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C6cc08797c1.Pdf Electronic Supplementary Material (ESI) for Chemical Communications. This journal is © The Royal Society of Chemistry 2017 Supplementary Information for Competition-based, quantitative chemical proteomics in breast cancer cells identifies new target profiles for sulforaphane James A. Clulow,a,‡ Elisabeth M. Storck,a,‡ Thomas Lanyon-Hogg,a,‡ Karunakaran A. Kalesh,a Lyn Jonesb and Edward W. Tatea,* a Department of Chemistry, Imperial College London, London, UK, SW7 2AZ b Worldwide Medicinal Chemistry, Pfizer, 200 Cambridge Park Drive, MA 02140, USA ‡ Authors share equal contribution * corresponding author, email: [email protected] Table of Contents 1 Supporting Figures..........................................................................................................................3 2 Supporting Tables.........................................................................................................................17 2.1 Supporting Table S1. Incorporation validation for the R10K8 label in the 'spike-in' SILAC proteome of the MCF7 cell line........................................................................................................17 2.2 Supporting Table S2. Incorporation validation for the R10K8 label in the 'spike-in' SILAC proteome of the MDA-MB-231 cell line ...........................................................................................17 2.3 Supporting Table S3. High- and medium- confidence targets of sulforaphane in the MCF7 cell line..............................................................................................................................................17 2.4 Supporting Table S4. High- and medium- confidence targets of sulforaphane in the MDA- MB-231 cell line................................................................................................................................24 3 Chemical synthesis methods ........................................................................................................36 3.1 General Information .............................................................................................................36 3.2 Synthesis of Probe 1 .............................................................................................................36 3.2.1 4-Hex-5-ynyloxy-benzaldehyde ....................................................................................37 3.2.2 2-(4-Chloro-butyl)-2-methyl-[1,3]dioxolane .................................................................37 3.2.3 2-(4-Phthalimido-butyl)-2-methyl-[1,3]dioxolane ........................................................37 3.2.4 2-(4-Amino-butyl)-2-methyl-[1,3]dioxolane .................................................................38 3.2.5 (4-hex-5-ynyloxy-benzyl)-[4-(2-methyl-[1,3]dioxolan-2-yl)-butyl]-amine.....................38 3.2.6 S-Ethyl (4-hex-5-ynyloxy-benzyl)-[4-(2-methyl-[1,3]dioxolan-2-yl)-butyl]- thiocarbamate ..............................................................................................................................38 3.2.7 S-Ethyl (4-hex-5-ynyloxy-benzyl)-(5-oxo-hexyl)-thiocarbamate ...................................39 3.2.8 S-Ethyl(4-hex-5-ynyloxy-benzyl)-(5-oxo-hexyl)-thiocarbamatesulfoxide (1) ................39 3.3 Synthesis of Probe 2 .............................................................................................................40 3.3.1 S-Ethyl (prop-2-ynyl)-[4-(2-methyl-[1,3]dioxolan-2-yl)-butyl]-thiocarbamate .............40 3.3.2 S-Ethyl (prop-2-ynyl)-(5-oxo-hexyl)-thiocarbamate......................................................41 3.3.3 S-Ethyl (prop-2-ynyl)-(5-oxo-hexyl)-thiocarbamate sulfoxide (2) .................................41 3.4 1H NMR, 13C NMR, HRMS and LC-MS data............................................................................42 4 Biological and biochemical methods ............................................................................................46 4.1 General methods ..................................................................................................................46 4.2 Cell culture............................................................................................................................46 4.3 In-cell competition-based assays for in-gel fluorescence and Western Blot analyses .........46 4.3.1 Compound treatment of cells and cell lysis..................................................................46 4.3.2 Copper catalysed azide-alkyne cycloaddition (CuAAC).................................................47 4.3.3 Gel electrophoresis and in-gel fluorescence.................................................................47 1 4.3.4 Affinity enrichment and Western Blot analysis ............................................................47 4.4 In-cell quantitative, competition-based assays for target identification by mass spectrometry ....................................................................................................................................48 4.4.1 Compound treatment of cells and cell lysis..................................................................48 4.4.2 Preparation of ‘spike-in’ SILAC cell lysates as quantification standards.......................49 4.4.3 CuAAC, affinity enrichment and on-bead reduction, alkylation and trypsin digest......49 4.4.4 LC-MS/MS .....................................................................................................................50 4.4.5 LC-MS/MS data analysis................................................................................................50 4.4.6 Abundance and bioinformatic analysis.........................................................................51 5 References....................................................................................................................................52 2 1 Supporting Figures MCF7 MDA-MB-231 4 4 o o i i t 2 t 2 n n a a i i r r e e L L c c / / n n H H 5 μM e 0 e 0 r r e e e e g g f f f f a a i i r r D D e -2 e -2 v v a a -4 -4 15 6 o i t o 10 i n 4 a i t r n a e i r L c / e n L 2 H c / e 5 n r e H e e 25 μM g f r e f a i e g 0 r f f a D e i r 0 v D e a v a -2 -5 -4 16 16 14 14 o o i 12 i 12 t t n n a a i 10 i 10 r r e e L 8 L 8 c c / / n n H H 100 μM e 6 e 6 r r e e e e g 4 g 4 f f f f a a i i r 2 r 2 D D e e v v a 0 a 0 -2 -2 -4 -4 16 14 Non-significant o Significant i 12 t n a i 10 r e L 8 c / Supporting Figure S1. Target profiles of 2 in competition with three concentrations of sulforaphane n H e 6 in MCF7 and MDA-MB-231 cells. Potential targets were identified as giving a statistically significant r e e g 4 f f change (t-test, S0 = 1, FDR = 0.01, shown in blue) in H/L ratio compared to samples treated with 2 a i r 2 D e alone. v a 0 -2 -4 3 MCF7 (a) 5 μM (b) 25 μM (c) 100 μM Supporting Figure S2. Volcano plots for 2 in competition with three concentrations of sulforaphane in MCF7. Medium-confidence targets were identified as giving a statistically significant change (t- test, S0 = 1, FDR = 0.01, shown in green) in H/L ratio compared to samples treated with 2 alone. The four high affinity and conserved targets MIF, KEAP1, ALDH9A1 and CPPED1 are highlighted to show distribution at each concentration. 4 MDA-MB-231 (a) 5 μM (b) 25 μM (c) 100 μM Supporting Figure S3. Volcano plots for 2 in competition with three concentrations of sulforaphane in MDA-MB-231 cells. Medium-confidence targets were identified as giving a statistically significant change (t-test, S0 = 1, FDR = 0.01, shown in green) in H/L ratio compared to samples treated with 2 alone. The four high affinity and conserved targets MIF, KEAP1, ALDH9A1 and CPPED1 are highlighted to show distribution at each concentration. 5 (a) MCF7 5 μM 25 μM 0 0 37 4 0 125 123 100 μM (b) MDA-MB-231 5 μM 25 μM 1 0 54 6 0 115 235 100 μM Supporting Figure S4. Medium- and high-confidence targets of sulforaphane in (a) MCF7 and (b) MDA-MB-231 cells. Medium-confidence targets were identified as giving a statistically significant (t- test, S0 = 1, FDR = 0.01) change in H/L ratio at one sulforaphane concentration. Medium-confidence targets identified at 5, 25, and 100 μM, or at 25 and 100 μM, were defined as high-confidence targets (highlighted green). 6 *** ) * * 7 0.4 F C M ( 0.2 e c n 0.0 a d n u -0.2 b A e v i -0.4 t a l e R -0.6 7 d 1 F e 3 C rv -2 e M s B n -M o A C D M Supporting Figure S5. Comparions of difference in relative abundance levels for conserved and cell line-specific high-confidence targets of sulforaphane. Relative abundance was calculated as described in materials and methods using intensity based absolute quantification (iBAQ) values for each cell line from proteomic analysis of the NCI-60 cell line panel.1 A relative abundance > 0 indicates a protein is present at higher levels in MCF7 cells than in MDA-MB-231 cells; conversely a relative abundance < 0 indicates a protein is present at higher levels in MDA-MB-231 cells than in MCF7 cells. Average relative abundance of targets indicates that cell line-specific targets show higher abundance within that cell line (* = p < 0.05, *** = p < 0.0001). 7 (a) MCF7 (b) MDA-MB-231 Upregulated Downregulated Supporting Figure S6. IPA heatmap analysis of up- and downregulated canonical pathways in (a) MCF7 and (b) MDA-MB-231 cell lines. Heatmaps are ordered by increasing or decreasing z-score trend, and coloured with orange indicating upregulation and blue indicating downregulation. 8 (a) Sulforaphane targets in apoptosis signalling in MCF7: (b) Sulforaphane targets in apoptosis signalling
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