Supporting Information Redefining the breast cancer exosome proteome by tandem mass tag quantitative proteomics and multivariate cluster analysis. David J Clarkǂ*1,2, William E Fondrieǂ2,3, Zhongping Liao4, Phyllis I Hanson5, Amy Fulton2, Li Mao1,2, Austin J Yang2 1 Department of Oncology and Diagnostic Sciences, University of Maryland School of Dentistry, Baltimore, MD 2 Marlene and Stewart Greenebaum Cancer Center, University of Maryland, Baltimore, MD 3Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, MD 4 Lily Research Laboratory, Eli Lily and Company, Indianapolis, IN 5Department of Cell Biology and Physiology, Washington University, St. Louis, MO ǂ These authors contributed equally to this work. *Corresponding author: [email protected] 410-706-6114 Table of Contents Figure S-1: TMT log2 protein ratios were plotted by 100K enrichment against Optiprep/100K enrichment. (pg. S-2) Figure S-2: Immunoblot of SEC fractions, and Venn diagram of SEC and TMT-SVM analysis. (pg. S-3) Table S-1 Protein identifications, TMT ratios, SVM Classification and SVM Probability of 10K, 100K, and Optiprep lysates derived from SKBR3B conditioned media. (pg. S-4) Table S-2. Plasma membrane markers and their respective SVM Classification (pg. S-75) Table S-3. Identified proteins from our SEC analysis with respective Protein Prophet probability (pg. S-76) S-1 Figure S-1. Known exosome markers localize to quadrant corresponding to 100K enrichment. TMT log2 protein ratios were plotted by 100K enrichment (x-axis) against Optiprep/100K enrichment (y-axis). Selected Exosome (red) and Non-exosome (blue) protein markers are annotated. S-2 Figure S-2. SEC fractions and LC-MS/MS analysis. (A) Immunoblot for known exosome markers ALIX and CD63 in the recovered 500 L SEC fractions. Fractions 9 and 10 were positive for both exosome markers. (B) Venn diagram showing overlap of the SVM exosome cluster and SVM non-exosome cluster with the SEC analysis. S-3 Table S-1. Protein identifications, TMT ratios, SVM Classification and SVM Probability of 10K, 100K, and Optiprep lysates derived from SKBR3B conditioned media. TMT ratios were calculated to the log base 2 prior to plotting and SVM cluster analysis Log 2 ratio Protein Uniprot TMT 130/129 TMT 131/129 TMT 131/130 SVM SVM Gene Symbol Protein Description Group Accession +/- SD +/- SD +/- SD 100K/1 Opti/10 Opti/100 Classification Probability 0K K K 1 A5D8V6 VPS37C Vacuolar protein sorting-associated protein 37C 0.886 +/- 0.094 0.974 +/- 0.396 1.064 +/- 0.334 -0.175 -0.038 0.09 Non-Exosome 0.57 2 B7ZAQ6-2 GPR89A Isoform 2 of Golgi pH regulator A 0.333 +/- 0.053 0.225 +/- 0.085 0.651 +/- 0.151 -1.585 -2.152 -0.62 Non-Exosome 0.90 3 O00186 STXBP3 Syntaxin-binding protein 3 1.188 +/- 0.172 0.507 +/- 0.033 0.44 +/- 0.092 0.248 -0.981 -1.184 Non-Exosome 0.73 4 O00220 TNFRSF10A Tumor necrosis factor receptor superfamily member 10A 0.835 +/- 0.205 0.755 +/- 0.145 1.008 +/- 0.421 -0.26 -0.405 0.011 Non-Exosome 0.73 5 O00232-2 PSMD12 Isoform 2 of 26S proteasome non-ATPase regulatory subunit 12 0.602 +/- 0.198 0.279 +/- 0.039 0.495 +/- 0.097 -0.731 -1.841 -1.014 Non-Exosome 0.92 6 O00442-2 RTCA Isoform 2 of RNA 3'-terminal phosphate cyclase 0.248 +/- 0.038 0.192 +/- 0.002 0.79 +/- 0.115 -2.01 -2.383 -0.341 Non-Exosome 0.85 7 O00487 PSMD14 26S proteasome non-ATPase regulatory subunit 14 0.483 +/- 0.067 0.219 +/- 0.014 0.466 +/- 0.094 -1.049 -2.19 -1.1 Non-Exosome 0.91 8 O00622 CYR61 Protein CYR61 0.944 +/- 0.021 0.323 +/- 0.057 0.343 +/- 0.068 -0.084 -1.632 -1.542 Non-Exosome 0.86 9 O14684 PTGES Prostaglandin E synthase 0.475 +/- 0.045 0.324 +/- 0.069 0.675 +/- 0.082 -1.074 -1.625 -0.568 Non-Exosome 0.93 10 O14763-2 TNFRSF10B Isoform Short of Tumor necrosis factor receptor superfamily member 10B 1.202 +/- 0.768 0.652 +/- 0.448 0.515 +/- 0.044 0.266 -0.616 -0.958 Non-Exosome 0.63 11 O14908-2 GIPC1 Isoform 2 of PDZ domain-containing protein GIPC1 0.640 +/- 0.180 0.590 +/- 0.110 1.054 +/- 0.468 -0.644 -0.761 0.075 Non-Exosome 0.87 12 O14949 UQCRQ Cytochrome b-c1 complex subunit 8 0.282 +/- 0.082 0.210 +/- 0.025 0.784 +/- 0.141 -1.824 -2.252 -0.35 Non-Exosome 0.88 13 O15347 HMGB3 High mobility group protein B3 0.265 +/- 0.015 0.140 +/- 0.060 0.543 +/- 0.257 -1.916 -2.837 -0.881 Non-Exosome 0.80 14 O43657 TSPAN6 Tetraspanin-6 2.473 +/- 0.908 2.482 +/- 1.226 0.95 +/- 0.147 1.306 1.312 -0.074 Exosome 0.88 15 O43677 NDUFC1 NADH dehydrogenase [ubiquinone] 1 subunit C1; mitochondrial 0.362 +/- 0.242 0.237 +/- 0.137 0.726 +/- 0.107 -1.467 -2.079 -0.462 Non-Exosome 0.91 16 O43854-2 EDIL3 Isoform 2 of EGF-like repeat and discoidin I-like domain-containing protein 3 3.947 +/- 0.580 3.450 +/- 0.616 0.87 +/- 0.028 1.981 1.787 -0.201 Exosome 0.87 17 O60524-3 NEMF Isoform 3 of Nuclear export mediator factor NEMF 0.968 +/- 0.032 0.585 +/- 0.505 0.588 +/- 0.502 -0.048 -0.773 -0.767 Non-Exosome 0.77 18 O60841 EIF5B Eukaryotic translation initiation factor 5B 0.656 +/- 0.044 0.285 +/- 0.065 0.43 +/- 0.07 -0.608 -1.811 -1.219 Non-Exosome 0.91 19 O60888-2 CUTA Isoform A of Protein CutA 0.830 +/- 0.440 0.260 +/- 0.110 0.338 +/- 0.047 -0.269 -1.943 -1.565 Non-Exosome 0.88 20 O75396 SEC22B Vesicle-trafficking protein SEC22b 0.576 +/- 0.041 0.509 +/- 0.226 0.916 +/- 0.458 -0.795 -0.975 -0.127 Non-Exosome 0.90 21 O75436-2 VPS26A Isoform 2 of Vacuolar protein sorting-associated protein 26A 0.668 +/- 0.372 0.289 +/- 0.041 0.578 +/- 0.261 -0.583 -1.792 -0.79 Non-Exosome 0.91 22 O75531 BANF1 Barrier-to-autointegration factor 0.421 +/- 0.051 0.169 +/- 0.009 0.405 +/- 0.027 -1.249 -2.563 -1.303 Non-Exosome 0.88 23 O75694-2 NUP155 Isoform 2 of Nuclear pore complex protein Nup155 0.766 +/- 0.297 0.359 +/- 0.147 0.464 +/- 0.012 -0.385 -1.48 -1.109 Non-Exosome 0.90 24 O75695 RP2 Protein XRP2 1.041 +/- 0.081 1.058 +/- 0.292 1.044 +/- 0.362 0.058 0.081 0.062 Exosome 0.56 25 O75718 CRTAP Cartilage-associated protein 0.481 +/- 0.161 0.150 +/- 0.095 0.277 +/- 0.106 -1.057 -2.734 -1.85 Non-Exosome 0.85 26 O75844 ZMPSTE24 CAAX prenyl protease 1 homolog 0.346 +/- 0.024 0.163 +/- 0.013 0.471 +/- 0.004 -1.532 -2.618 -1.087 Non-Exosome 0.86 27 O75915 ARL6IP5 PRA1 family protein 3 0.379 +/- 0.007 0.236 +/- 0.016 0.622 +/- 0.031 -1.399 -2.083 -0.686 Non-Exosome 0.91 S-4 28 O75935-2 DCTN3 Isoform 2 of Dynactin subunit 3 0.610 +/- 0.000 0.295 +/- 0.045 0.484 +/- 0.074 -0.713 -1.761 -1.048 Non-Exosome 0.92 29 O76003 GLRX3 Glutaredoxin-3 0.473 +/- 0.233 0.278 +/- 0.098 0.642 +/- 0.108 -1.079 -1.845 -0.64 Non-Exosome 0.93 30 O94826 TOMM70A Mitochondrial import receptor subunit TOM70 0.413 +/- 0.036 0.326 +/- 0.024 0.789 +/- 0.009 -1.277 -1.619 -0.341 Non-Exosome 0.93 31 O94874-2 UFL1 Isoform 2 of E3 UFM1-protein ligase 1 0.270 +/- 0.030 0.125 +/- 0.005 0.471 +/- 0.071 -1.889 -3 -1.087 Non-Exosome 0.78 32 O95084 PRSS23 Serine protease 23 2.591 +/- 0.140 1.330 +/- 0.053 0.514 +/- 0.007 1.374 0.411 -0.961 Exosome 0.86 33 O95810 SDPR Serum deprivation-response protein 0.520 +/- 0.140 0.368 +/- 0.108 0.822 +/- 0.428 -0.943 -1.444 -0.283 Non-Exosome 0.93 34 O95861-2 BPNT1 Isoform 2 of 3'(2');5'-bisphosphate nucleotidase 1 0.608 +/- 0.042 0.590 +/- 0.130 0.96 +/- 0.148 -0.717 -0.761 -0.059 Non-Exosome 0.87 35 O95881 TXNDC12 Thioredoxin domain-containing protein 12 0.235 +/- 0.041 0.107 +/- 0.004 0.466 +/- 0.065 -2.087 -3.224 -1.101 Non-Exosome 0.72 36 P00403 MT-CO2 Cytochrome c oxidase subunit 2 0.367 +/- 0.024 0.141 +/- 0.038 0.392 +/- 0.13 -1.446 -2.827 -1.35 Non-Exosome 0.83 37 P00734 F2 Prothrombin 1.992 +/- 0.130 0.637 +/- 0.100 0.318 +/- 0.03 0.994 -0.651 -1.654 Exosome 0.60 38 P00747 PLG Plasminogen 0.790 +/- 0.250 0.307 +/- 0.067 0.402 +/- 0.042 -0.34 -1.704 -1.315 Non-Exosome 0.90 39 P02792 FTL Ferritin light chain 1.022 +/- 0.178 0.461 +/- 0.066 0.453 +/- 0.014 0.032 -1.118 -1.142 Non-Exosome 0.81 40 P04040 CAT Catalase 0.597 +/- 0.310 0.340 +/- 0.175 0.571 +/- 0.003 -0.744 -1.556 -0.809 Non-Exosome 0.92 41 P04080 CSTB Cystatin-B 0.572 +/- 0.024 0.278 +/- 0.000 0.487 +/- 0.02 -0.807 -1.848 -1.039 Non-Exosome 0.92 42 P05121-2 SERPINE1 Isoform 2 of Plasminogen activator inhibitor 1 0.943 +/- 0.397 0.330 +/- 0.030 0.409 +/- 0.14 -0.084 -1.599 -1.291 Non-Exosome 0.86 43 P05161 ISG15 Ubiquitin-like protein ISG15 0.583 +/- 0.044 0.294 +/- 0.025 0.504 +/- 0.005 -0.778 -1.765 -0.988 Non-Exosome 0.92 44 P06400 RB1 Retinoblastoma-associated protein 0.295 +/- 0.035 0.165 +/- 0.005 0.565 +/- 0.05 -1.761 -2.599 -0.823 Non-Exosome 0.85 45 P07305-2 H1F0 Isoform 2 of Histone H1.0 0.320 +/- 0.050 0.095 +/- 0.035 0.322 +/- 0.16 -1.644 -3.396 -1.636 Non-Exosome 0.72 46 P07602-2 PSAP Isoform Sap-mu-6 of Prosaposin 0.416 +/- 0.024 0.191 +/- 0.021 0.465 +/- 0.076 -1.267 -2.385 -1.105 Non-Exosome 0.89 47 P07996 THBS1 Thrombospondin-1 1.992 +/- 0.862 0.605 +/- 0.102 0.346 +/- 0.098 0.994 -0.726 -1.53 Exosome 0.58 48 P08240-2 SRPR Isoform 2 of Signal recognition particle receptor subunit alpha 0.302 +/- 0.012 0.181 +/- 0.044 0.606 +/- 0.17 -1.729 -2.467 -0.723 Non-Exosome 0.87 49 P08572 COL4A2 Collagen alpha-2(IV) chain 0.480 +/- 0.000 0.415 +/- 0.085 0.865 +/- 0.177 -1.059 -1.269 -0.21 Non-Exosome 0.92 50 P08574 CYC1 Cytochrome c1; heme protein; mitochondrial 0.461 +/- 0.087 0.255 +/- 0.011 0.569 +/- 0.084 -1.117 -1.971 -0.813 Non-Exosome 0.92 51 P0C6C1 ANKRD34C Ankyrin repeat domain-containing protein 34C 1.275 +/- 0.415 0.990 +/- 0.490 0.729 +/- 0.147 0.35 -0.014 -0.457 Exosome 0.62 52 P0DME0 SETSIP Protein SETSIP 0.485 +/- 0.055 0.305 +/- 0.155 0.6 +/- 0.252 -1.044 -1.713 -0.736 Non-Exosome 0.93 53 P10606 COX5B Cytochrome c oxidase subunit