Supporting Online Material

Supplementary Information for

Photoaffinity-engineered protein scaffold for systematically exploring native phosphotyrosine signaling complexes in tumor samples

Bizhu Chu, An He, Yeteng Tian, Wan He, Peizhong Chen, Jintao Hu, Ruilian Xu, Wenbin Zhou, Mingjie Zhang, Pengyuan Yang, Shawn S. C. Li, Ying Sun, Pengfei Li, Tony Hunter, and Ruijun Tian

Ruijun Tian, Pengfei Li, and Ying Sun Email: [email protected], [email protected], [email protected]

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Materials and Methods 1. Trifunctional Probes Design and Chemical Synthesis 2. Preparation of Photo-pTyr-scaffold 3. Cell Culture 4. Tissue and Cell Lysate Preparation 5. Photo-pTyr-scaffold Photoreactive Crosslinking and Pull-down 6. SDS-PAGE and Western Blot Analysis 7. Immunohistochemical Analysis 8. Affinity Purification 9. MS Sample Preparation 10. MS Analysis 11. Data Analysis 12. Animal Studies Figs. S1 to S7 Quality control data S1 to S9 References for SI reference citations

1 www .pnas.org/cgi/doi/10.1073/pnas.1805633115

Supplementary Information Text Supplemental information includes supplemental materials and methods, seven figures, nine quality control data, and supplemental references.

Materials and Methods 1. Trifunctional Probes Design and Chemical Synthesis 1.1. Design of Trifunctional Probes The designed trifunctional probes comprise the following unique features: (1) an N- hydroxysuccinimide (NHS) ester group for react with the SH2 domains, which containing the free primary amines of lysine residues, without affecting the high selectivity and affinity toward pTyr proteins; (2) photoreactive groups for covalently crosslinking to pTyr protein complexes following exposure to mild UV light; (3) a biotin group for capturing crosslinked protein complexes, even under harsh washing conditions; and (4) flexible spacer arm which ensures efficient crosslinking of multi-protein complexes. Consequently, we used a modular strategy employing L-lysine as a trifunctional scaffold (Fig. 1). The aryl azide, benzophenone, and diazirine were chosen as photoreactive group, respectively. 1,13-diamino-4,7,10-trioxatridecane was used to link biotin group with L-lysine via amido bond. The photoreactive group was introduced into L-lysine skeleton via peptide linkage. The NHS group was tied to the side chain of L-lysine via a linker derived from dihydrofuran-2,5-dione and 6- aminohexanoic acid.

1.2. Synthesis of Trifunctional Probes Chemicals and materials were purchased from commercial sources, and used as received without further purification unless otherwise noted. Analytical TLC was carried out on Silica Gel 60 Å F254 plates with detection by a UV detector and/or by charring with 10% 4- (Dimethylamino)cinnamaldehyde in EtOH (w/v). Semi-preparative high performance liquid chromatography was used for the preparation of intermediates and the trifunctional probes. MS analysis was performed on a high-resolution Q-Exactive Orbitrap mass spectrometer (Thermo Fisher). NMR spectra were recorded on a 400 and 500 MHz machine with chemical shifts reported in ppm (δ) downfield from internal tetramethylsilane (TMS) reference. Signals are described as s (singlet), d (doublet), t (triplet), q (quintet) or m (multiplet), and the coupling constants were reported in Hz.

1.2.1. Synthesis of Probe 0

O O O O O O O H HN H H HN FmocHN N N O N NH HO N O N NH H 3 H H 3 H S H O S H c1 c2

NHBoc NHBoc O O O O O O O O O H H HN H H HN N N N HO N O N NH O N O N NH H 3 H H 3 H O S H O O S H O O c3 Probe 0 HN HN O O

Compound c1 was synthesized according to the reported procedure(1, 2). The characterizations of c1 were consistent with reported data. A mixture of c1 (896 mg, 1 mmol) and diethylamine (2.19 g, 30 mmol) in acetonitrile (ACN) was stirred at room temperature. Once the starting material c1 disappeared (monitored by TLC), the

solvent was removed. The residue was dissolved in dimethylformamide (DMF, 10 mL). Then N,N- diisopropylethylamine (258 mg, 2 mmol) and succinic anhydride (200 mg, 2 mmol) were added. The new mixture was stirred for over 12 h. Then DMF was removed, and the residue was separated - by silica gel to furnish c2 in 50% yield (387 mg). HRMS (m/z): [M-H] calcd for C35H61O11N6S 773.4125, found 773.4125. Trifluoroacetic acid (TFA, 1.026 g, 8.9 mmol) was added to a solution of c2 (70 mg, 0.09 mmol) in dichloromethane (5 mL) at 0 °C. TLC indicated the starting materials c2 disappeared after stirring for 2 h. The mixture was evaporated under vacuum. The mixture then was dissolved in DMF (5 mL), and triethylamine was added to adjust pH to larger than 9. 2,5-dioxopyrrolidin-1-yl 4-benzoylbenzoate (43.6 mg, 0.135 mmol) was then added, and the mixture was stirred at room temperature overnight. The solvent was removed under vacuum, and the residue was separated by - silica gel to furnish c3 in 59% yield (47 mg). HRMS (m/z): [M-H] calcd for C44H61O11N6S 881.4125, found 881.4130. To a solution of compound c3 (47 mg, 0.053 mmol), and hydroxysuccinimide (12 mg, 0.105 mmol) in DMF (5 mL), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI, 30 mg, 0.158 mmol) was added. The mixture was incubated overnight. When compound c3 disappeared (monitored by TLC), the solvent was removed under vacuum. The residue was separated and purified via semi-preparative high performance liquid chromatography to gain probe 0 with a yield 1 of 25% (13 mg). H NMR (500 MHz, DMF-d7) δ (ppm) 8.71 (t, J = 5.5 Hz, 1H), 8.17-8.12 (m, 3H), 7.90-7.83 (m, 5H), 7.76-7.73 (m, 2H), 7.64-7.61 (m, 2H), 6.42 (s, 1H), 6.32 (s, 1H), 4.48-4.45 (m, 1H), 4.36-4.28 (m, 2H), 3.59-3.57 (m, 5H), 3.48-3.38 (m, 7H), 3.24-3.18 (m, 5H), 2.99-2.92 (m, 5H), 2.72-2.69 (m, 3H), 2.16 (t, J = 7.5 Hz, 2H), 1.86-1.36 (m, 16H). 13C NMR (126 MHz, DMF- d7) δ (ppm) 196.3, 172.9, 172.4, 170.8, 169.7, 166.3, 163.7, 140.2, 139.1, 137.9, 133.6, 130.5, 130.3, 129.3, 128.0, 70.9, 70.6, 69.2, 69.1, 62.2, 60.6, 56.5, 54.0, 40.8, 40.2, 36.9, 36.8, 36.2, 32.8, 29.2, + 29.1, 26.8, 26.3, 26.2, 23.9. HRMS (m/z): [M+H] calcd for C48H66O13N7S 980.4434, found 980.4421.

1.2.2. Synthesis of Probe 1

Compound c4 was synthesized according to the reported procedure(1-3). The characterizations of c4 were consistent with reported data. TFA (1.026 g, 9 mmol) was added to a solution of c4 (100 mg, 0.09 mmol) in dichloromethane (5 mL) at 0 °C. TLC indicated the starting materials c4 disappeared after stirring for 2 h. The mixture was evaporated under vacuum. The mixture then was dissolved in DMF (5 mL), and triethylamine

was added to adjust pH to larger than 9. 2,5-dioxopyrrolidin-1-yl 4-benzoylbenzoate (43.6 mg, 0.135 mmol) was then added, and the mixture was incubated overnight. The solvent was removed under vacuum, and the residue was separated by silica gel to furnish c5 in 59% yield (65 mg). + HRMS (m/z): [M+Na] calcd for C62H95O14N9SNa 1244.6611, found 1244.6584. EDCI (30 mg, 0.158 mmol) was added to a solution of compound c5 (65 mg, 0.053 mmol), hydroxysuccinimide (12 mg, 0.105 mmol) in DMF (5 mL). The mixture was incubated overnight. When compound c5 disappeared (monitored by TLC), the solvent was removed under vacuum. The residue was separated and purified via semi-preparative high performance liquid chromatography 1 to gain probe 1 with a yield of 24% (17 mg). H NMR (500 MHz, DMF-d7) δ 8.71 (br s, 1H), 8.13 (d, J = 8.0 Hz, 2H), 8.01-7.83 (m, 7H), 7.75-7.73 (m, 4H), 7.62 (t, J = 7.5 Hz, 2H), 6.42 (s, 1H), 6.33 (s, 1H), 4.48-4.46 (m, 1H), 4.30-4.22 (m, 2H), 3.66-3.57 (m, 7H), 3.49-3.39 (m, 8H), 3.23- 3.20 (m, 5H), 3.17-3.12 (m, 6H), 2.95-2.93 (m, 3H), 2.72-2.68 (m, 4H), 2.56-2.42 (m, 4H), 2.18- 13 2.12 (m, 6H), 1.78-1.55 (m, 18H), 1.51-1.28 (m, 16H). C NMR (126 MHz, DMF-d7) δ 196.2, 172.9, 172.8, 172.6, 172.5, 170.9, 169.8, 166.3, 162.9, 162.7, 162.5, 140.2, 139.1, 137.8, 133.6, 130.5, 130.3, 129.3, 128.0, 70.9, 70.6, 69.2, 62.2, 60.6, 56.5, 54.0, 40.8, 40.3, 39.4, 39.3, 39.2, 37.0, 36.8, 36.4, 36.2, 32.3, 31.8, 31.6, 31.0, 30.0, 29.9, 29.2, 29.1, 27.2, 26.5, 26.4, 26.2, 26.1, 26.0, + 25.0, 23.9. HRMS (m/z): [M+H] calcd for C66H99O16N10S 1319.6956, found 1319.6957.

1.2.3. Synthesis of Probe 2

TFA (1.026 g, 9 mmol) was added to a solution of c4 (100 mg, 0.09 mmol) in dichloromethane (5 mL) at 0 °C. TLC indicated the starting material c4 disappeared after stirring for 2 h. The mixture was evaporated under vacuum. The mixture then was dissolved in DMF (5 mL), and triethylamine was added to adjust pH to larger than 9. 2,5-dioxopyrrolidin-1-yl 4-azidobenzoate (35.2 mg, 0.14 mmol) was then added, and the mixture was stirred at room temperature overnight. The solvent

was removed under vacuum, and the residue was separated by silica gel to furnish c6 in 61% yield - (63 mg). HRMS (m/z): [M-H] calcd for C55H89O13N12S 1157.6398, found 1157.6417. EDCI (30 mg, 0.158 mmol) was added to a solution of compound c6 (61 mg, 0.053 mmol), hydroxysuccinimide (12 mg, 0.106 mmol) in DMF (5 mL). The mixture was stirred at room temperature overnight. When compound c6 disappeared (monitored by TLC), the solvent was removed under vacuum. The residue was separated and purified via semi-preparative high performance liquid chromatography to gain compound probe 2 with a yield of 24% (16 mg). 1H NMR (500 MHz, DMF-d7) δ 8.50 (s, 1H), 8.03-8.01 (m, 3H), 7.97-7.90 (m, 2H), 7.77-7.73 (m, 3H), 7.24-7.21 (m, 2H), 6.43 (s, 1H), 6.34 (s, 1H), 4.48-4.46 (m, 1H), 4.31-4.22 (m, 2H), 3.66-3.57 (m, 9H), 3.49-3.45 (m, 6H), 3.24-3.19 (m, 6H), 3.16-3.11 (m, 5H), 2.93-2.91 (m, 5H), 2.71-2.68 (m, 2H), 2.54-2.43 (m, 4H), 2.18-2.12 (m, 6H), 1.75-1.67 (m, 7H), 1.61-1.26 (m, 27H). 13C NMR (126 MHz, DMF-d7) δ 172.9, 172.8, 172.6, 172.1, 172.0, 171.5, 171.0, 170.8, 169.8, 166.1, 163.7, 143.2, 132.4, 129.8, 119.5, 70.9, 70.6, 69.2, 62.2, 60.6, 56.5, 54.0, 51.8, 40.8, 40.2, 39.3, 39.2, 38.2, 37.0, 36.8, 36.4, 36.2, 34.2, 32.3, 31.8, 31.6, 31.0, 29.2, 29.1, 27.2, 26.5, 26.4, 26.2, 26.1, 25.0, 23.9. + HRMS (m/z): [M+H] calcd for C59H94O15N13S 1256.6708, found 1256.6705.

1.2.4. Synthesis of Probe 3

O O O O H H HN HO N N N O N NH H H 3 H O O S H

c4 O O O O H H HN HN O HO N N N O N NH H H 3 H O O S H

O c7 NHBoc HN O N H O O O O O H H HN O N O N N N O N NH H N N H H 3 H N O O S H N O H O

HN O Probe 3

O H N N N N H O

TFA (1.026 g, 9 mmol) was added to a solution of c4 (100 mg, 0.09 mmol) in dichloromethane (5 mL) at 0 °C. TLC indicated the starting materials c4 disappeared after stirring for 2 h. The mixture was evaporated under vacuum. The mixture then was dissolved in DMF (5 mL), and triethylamine was added to adjust pH to larger than 9. 2,5-dioxopyrrolidin-1-yl 3-(3-methyl-3H-diazirin-3- yl)propanoate (30 mg, 0.135 mmol) was then added, and the mixture was incubated overnight. The solvent was removed under vacuum, and the residue was separated by silica gel to furnish c7 in 61% + yield (61 mg). HRMS (m/z): [M+H] calcd for C53H94O13N11S 1124.6748, found 1124.6478.

EDCI (30 mg, 0.158 mmol) was added to a solution of compound c7 (60 mg, 0.053 mmol), hydroxysuccinimide (12 mg, 0.105 mmol) in DMF (5 mL). The mixture was stirred at room temperature overnight. When compound c7 disappeared (monitored by TLC), the solvent was removed under vacuum. The residue was separated and purified via semi-preparative high performance liquid chromatography to gain compound probe 3 with a yield of 25% (16 mg). 1H NMR (500 MHz, DMF-d7) δ 8.12-8.04 (s, 1H), 7.97-7.95 (m, 1H), 7.93-7.91 (m, 1H), 7.84-7.83 (m, 1H), 7.78-7.73 (m, 3H), 6.43-6.34 (m, 2H), 4.48-4.46 (m, 1H), 4.31-4.29 (m, 1H), 4.27-4.22 (m, 1H), 3.59-3.57 (m, 3H), 3.54-3.53 (m, 5H), 3.49-3.45 (m, 5H), 3.24-3.11 (m, 13H), 2.93-2.91 (m, 4H), 2.73-2.69 (m, 3H), 2.56-2.43 (m, 4H), 2.18-2.12 (m, 6H), 2.08-2.06 (m, 2H), 1.78-1.51 13 (m, 19H), 1.50-1.27 (m, 17H), 1.01 (s, 3H). C NMR (126 MHz, DMF-d7) δ 172.9, 172.8, 172.7, 172.6, 171.5, 171.0, 170.9, 169.8, 163.7, 70.9, 70.7, 69.2, 62.3, 60.6, 56.6, 54.0, 51.9, 51.7, 40.8, 39.5, 39.4, 39.3, 37.0, 36.9, 36.4, 36.2, 32.3, 31.8, 31.7, 31.0, 30.9, 30.7, 29.3, 29.1, 27.2, 26.5, + 26.4, 26.3, 26.1, 25.0, 24.0, 19.6. HRMS (m/z): [M+H] calcd for C57H97O15N12S 1221.6912, found 1221.6886.

2. Preparation of Photo-pTyr-scaffold 2.1. Expression and Purification of Recombinant Proteins The following plasmids were transformed into BL21 cells, including GST-tagged NPDZ1 (4, 5), His-tagged SAM-PBM (5), His-tagged Cad23-cyto (4, 6), GST-tagged N-PI3K, GST-tagged GRB2 SH2 domain, GST-tagged SHC1 SH2 domain (provided by Prof. Y. Zheng, National Center for Protein Sciences, Beijing, China) and GST-tagged Src SH2 domain superbinder. The overnight cultured BL21 cells were 1:500 diluted with fresh LB medium containing 50 mg/L ampicillin or kanamycin. The cells were then grown at 37 °C to an OD600 of 0.6. Isopropyl β-D-1- thiogalactopyranoside (IPTG, 0.25 mM) was then added into the medium for induction and culture for another 12 h at 16 °C. GST-tagged protein purification: one liter of BL21 cells were harvested by centrifugation at 6,000 × g for 30 min, wash with 1 × PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.7 mM KH2PO4, pH 7.4) once and centrifuge again. The cell pellet was resuspended in the lysis buffer of 1% (v/v) Triton X-100, 1.5 mM DL-dithiothreitol (DTT), 2 mM ethylenediaminetetraacetic acid (EDTA) in 1 × PBS, pH 8.0 and then sonicated 30 times for 5 s each at a power of 400 W, with a 15 s incubation on ice between each sonication. The obtained cell lysate was then cleaned up by centrifugation at 14,000 × g for 30 min at 4 °C and then loaded onto a Glutathione Sepharose 4B column (5 mL, GE Healthcare). The column was washed with two column volumes of the lysis buffer and eluted with elution buffer (50 mM Tris-HCl, 150 mM NaCl, and 10 mM reduced glutathione, pH 8.0). The elution buffer containing the expressed proteins were exchanged to the HEPES buffer (50 mM HEPES, 150 mM NaCl, 1 mM DTT, and 1 mM EDTA), pH 8.0 by ultrafiltration with a 3 kDa cut-off Amicon Ultra centrifugal filter device (Merck Millipore). The protein concentration was measured using the Bradford assay and adjusted to 1 mg/mL with the HEPES buffer. His-tagged protein purification: one liter of BL21 cells were harvested by centrifugation and washed with 1 × PBS. The cell pellet was resuspended in the lysis buffer of 50 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 8.0. After sonication and centrifugation as described above, the

cell lysate was loaded onto a Ni-NTA column (5 mL, GE Healthcare). The column was washed with two column volumes of washing buffer (20 mM Tris-HCl, 500 mM NaCl, and 50 mM imidazole, pH 8.0) and eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, and 1 M imidazole, pH 8.0). The elution buffer containing the expressed proteins were exchanged to the HEPES buffer and adjusted to 1 mg/mL as described above.

2.2. Trifunctional Probe NHS group Labeling and Optimization Photo-pTyr-scaffold preparation: fifty microliters of bait protein (N-PI3K, NPDZ1, SHC1 SH2 domain, GRB2 SH2 domain, and Src SH2 domain superbinder, 1 mg/mL in the HEPES buffer) was treated with 0.5 µL of probe (100 × stock in DMSO, protein-to-probe ratio of 1:10) or DMSO at room temperature for 1 min. Five microliters of 1 M glycine were then added to stop the reaction. The hydrolyzed and non-reacted probe was removed by ultrafiltration with a 3 kDa cut-off Amicon Ultra centrifugal filter device. As shown in Fig. S1A, the NHS group labeling efficiency optimization was done as follows: fifty microliters of the N-PI3K (1 mg/mL in the HEPES buffer) was treated with 0.5 µL of probe 1 (100 × stock in DMSO) or DMSO at room temperature for 1 to 10 min, with different protein-to-probe ratio (1:2 to 1:30). Five microliters of 1 M glycine were then added to stop the reaction and samples were subsequently boiled with 2 × SDS-PAGE loading buffer for SDS-PAGE or WB analysis. The Coomassie brilliant blue staining results shown that probe 1 treatment lead to a clear N-PI3K protein shift in a time- and ratio-dependent manner. This result was also confirmed by WB analysis with streptavidin-HRP. Next, we want to know whether the NHS group labeling of bait protein will affect its binding affinity to prey proteins. As shown in Fig. S1C, we used N-PI3K for the test as its active form will capture pTyr proteins. Fifty microliters of the N-PI3K was engineered by probe 1 at room temperature for 1 min as described above. Five microliters of 1 M glycine were then added to stop the reaction. The hydrolyzed and non-reacted probe was removed by ultrafiltration with a 3 kDa cut-off Amicon Ultra centrifugal filter device. The GST-tagged and probe 1-engineered N-PI3K was incubated with 100 μg of freshly isolated HeLa cell total lysate which was stimulated by 1 mM pervanadate (PV) for boosting up tyrosine phosphorylation. The result shown that the NHS group labeling did not affect the N-PI3K binding affinity to pTyr proteins with a protein-to-probe ratio of less than 1:30. NHS group labeling time of 1 min at a protein-to-probe ratio of 1:10 was therefore used throughout the study for all five probes (probe 0-3 and Sulfo-SBED).

3. Cell Culture The human cervical cancer cell lines HeLa, human breast cancer cell lines MDA-MB-468, MDA- MB-231 and MCF-7 were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 1% (v/v) penicillin/streptomycin (Gibco). Human breast cancer cell lines BT-474 and SK-BR-3 were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco) supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin. Human breast cell lines MCF 10A were cultured in RPMI 1640 Medium supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin. Human Mammary Fibroblasts (HMF) were cultured in Fibroblast Medium (FM) (ScienCell) supplemented with 2% (v/v) FBS (ScienCell), 1% (v/v) fibroblast growth supplement (FGS, ScienCell) and 1% (v/v) penicillin/streptomycin (ScienCell). The cell lines were maintained at 37 °C in 5% CO2. For stable isotope labeling with amino acids in cell culture (SILAC) labeling, the HeLa cells were cultured in three different SILAC mediums: L-lysine and L-arginine free DMEM medium (Caisson) supplied with 10% (v/v) dialyzed FBS (Thermo Fisher), 1% (v/v) penicillin/streptomycin, 50 mg/L normal 2 15 13 L-lysine and L-arginine (Light), or L-[ H4]-lysine and L-[ N4]-arginine (Medium), or L-[ C6, 15 13 15 N2]-lysine and L-[ C6, N4]-arginine (Heavy) (Thermo Fisher). The cells were cultured for at least ten passages to ensure full incorporation. For the conditioned medium stimulation, the confluent cells (MDA-MB-468) were washed twice with FBS-free medium, and starve the cells

with FBS-free medium for 24 h at 37 °C. The conditioned medium was collected, then cleaned up by centrifugation at 1,000 × g. HMF also cultured with FBS-free medium for 24 h in the presence and absence of crenolanib (0.5 μM, 12 h, Selleck) at 37 °C, then treated with conditioned medium or 100 ng/mL PDGFB for 5 min.

4. Tissue and Cell Lysate Preparation Human breast cancer tissue samples were obtained from Shenzhen People’s Hospital and then de- identified to protect patient confidentiality. All patients provided written informed consent, and all human studies were approved by the institutional review board. The samples were stored in liquid nitrogen. Fifty milligrams of human breast cancer tissue samples were washed with ice-cold 1 × PBS and then lysed by using PowerLyzer Bead-Based Homogenizer (MOBIO) for 4 rounds of 10 s at a speed of 3,500 rpm, with a 60 s incubation on ice between rounds. Ice-cold NP-40 buffer [50 mM Tris-HCl, 150 mM NaCl, and 1% (v/v) NP-40, pH 7.4] containing freshly added protease and phosphatase inhibitor cocktails (Roche) was used as the lysis buffer. The obtained cell lysate was sonicated 4 times for 5 s each at a power of 200 W, with a 10 s incubation on ice between each sonication. The lysate was then clarified by centrifugation at 14,000 × g for 10 min at 4 °C. The protein concentration was measured using the bicinchoninic acid (BCA) assay. When the cells reached 80% confluence, starve the cells with FBS-free medium for 4 h at 37 °C, then cells were treated with 100 ng/mL EGF or PDGFB for 5 min or 1 mM PV for 10 min. The cells were washed twice with ice-cold 1 × PBS. Total cell lysates were prepared by scraping cells from the petri dishes in ice-cold NP-40 buffer as described above. The obtained cell lysate was sonicated 4 times for 5 s each at a power of 200 W, with a 10 s incubation on ice between each sonication. The lysate was then clarified by centrifugation at 14,000 × g for 10 min at 4 °C. The protein concentration was measured using the BCA assay.

5. Photo-pTyr-scaffold Photoreactive Crosslinking and Pull-down Fifty micrograms of Photo-pTyr-scaffold was prepared as described in section 2.2. The Photo- pTyr-scaffold was incubated with the prey protein or freshly isolated total lysate in NP-40 buffer. Samples were incubated for 2 h at 4 °C with end-over-end rotation to allow the protein complexes to form. Then, complexes were placed in a quartz colorimetric cuvette and subjected to UV irradiation for 30 min in the UVP CL-1000L UV Crosslinker (365 nm, UVP) at a distance of ~3 cm on ice. After UV irradiation, the samples were incubated with 30 μL of streptavidin beads (GE Healthcare) for 2 h at 4 °C with end-over-end rotation. The beads were washed three times with 1 mL of harsh modified RIPA buffer [50 mM Tris-HCl, 1 M NaCl, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, and 1% (w/v) SDS, pH 7.4], and subsequently the samples were subjected to WB or MS analysis. The benzophenone group photoreactive crosslinking efficiency optimization was done as follows (related to Fig. S1D): The NHS group of probe 1 was first blocked by treating 0.5 µL of probe 1 (100 × stock in DMSO) with 5 µL 1 M glycine at room temperature for 60 min in the dark. Fifty microliters of the N-PI3K (1 mg/mL in the HEPES buffer, protein-to-probe ratio of 1:10) was then added to the buffer. The solution was irradiated for 10 to 60 min and subsequently boiled with 2 × SDS-PAGE loading buffer for WB analysis. The result shown that after UV irradiating for 10 to 60 min, the probe 1 can covalently bind to N-PI3K. For ensuring complete UV crosslinking and reported by other study (7), 30 min of UV irradiation was used throughout the study for all five probes (probe 0-3 and Sulfo-SBED).

6. SDS-PAGE and Western Blot Analysis The numbers of labeled probe 1 on each bait protein (related to Fig. S1B) was characterized by SDS-PAGE using PROTEAN® Ⅱ xi cell electrophoresis instrument (Bio-Rad, gel size: 16 × 20 cm), with an initial voltage of 200 V. The voltage was increased to 500 V when the sample has

completely entered into the separating gel. The SDS-PAGE was visualized by Coomassie brilliant blue staining. Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Roche). Membranes were blocked in 1 × TBST buffer [20 mM Tris-HCl, 138 mM NaCl, and 0.1% (v/v) Tween-20, pH 7.6] containing 5% (w/v) BSA at room temperature for 2 h and incubated with a primary antibody in 5% (w/v) BSA overnight at 4 °C, followed by an incubation with an HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology). Proteins were visualized using a Western ECL Substrate kit (Bio-Rad). The primary antibodies used in this study were: streptavidin-HRP (Thermo Fisher), anti-4G10 (Merck Millipore), anti-His (Cell Signaling Technology), anti-EGFR (Cell Signaling Technology), anti-ERBB2 (Cell Signaling Technology), anti-PDGFRB (Cell Signaling Technology), anti-p- STAT3 (Cell Signaling), anti-p-AKT (Cell Signaling), anti-AKT (Cell Signaling), anti-p-ERK 1/2 (Cell Signaling Technology), anti-ERK 1/2 (Cell Signaling Technology) and anti-β-actin (Beyotime).

7. Immunohistochemical Analysis Mice and patient tissue samples were fixed with 10% (w/v) buffered formalin for 12 h and embedded in paraffin. The paraffin-embedded slides (4 μm thick) were deparaffinized in xylene and then rehydrated using a graded ethanol series. Slides were processed for antigen retrieval using EDTA buffer (DAKO) in a pressure cooker at 100 °C for 5 min. After endogenous peroxidase activity was blocked with 3% (w/v) hydrogen peroxide, the slides were immunostained with the ERBB2 or PDGFRB primary antibody (Cell Signaling Technology) overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibody (DAKO). The signal was detected using a 3,3’-diaminobenzidine (DAB) detection kit (DAKO). Slides were counterstained with hematoxylin, dehydrated, mounted and analyzed by light microscopy. The slides were subsequently scored by visual assessment as “negative (-)”, “weak (1+)”, “moderate (2+)” or “strong (3+)”, according to the staining intensities of PDGFRB. In addition, quantification of PDGFRB were scored using the H-score according to staining intensities and percentage of positive cells within the whole tissue section (200 × magnification). For each mouse, three randomly fields were counted.

8. Affinity Purification 8.1. GST Pull-down Equimolar amounts of either GST-NPDZ1 and His-SAM-PBM, or GST-NPDZ1 and His-Cad23- cyto (0.6 nmol each) were mixed in 300 μL of NP-40 buffer. For SH2 domain-related experiments, the GST-tagged N-PI3K was incubated with 100 μg of freshly isolated total lysate in NP-40 buffer. Samples were incubated for 2 h at 4 °C with end-over-end rotation to allow the complexes to form. Samples were incubated with 30 μL of glutathione beads for 2 h at 4 °C with end-over-end rotation to pull-down the protein complexes. The beads were washed three times with 1 mL of RIPA buffer [50 mM Tris-HCl, 150 mM NaCl, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS, pH 7.4] and subsequently boiled with 2 × SDS-PAGE loading buffer for SDS-PAGE or WB analysis.

8.2. Streptavidin Pull-down Optimization of pull-down with streptavidin beads (related to Fig. S1E): fifty microliters of the N- PI3K was labeled with probe 1 as described in section 2.2. After removed the hydrolyzed and non- reacted probe by ultrafiltration, the probe 1-engineered N-PI3K was incubated with 30 μL of streptavidin beads in 300 μL of NP-40 buffer for 2 h at 4 °C with end-over-end rotation. The beads were washed three times with 1 mL of harsh modified RIPA buffer, and subsequently the beads, flow-through and wash buffers were detected by Coomassie brilliant blue staining and WB. The result shown that all probe 1-engineered N-PI3K was efficiently captured by 30 μL streptavidin

beads even under harsh washing conditions. Thirty microliters of the streptavidin beads were therefore adopted for the streptavidin pull-down. For NPDZ1 domain-related experiments (related to Fig. 2A and S2B), fifty microliters of the NPDZ1 domain were labeled with probe 1 as described in section 2.2. The probe 1-coupled NPDZ1 was incubated with SAM-PBM or Cad23-cyto (equimolar amounts) in 300 μL of the NP-40 buffer. Samples were incubated for 2 h at 4 °C with end-over-end rotation to allow the protein complexes to form. After UV irradiation for 30 min, the complexes were incubated with 30 μL of streptavidin beads for 2 h at 4 °C with end-over-end rotation. The beads were washed three times with 1 mL of harsh modified RIPA buffer and subsequently boiled with 2 × SDS-PAGE loading buffer for SDS- PAGE or WB analysis.

9. MS Sample Preparation The streptavidin pull-down for Photo-pTyr-scaffold approach was performed as described in section 5, with the exception that the Photo-pTyr-scaffold was incubated with 1.4 mg of freshly isolated total lysate in NP-40 buffer. After the streptavidin pull-down, the beads were washed three times with 1 mL of harsh modified RIPA buffer and then washed with 1 mL of 50 mM ammonium bicarbonate (ABC). Subsequently, the samples were reduced with 10 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) in 50 mM ABC at room temperature for 15 min and alkylated with 30 mM iodoacetamide (IAA) in 50 mM ABC for 30 min at room temperature in dark. After an additional 5 mM TCEP was added and the samples were incubated for 15 min, the beads were washed with 1 mL of 50 mM ABC. Then, the beads were incubated with 1 μg of trypsin (Promega) in 50 mM ABC overnight at 37 °C. The digested peptides were collected by washing the beads with 1% (v/v) formic acid (FA), and the peptides were subjected to StageTip C18 desalting before MS analysis. In-gel digestion was performed as described below (related to Fig. 2A). After Coomassie brilliant blue staining, bands from the SDS-PAGE gel were cut into small fragments of approximately 1 mm3 and transferred to Eppendorf tubes. The gel fragments were washed briefly with 50 mM ABC and shrunk with 50% (v/v) ACN in 25 mM ABC for 15 min at room temperature. After the samples were lyophilized to dryness, the gel fragments were swelled in 10 mM TCEP in 50 mM ABC at room temperature for 15 min. The samples were then alkylated by incubating them with 100 mM IAA and 50 mM ABC for 30 min at room temperature in dark. After washing and drying with speedvac, the samples were subjected to a tryptic digestion overnight at 37 °C. Finally, the digested peptides were harvested by washing the gel bands with 5% (v/v) FA and 50% (v/v) ACN, and the samples were then subjected to StageTip C18 desalting prior to the MS analysis. Total cell lysates were digested using simple and integrated spintip-based proteomics technology (SISPROT), which was described in our previous report (8) and briefly summarized here. The SISPROT tip contains strong cation exchange (SCX) beads and C18 membrane at the bottom. The SISPROT tip was firstly equilibrated with methanol and 10 mM potassium citrate buffer (pH 3.0). The protein samples (10 μg) were acidified to pH 3.0 and loaded onto the tip by centrifugation at 300 × g for 5 min. After the tip was washed with 20% (v/v) ACN in 8 mM potassium citrate buffer (pH 3.0), the proteins were reduced by infusing 10 mM TCEP in 9 mM potassium citrate buffer (pH 3.0) into the tip with a syringe and incubating the samples at room temperature for 15 min. The tip was then washed with water before an infusion with trypsin (2 μg/μL, 8 μg) in 10 mM IAA and 100 mM Tris-HCl, pH 8.0, and incubated at room temperature for 60 min in dark. Then, the digested peptides were transferred from the SCX beads to the C18 membrane by centrifugation at 400 × g for 5 min in the presence of 200 mM ABC. After a desalting wash with 1% (v/v) FA at 1,500 × g for 1 min, the peptides were eluted with 80% (v/v) ACN in 0.5% (v/v) acetic acid at 400 × g for 5 min. Finally, the eluted peptides were lyophilized to dryness. For the secretome analysis (related to Fig. 6D), the confluent cells (BT-474, MDA-MB-468 and MDA-MB-231) were washed twice with FBS-free medium, and starve the cells with FBS-free medium for 24 h at 37 °C. The conditioned medium was collected, then cleaned up by

centrifugation at 1,000 × g. The supernatant was concentrated and exchanged to the 1 × PBS buffer by ultrafiltration with a 3 kDa cut-off Amicon Ultra centrifugal filter device. After the samples were lyophilized to dryness, the proteins were dissolved in 8 M urea buffer. Subsequently, the protein samples (20 μg) were reduced with 5 mM TCEP at room temperature for 30 min and alkylated with 15 mM IAA for 30 min at room temperature in dark. After an additional 5 mM TCEP was added and the samples were incubated for 15 min, the 8 M urea buffer were diluted to 1 M with 100 mM Tris (pH 8.0) buffer. Then, the samples were incubated with 1 μg of trypsin overnight at 37 °C. The digested peptides were subjected to StageTip C18 desalting before MS analysis.

10. MS Analysis Peptides were resuspended in 15 μL 0.1% (v/v) FA. The samples were analyzed with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher, for the probe spacer arm analysis, background labeling analysis, and Src superbinder Photo-pTyr-scaffold enriched proteins from EGF stimulated HeLa cell lines, related to Fig. 2E, 3A, 3C-D and 4A) or Orbitrap Fusion mass spectrometer (Thermo Fisher, for the SILAC samples, breast cancer cell lines, tissue samples and secretome analysis, related to Fig. S3C-D, 4C-D, 5C and 6D) directly coupled to an Easy-nLC 1000 nano HPLC system (Thermo Fisher). The nano HPLC separation system consisted of an analytical column with an integrated spray tip (100 μm inner diameter × 20 cm) packed with 1.9 μm / 120 Å ReproSil-Pur C18 resin (Dr. Maisch GmbH). The buffers used for separation were 0.1% (v/v) FA in water (buffer A) and 0.1% (v/v) FA in ACN (buffer B). Samples (5 μL) were loaded onto the analytical column and separated at a flow rate of 200 nL/min. The gradient was set as follows: from 3 to 7% buffer B in 2 min, from 7 to 22% buffer B in 100 min, from 22 to 35% buffer B in 20 min, from 35 to 90% buffer B in 2 min, holding at 90% buffer B for 6 min, declining to 3% buffer B in 2 min, and holding at 3% buffer B for 13 min. For SILAC samples, the separation gradient times were increased to 200 min from 7 to 22% buffer B and 40 min from 22 to 35% buffer B, respectively. For the MS scans, the full MS scan range was set to 350-1,550 m/z with a mass resolution of 70,000 (Q-Exactive Orbitrap) or 12,000 (Orbitrap Fusion). The MS/MS spectra were acquired in data- dependent acquisition mode with the 10 most intense ions (Q-Exactive Orbitrap) or the Top Speed method (3 s, Orbitrap Fusion). Tandem MS was performed in the ion trap mass analyzer using an isolation window of 1.6 Da by quadrupole mass analyzer and high-energy collision-induced dissociation fragmentation with normalized collision energy of 27 (Q-Exactive Orbitrap) or 30 (Orbitrap Fusion).

11. Data Analysis The raw data files were searched with MaxQuant software (version 1.5.5.1) (9) against the human UniProt FASTA database (70,956 entries, downloaded on Dec 9, 2016). The false discovery rate (FDR) evaluation was done by searching a reverse database and was set to 0.01 for proteins and peptides. All statistical and bioinformatics analyses were performed with the Perseus software (version 1.5.5.3, as part of the MaxQuant environment) (10), Microsoft Excel or the R language (version 3.4.0). The following parameters were used for the label-free quantification (LFQ) analysis: cysteine carbamidomethylation was set as a fixed modification, whereas methionine oxidation, glutamine deamidation, serine/threonine/tyrosine phosphorylation were set as variable modifications. The maximum number of missed cleavages was set to two for trypsin digestion. The “Match between runs” and “Re-quantify” options were selected, and the other parameters were set to default values. Contaminants, proteins identified by reverse identification, proteins identified only by site and proteins with ≤ 2 unique peptides were excluded from further analysis. For the LFQ analysis of data obtained from the HeLa cell lines, breast cancer cell lines and secretome, protein groups identified with “3” valid values “in at least one group” were reserved, and missing values were assigned an artificial value sampled from a normal distribution (width = 0.3, down-shift = 1.8) (11). Proteins identified with an FDR = 0.05 (HeLa cell lines samples) or 0.01 (breast cancer cell lines

samples) and s0 = 2 were considered differentially regulated. For the LFQ analysis of data obtained from tissue samples, protein groups that were quantified in only one of twelve pull-down samples were filtered out. Fold changes (log2 ratios of the mean of the pull-down samples/the mean of the total cell lysate samples) were calculated and the fold changes > 1 were considered differentially enriched. The following parameters were used for the SILAC analysis: Lys4 and Arg4 were set as medium (M) labels; Lys8 and Arg10 were set as heavy (H) labels; the enzyme specificity was set to trypsin; the maximum number of missed cleavages was set to two; cysteine carbamidomethylation was set as a fixed modification, whereas methionine oxidation and protein N-terminal acetylation were set as variable modifications. The “Match between runs” and “Re-quantify” options were selected, and the other parameters were set to default values. Contaminants, proteins identified by reverse identification, proteins identified only by site and proteins with ≤ 2 unique peptides were excluded from further analysis using Perseus software. Fold changes (t-test difference, log2 ratios of the mean of the normalized H/M ratio from three replicates) were calculated and plotted against the -log10 of the p-values derived from a t-test. Only proteins showing significant differences by t-test (FDR = 0.05, s0 = 2) were considered differentially regulated. The raw data related to Dataset S1 were searched against the human UniProt FASTA database as described above using Sequest HT (12) node integrated within the Proteome Discoverer software (version 1.4, Thermo Fisher). The precursor and fragment mass tolerances were set to 5 ppm and 0.6 Da, respectively. Cysteine carbamidomethylation was set as a fixed modification, whereas methionine oxidation, glutamine deamidation were set as variable modifications. The maximum number of missed cleavages was set to two for trypsin digestion. FDR of peptide spectrum matches (PSMs) and identified peptides were determined by searching the forward and reverse database with Sequest HT node and were validated by the Percolator algorithm (13) at 1% based on q-values. For the hierarchical clustering analysis of the identified pTyr proteins (related to Fig. S7A), the pTyr proteome dataset reported by Bian et al. (4,558 pTyr proteins) (14) were analyzed against identified proteins from the breast cancer tissue samples. The reason for selecting this dataset is that (1) this is the largest pTyr proteome dataset from nine different cell lines; (2) the authors also adopted the Src superbinder as their enrichment approach but at peptide level. Although we also analyzed the pTyr proteins from the PhosphoSitePlus database, we did not adopt the analysis results due to the significant redundancy and various difference resources of the data. For the hierarchical clustering analysis of the identified kinases (related to Fig. 5E), 518 previously reported kinases (15) were analyzed against identified proteins from the breast cancer tissue samples. Hierarchical clustering of the identified proteins was performed on log-transformed LFQ intensities using the Euclidean distance, and missing values were artificially assigned a value of 1/10 of the minimum intensity. Gene ontology (GO) terms for specific identified proteins were performed using DAVID software (version 6.8) (16), and terms were categorized based on their biological process (BP) or molecular function (MF). The top 15 terms (related to Fig. S6) or terms FDR < 0.02 after Benjamini correction (17) (related to Fig. S7A) were kept for the data analysis. Protein-protein interactions map was analysis with STRING (version 10.0) (18). To ensure high confidence, only interactions assigned as experimentally verified were used and with a confidence score greater than 0.5. Finally, the association map was visualized using the Cytoscape (version 3.4.0) (19).

12. Animal Studies All animal procedures were approved by the Institutional Animal Care and Use Committee at Southern University of Science and Technology. Twelve 8-week-old female MMTV-PyMT mice were divided into 6 groups. The two mice in each group were littermates. Each group of mice were treated with Crenolanib (20 mg/kg, Selleck, dissolved in vehicle) or vehicle [5% (v/v) glycerol formal, Sigma] intraperitoneally once daily for 35 days. Once palpable, tumor sizes were measured with a digital caliper every five day, and the volumes were calculated using the formula (length ×

width2)/2. Data were reported as means ± SE. At the end of crenolanib treatment, mice were sacrificed, tumors were harvested and weighted.

Figs. S1 to S7

Fig. S1. Supporting data related to Fig. 1. (A) The labeling efficiency of the NHS group of probe 1 was optimized using the purified N-terminal SH2 domain of PI3K kinase p85α (termed as N- PI3K) and incubated for different times (1 to 10 min) and protein-to-probe ratios (1:2 to 1:30). (B) The labeling efficiency and stoichiometry of probe 1 to various bait proteins used in this study as characterized with a 16 × 20 cm SDS-PAGE gel for different times (1 to 10 min) at a protein-to- probe ratio of 1:10. (C) The binding activity of the probe 1-engineered N-PI3K was assessed using GST and streptavidin pull-down of pTyr proteins. The GST-tagged and probe 1-engineered N-PI3K with different protein-to-probe ratio were incubated with pervanadate (PV)-stimulated HeLa cell lysate. Enriched pTyr proteins were detected by WB analysis with a 4G10 antibody. (D) The benzophenone group photoreactive labeling efficiency was optimized. The NHS group of probe 1 was first blocked in a reaction with 1 M glycine. The N-PI3K was incubated with probe 1 at a protein-to-probe ratio of 1:10 and then irradiated with 365 nm UV light for the indicated amount of time. (E) The pull-down efficiency of the streptavidin beads was examined. The probe 1- engineered N-PI3K was incubated with streptavidin beads, then the beads were washed three times with the harsh modified RIPA buffer, subsequently the beads, flow-through and wash buffers were detected.

Fig. S2. Supporting data related to Fig. 2A-B. (A) Schematic diagram showing the domain organization of the NPDZ1 domain of Harmonin (green), the SAM-PBM domains of Sans (red) and the cytoplasmic domain of Cadherin 23 (Cad23-cyto, orange). (B) An extended presentation of Fig. 2A with both Coomassie brilliant blue staining and WB results using streptavidin-HRP and anti-His antibody. As both SAM-PBM and Cad23-cyto contain the His tag, this extended result further confirms the inter-molecular crosslinking of the NPDZ1-SAM-PBM and NPDZ1-Cad23- cyto protein complexes by UV irradiation. The arrow indicates non-specific staining of proteins, and the asterisk indicates specifically stained proteins. (C) GST pull-down validation of the data related to Fig. 2A. GST-tagged NPDZ1 was pulled down using glutathione beads, and the associated proteins were visualized by both Coomassie brilliant blue staining and WB analysis. (D) UV irradiation-induced NPDZ1 intramolecular crosslinking, as detected by Coomassie brilliant blue staining. (E) Peptide sequences of NPDZ1, SAM-PBM and Cad23-cyto identified from the indicated region of the gel shown in Fig. 2A.

Fig. S3. Supporting data related to Fig. 2F. (A) HeLa cells were treated with EGF for 5 min or PV for 10 min. Cell lysates were analyzed by WB analysis with the indicated antibodies. (B) Schematic of a stable isotope labeling with amino acids in cell culture (SILAC)-based chemical proteomics workflow. The SHC1 SH2 or GRB2 SH2 domain-based Photo-pTyr-scaffold were incubated with EGF stimulated HeLa cell lysate. The EGF-activated, pTyr-dependent protein complexes were then crosslinked, enriched, and identified by using MS. Volcano plots of the enriched proteins identified by the (C) SHC1 SH2 domain-based Photo-pTyr-scaffold and (D) GRB2 SH2 domain-based Photo- pTyr-scaffold. The identified EGFR signaling protein complexes are highlighted in red (FDR = 0.05, s0 = 2, two sample t-test, n = 3). Large amounts of other proteins are identified due to non- specific crosslinking or enrichment, which are well differentiated by the SILAC-based quantification.

Fig. S4. Supporting data related to Fig. 3. (A) An extended presentation of Fig. 3B with the identified peptides of the enriched proteins from the EGF stimulated HeLa cell lines. Error bars represent means ± SE from three independent experiments. Streptavidin and N-PI3K were chosen as loading controls. (B) The chemical structure of the Sulfo-SBED probe. (C) The labeling activity of probe 1 and Sulfo-SBED were assessed using streptavidin pull-down. The engineered N-PI3K by both probe 1 and Sulfo-SBED was incubated with EGF stimulated HeLa cell lysate. Enriched proteins after UV irradiation were detected. The asterisk indicates dimeric and trimeric bait protein.

Fig. S5. Supporting data related to Fig. 4. (A) The LFQ intensities of the respective proteins between Src superbinder and N-PI3K based Photo-pTyr-scaffold. Error bars represent means ± SE from three independent experiments. *p < 0.05, **p < 0.01, #p > 0.05. (B) Volcano plots of differentially expressed pTyr protein complexes from MDA-MB-231 vs. BT-474 breast cancer cell lines. The probe 1-engineered Src superbinder were incubated with normal cultured BT-474 or MDA-MB-231 cell lysates respectively, enriched proteins were detected with MS analysis. Red and green dots indicate significant enrichment proteins (FDR = 0.01, s0 = 2, two sample t-test, n = 3). (C) WB validation of the capture of native EGFR and ERBB2 from three unstimulated breast cancer cell lines. The probe 1-engineered Src superbinder were incubated with three normal cultured breast cancer cell lysates respectively. Enriched proteins were detected with both Coomassie brilliant blue staining and WB analysis with anti-EGFR and anti-ERBB2.

Fig. S6. Supporting data related to Fig. 4C-D. (A) GO annotations for biological processes (BP) enriched in MDA-MB-468 cells compared to MDA-MB-231 cells. (B) GO annotations for biological processes enriched in BT-474 cells compared to MDA-MB-231 cells. Biological processes related to the EGFR and ERBB2 signaling pathways are highlighted in red.

Fig. S7. Supporting data related to the MS data of Fig. 5. (A) Hierarchical clustering of pTyr proteins identified by the Src superbinder Photo-pTyr-scaffold approach or directly from the indicated cell lysates, and highlighted molecular functions annotated by GO for corresponding clusters. (B) Enriched protein-protein interactions map of the identified kinases. The thickness of the edge indicates the STRING confidence for protein interaction predication.

Quality Control Data S1 to S9

Quality control data S1. Supporting data related to Fig. 2E. (A) Results of the MS identification from probe 1-engineered N-PI3K. The number of peptides, unique peptides and protein groups from the three biological replicates are illustrated. (B) The LFQ quantification accuracy of the above MS data was evaluated. Pearson’s correlation coefficients between pairs of the three biological replicates are displayed. Exp.2 = EGF-, UV+; Exp.3 = EGF+, UV+.

Quality control data S2. Supporting data related to Fig. 3A. (A) Results of the MS identification from probe 0-engineered N-PI3K. The number of peptides, unique peptides and protein groups from the three biological replicates are illustrated. (B) The LFQ quantification accuracy of the above MS data was evaluated. Pearson’s correlation coefficients between pairs of the three biological replicates are displayed. Exp.2 = EGF-, UV+; Exp.3 = EGF+, UV+.

Quality control data S3. Supporting data related to Fig. 3C-D. (A) Results of the MS identification from probe 1-engineered N-PI3K. The number of peptides, unique peptides and protein groups from the three biological replicates are illustrated. (B) The LFQ quantification accuracy of the above MS data was evaluated. Pearson’s correlation coefficients between pairs of the three biological replicates are displayed. Exp.1 = EGF-, UV-; Exp.2 = EGF-, UV+; Exp.3 = EGF+, UV+.

Quality control data S4. Supporting data related to Fig. 3C-D. (A) Results of the MS identification from probe 2-engineered N-PI3K. The number of peptides, unique peptides and protein groups from the three biological replicates are illustrated. (B) The LFQ quantification accuracy of the above MS data was evaluated. Pearson’s correlation coefficients between pairs of the three biological replicates are displayed. Exp.1 = EGF-, UV-; Exp.2 = EGF-, UV+; Exp.3 = EGF+, UV+.

Quality control data S5. Supporting data related to Fig. 3C-D. (A) Results of the MS identification from probe 3-engineered N-PI3K. The number of peptides, unique peptides and protein groups from the three biological replicates are illustrated. (B) The LFQ quantification accuracy of the above MS data was evaluated. Pearson’s correlation coefficients between pairs of the three biological replicates are displayed. Exp.1 = EGF-, UV-; Exp.2 = EGF-, UV+; Exp.3 = EGF+, UV+.

Quality control data S6. Supporting data related to Fig. 4A. (A) Results of the MS identification related to Fig. 4A. The number of peptides, unique peptides and protein groups from the three biological replicates are illustrated. (B) The LFQ quantification accuracy of the above MS data was evaluated. Pearson’s correlation coefficients between pairs of the three biological replicates are displayed. Exp.2 = EGF-, UV+; Exp.3 = EGF+, UV+.

Quality control data S7. Supporting data related to Fig. 4C-D and S5B. (A) Results of the MS identification related to Fig. 4C-D and S5B. The probe 1-engineered Src superbinder was incubated with three normal cultured breast cancer cell lysates respectively, enriched proteins were detected with MS analysis. The number of peptides, unique peptides and protein groups from the three biological replicates are illustrated. (B) The LFQ quantification accuracy of the above MS data was evaluated. Pearson’s correlation coefficients between pairs of the three biological replicates are displayed.

Quality control data S8. Supporting data related to the MS data of Fig. 5. (A) The number of peptides, unique peptides and protein groups identified from twelve breast cancer patient samples. Lysate: total lysate without enrichment. (B) Pearson’s correlation coefficients of the identified proteins and LFQ intensities across the tissue samples from the twelve patients.

Quality control data S9. Supporting data related to the MS data of Fig. 6D. (A) The number of peptides, unique peptides and protein groups identified from the three biological replicates. (B) Pearson’s correlation coefficients of the identified proteins and LFQ intensities across the tissue samples from the three biological replicates.

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