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Supporting Information a Novel Strategy for Facile Serum Exosome Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2018 Supporting Information A novel strategy for facile serum exosome isolation based on specific interactions between phospholipid bilayers and TiO2 Fangyuan Gao, ‡a Fenglong Jiao, ‡ab Chaoshuang Xia,a Yang Zhao,a Wantao Ying,a Yuping Xie,a Xiaoya Guan, c Ming Tao, d Yangjun Zhang, *a Weijie Qin, *a Xiaohong Qian, *a a. State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing Proteome Research Center. E-mail: [email protected]; [email protected]; [email protected]. b. School of Life Science and Technology, Beijing Institute of Technology. c. Peking University Cancer Hospital. d. Peking University Third Hospital. ‡ These authors contributed equally. 1 Table of Contents 1. Supplemental Fig. S1 Isolation and characterization of exosomes from HeLa cells culture medium. 2. Supplemental Fig.S2 SDS-PAGE analysis (silver stained) of the proteins isolated from model sample by TiO2 microspheres. 3. Supplemental Fig. S3 SDS-PAGE images of (A) α-casein (1 μg), β-casein (1 μg) and horseradish peroxidase (1 μg). (B) the model exosomal proteins (2 μg), mixture of the model exosomal proteins (2 μg)+α-casein (1 μg)+ β-casein (1 μg)+horseradish peroxidase (1 μg), and mixture of the model exosomal protein (2 μg)+α-casein (1 μg)+β-casein (1 μg)+horseradish peroxidase (1 μg) after isolated by TiO2. 4. Supplemental Fig. S4 SDS-PAGE image of the proteins extracted from the exosomes isolated by TiO2 in three replicates (Lane 1, 3 and 5) and the corresponding residual proteins on TiO2 after exosome elution (Lane2, 4, 6). 5. Supplemental Fig. S5 Exosome enrichment effect by using (A) different TiO2 amounts and (B) different incubation time (evaluated by Western Blot analysis of TSG101). 6. Supplemental Fig. S6 Exosome isolation using different commercial titanium dioxide particles. 7 Supplemental Fig. S7 Schematic of the TiO2-based method for exosomes isolation and downstream analyses. 8 Supplemental Fig. S8 Gene Ontology analysis of cellular components of proteins identified in exosomes obtained by different methods. 9. Supplemental Table S1 Brief Comparison of Various Exosome Isolation Methods. 10. Supplemental Table S2 Numbers of protein groups identified under different conditions. 11. Supplemental Table S3 Significantly regulated serum exosomal proteins in pancreatic cancer. 12. References 2 A B D HeLa cell culture medium kDa M Cell Exo 180 300 g, 10 min cells 130 95 Supernatant 72 55 2,000 g, 10 min 43 dead cells Supernatant 34 26 cells debris 10,000 g, 30 min 100 nm Supernatant 17 C 1E+0 100,000 g, 70 min 9E-1 e v 8E-1 i Crude exosomes t a E l 7E-1 e R 6E-1 Cell Exo wash in PBS 100,000 g, 70 min n o i t 5E-1 CD81 a r t n 4E-1 e CD81 c TSG101 n 3E-1 wash in PBS 100,000 g, 70 min o C 2E-1 PHB1 Exosomes 1E-1 0E+0 β-actin 5 10 100 1000 2000 Diameter/nm Supplemental Fig. S1 Isolation and characterization of exosomes from HeLa cells culture medium. (A) Workflow for isolation of exosomes using ultracentrifugation. (B) TEM image of exosomes. (C) NTA measurement of the size distribution of exosomes. (D) Coomassie Brilliant Blue stained 12% SDS- PAGE analysis of HeLa cell lysates and exosome proteins. (E) Western blotting analysis of common exosome markers (TSG101 and CD81), mitochondrial marker (PHB1) and internal control (β-actin). As shown in Fig. S1B, the exosomes exhibited typical cup-shaped vesicles structure. Nanoparticle tracking analysis (NTA) showed that the size distribution of the exosomes is from 45 nm to 225 nm and centered at 122 nm, which was consistent with the typical size range of exosomes (30-150 nm) (Fig. S1C). To further investigate the performance of model exosomes preparations, the proteins extracted from HeLa cell lysates and from model exosome samples were separated by SDS-PAGE and stained with Coomassie Brilliant Blue, respectively. The distinct protein profiles of the two types of samples were shown in Fig. S1D. Clearly visible differences could be observed between 43 kDa to 95 kDa and small molecular weight range (less than 17 kDa). Then, the expression of exosomal proteins in HeLa cell lysates and model exosome samples was examined by western blotting analysis, respectively. As shown in Fig. S1E, the commonly used exosomal marker proteins, TSG101 and CD81, were highly enriched in the model exosome samples compared with HeLa cell lysates. Moreover, the mitochondrial marker, prohibitin-1 (PHB1) was not detected in the exosome fraction. The results indicated that the isolated exosomes had high purity by avoiding the interference by organelles such as mitochondria. 3 k D a 18 1 0 30 Marker 95 Supplemental 7 2 Model sample 55 43 3 Supernatant Fig. 4 2 S2 6 First wash SDS-PAGE 1 7 Second wash analysis Third wash (silver Elute stained) k D of a Marker the 18 1 0 proteins 30 9 5 Lane 1 72 isolated 55 43 Lane 2 from 3 model 4 Lane 3 26 sample Lane 4 1 7 by Lane 5 TiO 2 microspheres. Lane 6 4 le P p R le m H p sa + m + in sa e se a 2 e om iO m s -C o o β T s x + m o e n o in n ex el i r er e ei er l d se f k s s k e o a te r a a P r d C u a -C -C R a o M - l M α β H M M α E kDa kDa + 180 180 130 130 95 95 72 72 55 55 43 43 HRP 34 34 α-Casein 26 β-Casein 26 17 17 (A) (B) Supplemental Fig. S3 SDS-PAGE images of (A) α-casein (1 μg), β-casein (1 μg) and horseradish peroxidase (1 μg). (B) the model exosomal proteins (2 μg), mixture of the model exosomal proteins (2 μg)+α-casein (1 μg)+ β-casein (1 μg)+horseradish peroxidase (1 μg), and mixture of the model exosomal protein (2 μg)+α-casein (1 μg)+β-casein (1 μg)+horseradish peroxidase (1 μg) after isolated by TiO2 5 le p t h n as h m ta sh as r sa a a w e l n w d w k e er t n d e r d p s o ir t a o u ir ec h lu kDa M M S F S T E 180 130 95 72 55 43 34 26 17 r e 1 2 3 4 5 6 k e e e e e e r n n n n a a a an a an a M L L L L L L kDa 180 130 95 72 55 43 34 26 17 Supplemental Fig. S4 SDS-PAGE image of the proteins extracted from the exosomes isolated by TiO2 in three replicates (Lane 1, 3 and 5) and the corresponding residual proteins on TiO2 after exosome elution (Lane2, 4, 6). 6 p up u o ro gr g l n l g g o i in o g g g r in in in r m m t m m t .5 m m m 0 n .5 m m m 0 n 0 1 2 5 1 co 0 1 2 5 1 co Replicate 1 Replicate 1 Replicate 2 Replicate 2 Replicate 3 Replicate 3 A B Supplemental Fig. S5 Exosome enrichment effect by using (A) different TiO2 amounts and (B) different incubation time (evaluated by Western Blot analysis of TSG101). 7 TiO enriched exosome proteins A 2 mixture of rutile UC rutile anatase and anatase Marker sample ＜5 μm ~100 nm ~25 nm ~5 μm ~100 nm a b c d e kDa 180 130 95 72 55 43 34 26 17 the TiO2 used in our method B Supplemental Fig. S6 Exosome isolation using different commercial titanium dioxide particles. (A) SDS-PAGE image of the proteins extracted from the exosomes isolated by different titanium dioxide particles, (B) SEM images of different titanium dioxide particles. 8 Supplemental Fig. S7 Schematic of the TiO2-based method for exosomes isolation and downstream analyses 9 16 0 E x o 1 s 40 o m T ) es i e 1 O lu 20 2 va - 10 (P 0 g 10 o 8 -l 0 U 6 lt 0 ra c en 40 t ri fu g 20 a t io n 0 E E x Supplemental x o extracellular exosome o s so o extracellular space m m extracellular region e e s P blood microparticle r extracellular matrix ec ip platelet alpha granule lumen i Fig. ta very-low-density lipoprotein particle t io S8 high-density lipoprotein particle n chylomicron Gene R platelet dense granule lumen ea g E e Ontology x n blood microparticle o t so extracellular region m extracellular exosome es analysis extracellular space platelet alpha granule lumen immunoglobulin complex, circulating of membrane attackcomplex cellular very-low-density lipoprotein particle spherical high-density lipoprotein particle high-density lipoprotein particle components bloodmicroparticle extracellular region extracellular exosome extracellular space of platelet alphagranulelumen proteins high-densitylipoproteinparticle very-low-densitylipoproteinparticle extracellular matrix identified immunoglobulincomplex, circulating chylomicron in exosomes obtained by different methods 10 Supplemental Table S1. Brief Comparison of Various Exosome Isolation Methods1 Methods Assay time Exosome recovery, % Ultracentrifugation 5-9 h 2-80% (blood, serum or plasma) Density Gradient Centrifugation 16-90 h 10% (plasma or serum) Size Exclusion Chromatograph 0.3 h 40-90% (plasma) Ultrafiltration 0.5 h 10-80% (plasma) Immuno-affinity 4-20 h / Precipitation based on polymer 0.25-12 h 90% (a mixture of EVs and serum proteins) TiO2-based method 5 min for incubation, 30 min for all steps (filtration needs less than 1 ≈80% (serum) min, washing steps cost about 4 min and on microspheres exosome 93.4% (model exosomes) lysis takes about 20 min) 11 Supplemental Table S2.
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