. M. Richards, 3-Trifluoromethyl-3-phenyldiazirine. A new carbene
generating group for photolabeling reagents. J. Biol. Chem. 255, 3313–3318 (1980).
Medline
26. D. M. Arias-Rotondo, J. K. McCusker, The photophysics of photoredox catalysis: A roadmap
for catalyst design. Chem. Soc. Rev. 45, 5803–5820 (2016). doi:10.1039/C6CS00526H
Medline
27. J. Wang, J. Kubicki, H. Peng, M. S. Platz, Influence of solvent on carbene intersystem
crossing rates. J. Am. Chem. Soc. 130, 6604–6609 (2008). doi:10.1021/ja711385t
Medline
28. A. Singh, K. Teegardin, M. Kelly, K. S. Prasad, S. Krishnan, J. D. Weaver, Facile synthesis
and complete characterization of homoleptic and heteroleptic cyclometalated Iridium(III)
complexes for photocatalysis. J. Organomet. Chem. 776, 51–59 (2015).
doi:10.1016/j.jorganchem.2014.10.037
29. L. Y. Bourguignon, S. J. Singer, Transmembrane interactions and the mechanism of capping
of surface receptors by their specific ligands. Proc. Natl. Acad. Sci. U.S.A. 74, 5031–5035
(1977). doi:10.1073/pnas.74.11.5031 Medline
30. L. Y. Bourguignon, Biochemical analysis of ligand-induced receptor patching and capping
using a novel immunolactoperoxidase iodination technique. J. Cell Biol. 83, 649–656
(1979). doi:10.1083/jcb.83.3.649 Medline
31. M. L. Hermiston, Z. Xu, A. Weiss, CD45: A critical regulator of signaling thresholds in
immune cells. Annu. Rev. Immunol. 21, 107–137 (2003).
doi:10.1146/annurev.immunol.21.120601.140946 Medline
32. D. Szklarczyk, A. L. Gable, D. Lyon, A. Junge, S. Wyder, J. Huerta-Cepas, M. Simonovic,
N. T. Doncheva, J. H. Morris, P. Bork, L. J. Jensen, C. V. Mering, STRING v11: Protein-
protein association networks with increased coverage, supporting functional discovery in
genome-wide experimental datasets. Nucleic Acids Res. 47 (D1), D607–D613 (2019).
Medline
33. V. Hung, N. D. Udeshi, S. S. Lam, K. J. Loh, K. J. Cox, K. Pedram, S. A. Carr, A. Y. Ting,
Spatially resolved proteomic mapping in living cells with the engineered peroxidase
APEX2. Nat. Protoc. 11, 456–475 (2016).
34. H. O. Alsaab, S. Sau, R. Alzhrani, K. Tatiparti, K. Bhise, S. K. Kashaw, A. K. Iyer, PD-1 and
PD-L1 Checkpoint signaling inhibition for cancer immunotherapy: Mechanism,
combinations, and clinical outcome. Front. Pharmacol. 8, 561 (2017).
doi:10.3389/fphar.2017.00561 Medline
35. C. A. van der Weyden, S. A. Pileri, A. L. Feldman, J. Whisstock, H. M. Prince,
Understanding CD30 biology and therapeutic targeting: A historical perspective
providing insight into future directions. Blood Cancer J. 7, e603 (2017).
doi:10.1038/bcj.2017.85 Medline
36. O. Zenarruzabeitia, J. Vitallé, C. Eguizabal, V. R. Simhadri, F. Borrego, The biology and
disease relevance of CD300a, an inhibitory receptor for phosphatidylserine and
phosphatidylethanolamine. J. Immunol. 194, 5053–5060 (2015).
doi:10.4049/jimmunol.1500304 Medline
37. J. R. James, R. D. Vale, Biophysical mechanism of T-cell receptor triggering in a
reconstituted system. Nature 487, 64–69 (2012). doi:10.1038/nature11220 Medline
38. C. B. Carbone, N. Kern, R. A. Fernandes, E. Hui, X. Su, K. C. Garcia, R. D. Vale, In vitro
reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase. Proc.
Natl. Acad. Sci. U.S.A. 114, E9338–E9345 (2017). doi:10.1073/pnas.1710358114
Medline
39. M. L. Dustin, The immunological synapse. Cancer Immunol. Res. 2, 1023–1033 (2014).
doi:10.1158/2326-6066.CIR-14-0161 Medline
40. I. V. Pinchuk, E. J. Beswick, V. E. Reyes, Staphylococcal enterotoxins. Toxins (Basel) 2,
2177–2197 (2010). doi:10.3390/toxins2082177 Medline
41. M. C. Montoya, D. Sancho, G. Bonello, Y. Collette, C. Langlet, H. T. He, P. Aparicio, A.
Alcover, D. Olive, F. Sánchez-Madrid, Role of ICAM-3 in the initial interaction of T
lymphocytes and APCs. Nat. Immunol. 3, 159–168 (2002). doi:10.1038/ni753 Medline
42. C. White, R. Oslund, O. Fadeyi, T. Reyes Robles, Flow cytometry data files for article:
Microenvironment mapping via Dexter energy transfer on immune cells. Harvard
Dataverse (2020); doi:10.7910/DVN/O6O04O.
43. C. White, Merck/Photoproximity_Labeling: Photoproximity_Labeling_v1.0. Zenodo (2020);
doi:10.5281/zenodo.3608233.
44. J. I. Day, K. Teegardin, J. Weaver, J. Chan, Advances in photocatalysis: A microreview of
visible light mediated ruthenium and iridium catalyzed organic transformations. Org.
Process Res. Dev. 20, 1156–1163 (2016). doi:10.1021/acs.oprd.6b00101 Medline
45. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, ed. 3, 2006).
46. A. B. Pangborn, M. A. Giardello, R. H. Grubbs, R. K. Rosen, F. J. Timmers, Safe and
Convenient Procedure for Solvent Purification. Organometallics 15, 1518–1520 (1996).
doi:10.1021/om9503712
47. S. Le Saux et al., Generation of Immuno-Modulator Receptors (IMR)-transduced human JY
cell lines to test combo immune-therapies. J. Immunol. 198, 204 (2017).
48. B. Bhagwat, H. Cherwinski, M. Sathe, W. Seghezzi, T. K. McClanahan, R. de Waal Malefyt,
A. Willingham, Establishment of engineered cell-based assays mediating LAG3 and PD1
immune suppression enables potency measurement of blocking antibodies and
assessment of signal transduction. J. Immunol. Methods 456, 7–14 (2018).
doi:10.1016/j.jim.2018.02.003 Medline
49. H. Yu, L. Xiao, X. Yang, L. Shao, Controllable access to multi-substituted imidazoles via
palladium(ii)-catalyzed C-C coupling and C-N condensation cascade reactions. Chem.
Commun. 53, 9745–9748 (2017). doi:10.1039/C7CC05315K Medline
50. L. A. Terry, M. H. Brown, P. C. Beverley, The monoclonal antibody, UCHL1, recognizes a
180,000 MW component of the human leucocyte-common antigen, CD45. Immunology
64, 331–336 (1988). Medline
51. A. Miyagawa-Yamaguchi, N. Kotani, K. Honke, Each GPI-anchored protein species forms a
specific lipid raft depending on its GPI attachment signal. Glycoconj. J. 32, 531–540
(2015). doi:10.1007/s10719-015-9595-5 Medline
52. J. K. Eng, A. L. McCormack, J. R. Yates, An approach to correlate tandem mass spectral
data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass
Spectrom. 5, 976–989 (1994). doi:10.1016/1044-0305(94)80016-2 Medline
53. E. L. Huttlin, M. P. Jedrychowski, J. E. Elias, T. Goswami, R. Rad, S. A. Beausoleil, J.
Villén, W. Haas, M. E. Sowa, S. P. Gygi, A tissue-specific atlas of mouse protein
 phosphorylation and expression. Cell 143, 1174–1189 (2010).
doi:10.1016/j.cell.2010.12.001 Medline
54. J. E. Elias, S. P. Gygi, Target-decoy search strategy for increased confidence in large-scale
protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).
doi:10.1038/nmeth1019 Medline
55. R Development Core Team, R (R foundation for statistical computing Vienna, Austria,
2011).
56. H. Wickham, ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag, 2016).
57. M. E. Ritchie, B. Phipson, D. Wu, Y. Hu, C. W. Law, W. Shi, G. K. Smyth, limma powers
differential expression analyses for RNA-sequencing and microarray studies. Nucleic
Acids Res. 43, e47 (2015). doi:10.1093/nar/gkv007 Medline
58. Y. Benjamini, Y. Hochberg, Controlling the false discovery rate: A practical and powerful
approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 57, 289–300 (1995).