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.

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

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.

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

Supplemental Fig. S2 SDS-PAGE analysis (silver stained) of the proteins isolated from model sample by TiO2 microspheres. 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

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

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

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).

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).

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

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.

Supplemental Fig. S7 Schematic of the TiO2-based method for exosomes isolation and downstream analyses

TiO2 Ultracentrifugation Exosome Precipitation Reagent Exosomes 160

140

120 ) e

u Exosomes Exosomes l

a 100 v - P (

0 80 1 g o l

- 60

e e n e x n e e n n e n e e n g x e e e m c o cl ri e cl cl o e cl o m c e n le cl cl cl le n e e n le le ix g n o a gi ti t m ti ti r m ti gi o a m ti p ti ti ti ic io m ac e ic ic r in o s sp e r a u r r ic u r e s sp u la r r r t g o p m t t at t cr o r r a m l a a m l a r o r l u m a a a r e os s lu r r la i ex a r p r le p p o le p r ex a le c co p p p pa r x r pa pa m u m r l la ro la u n n yl u ro la r l u ir n n n o ar e la le r rc lo a llu u ic u n ei ei h n ic u a llu n c ck ei ei ei r l r u u in in la i y ul e ll ll a t t c a ll ul e a , a t t t ic lu la ll n te te lu , c h ll c ce m e gr ro ro gr m ce ll c gr ex tt ro ro ro m el u ce ra o o l x c e ra a d ac p p d a e ra l a p p p c ll a g r r ce le c t r o r a o o se o r c t a p e o o o d a ce r a p p a p ra ex xt lo t h ip ip n lo xt ra ex h m n ip ip ip oo tr a xt h o o tr t e b ex lp l l e b e t lp o a l l l l x r e p ip ip x om ex a y y d ex a c br y y y b e xt al l l e c et it it et et in it it it e t ty ty n l s s l l ul em s s s le si si li te en en te te b en en en e n n u la d d la la o m d d d at e e b p - h- p p gl - h- h- l -d -d lo ow g o ow g g p h w g -l hi n -l hi hi ig lo no y u y l h - u r m r a ry ve m ve ic e m i r v im he sp

Supplemental Fig. S8 Gene Ontology analysis of cellular components of proteins identified in exosomes obtained by different methods

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)

Supplemental Table S2. Numbers of protein groups identified under different conditions.

Volumn LC-MS condition Data Searching softeware Identified proteins groups in three replicate experiments (μL) 1 15 cm column, 78 min gradient MaxQuant 1.5.2.8 308 (212, 248, 248) 100 15 cm column, 78 min gradient MaxQuant 1.5.2.8 384 (294, 282, 319) 200 25 cm column, 120 min gradient Proteome Discoverer 2.0 889 (549, 567, 588)

Supplemental Table S3. Significantly regulated serum exosomal proteins in pancreatic cancer.

logF Reported in Pancreatic Cancer No. Gene.Symbol Uniprot ID Protein Gene adj.P.Val C (reference shown)

1 KV306_HUMAN P01624 Immunoglobulin kappa variable 3-15 IGKV3-15 7.74 1.49E-05 2 S10A9_HUMAN P06702 Protein S100-A9 S100A9 7.55 4.50E-09 2 3 H2B1H_HUMAN Q93079 Histone H2B type 1-H HIST1H2BH 7.50 2.97E-05 4 S10A6_HUMAN P06703 Protein S100-A6 S100A6 7.14 1.49E-05 2, 3 5 PROF1_HUMAN P07737 Profilin-1 PFN1 6.41 7.50E-06 2, 4, 5 6 LEAP2_HUMAN Q969E1 Liver-expressed antimicrobial peptide 2 LEAP2 6.32 3.92E-06 Putative aspartate aminotransferase, 7 AATC2_HUMAN Q8NHS2 GOT1L1 6.09 2.55E-07 cytoplasmic 2 Immunoglobulin lambda-like polypeptide 8 IGLL5_HUMAN B9A064 IGLL5 5.97 1.49E-05 5 9 S10A7_HUMAN P31151 Protein S100-A7 S100A7 5.44 2.93E-04 10 RAN_HUMAN P62826 GTP-binding nuclear protein Ran RAN 5.44 3.43E-04 6 11 KPRP_HUMAN Q5T749 Keratinocyte proline-rich protein KPRP 5.18 1.63E-03 12 CALL5_HUMAN Q9NZT1 Calmodulin-like protein 5 CALML5 5.14 4.06E-03 7 13 SBSN_HUMAN Q6UWP8 Suprabasin SBSN 5.10 1.86E-04 14 THIO_HUMAN P10599 Thioredoxin TXN 4.95 4.53E-03 8 15 SPR1B_HUMAN P22528 Cornifin-B SPRR1B 4.92 3.24E-03 16 PRDX2_HUMAN P32119 Peroxiredoxin-2 PRDX2 4.75 4.53E-03 9 17 LV301_HUMAN P01714 Immunoglobulin lambda variable 3-19 IGLV3-19 4.66 7.62E-03 18 FIBG_HUMAN P02679 Fibrinogen gamma chain FGG 4.59 4.57E-05 4, 6 19 CBG_HUMAN P08185 Corticosteroid-binding globulin SERPINA6 4.57 1.28E-03 20 SHBG_HUMAN P04278 Sex hormone-binding globulin SHBG 4.49 2.00E-03 10 Peptidyl-prolyl cis-trans isomerase 21 FKB1A_HUMAN P62942 FKBP1A 4.48 4.14E-03 FKBP1A 22 CYTA_HUMAN P01040 Cystatin-A CSTA 4.46 3.64E-03 23 CSRP1_HUMAN P21291 Cysteine and glycine-rich protein 1 CSRP1 4.45 9.88E-03 24 S10A4_HUMAN P26447 Protein S100-A4 S100A4 4.31 4.14E-03 11 25 PIP_HUMAN P12273 Prolactin-inducible protein PIP 4.29 1.08E-03 12, 13 Myeloid cell nuclear differentiation 26 MNDA_HUMAN P41218 MNDA 4.16 6.42E-03 antigen 27 LCN1_HUMAN P31025 Lipocalin-1 LCN1 4.15 2.76E-03 14 28 PIGR_HUMAN P01833 Polymeric immunoglobulin receptor PIGR 4.05 2.31E-03 15, 16 29 LDHB_HUMAN P07195 L-lactate dehydrogenase B chain LDHB 3.89 2.53E-04 17 30 FBX50_HUMAN Q6ZVX7 F-box only protein 50 NCCRP1 3.81 1.39E-03 18 31 GPV_HUMAN P40197 Platelet glycoprotein V GP5 3.75 4.14E-03 19 Adipocyte plasma membrane-associated 32 APMAP_HUMAN Q9HDC9 APMAP 3.67 9.07E-05 protein 33 DCD_HUMAN P81605 Dermcidin DCD 3.67 3.11E-06 20 34 FLNA_HUMAN P21333 Filamin-A FLNA 3.66 8.46E-03 21 35 BASP1_HUMAN P80723 Brain acid soluble protein 1 BASP1 3.56 3.18E-03 Protein-glutamine gamma- 36 TGM3_HUMAN Q08188 TGM3 3.55 1.38E-03 glutamyltransferase E 37 CFAI_HUMAN P05156 Complement factor I CFI 3.53 1.85E-03 22 Deleted in malignant brain tumors 1 38 DMBT1_HUMAN Q9UGM3 DMBT1 3.52 4.09E-04 23 protein 39 ELNE_HUMAN P08246 Neutrophil elastase ELANE 3.41 8.49E-03 24 40 F13B_HUMAN P05160 Coagulation factor XIII B chain F13B 3.37 1.63E-03 41 K2C78_HUMAN Q8N1N4 Keratin, type II cytoskeletal 78 KRT78 3.27 6.18E-03 42 TCPG_HUMAN P49368 T-complex protein 1 subunit gamma CCT3 3.24 4.53E-03 6 43 HORN_HUMAN Q86YZ3 Hornerin HRNR 3.21 4.54E-03 25 44 DSC1_HUMAN Q08554 Desmocollin-1 DSC1 3.21 2.85E-03 26 Eukaryotic translation initiation factor 2 45 IF2A_HUMAN P05198 EIF2S1 3.17 1.63E-03 subunit 1 46 IGJ_HUMAN P01591 Immunoglobulin J chain JCHAIN 3.14 4.38E-03 47 MYH9_HUMAN P35579 Myosin-9 MYH9 2.97 8.46E-03 2

48 CO4B_HUMAN P0C0L5 Complement C4-B C4B 2.86 3.23E-04 5 49 CO6_HUMAN P13671 Complement component C6 C6 2.83 1.40E-04 5 50 DESP_HUMAN P15924 Desmoplakin DSP 2.76 6.71E-03 51 H2A1J_HUMAN Q99878 Histone H2A type 1-J HIST1H2AJ 2.75 7.36E-03 52 MOES_HUMAN P26038 Moesin MSN 2.71 6.18E-03 27 53 CO7_HUMAN P10643 Complement component C7 C7 2.63 1.89E-05 54 FILA_HUMAN P20930 Filaggrin FLG 2.56 3.72E-04 Complement component C8 gamma 55 CO8G_HUMAN P07360 C8G 2.48 9.07E-05 chain Tyrosine-protein phosphatase non- 56 PTN6_HUMAN P29350 PTPN6 2.46 6.78E-03 receptor type 6 57 C4BPB_HUMAN P20851 C4b-binding protein beta chain C4BPB 2.39 1.85E-03 58 ACTB_HUMAN P60709 Actin, cytoplasmic 1 ACTB 2.21 1.42E-03 4 59 CO5_HUMAN P01031 Complement C5 C5 2.14 4.79E-05 15, 22 60 PLTP_HUMAN P55058 Phospholipid transfer protein PLTP -2.04 2.27E-03 61 PCYOX_HUMAN Q9UHG3 Prenylcysteine oxidase 1 PCYOX1 -3.57 4.14E-03 62 DNJC3_HUMAN Q13217 DnaJ homolog subfamily C member 3 DNAJC3 -4.17 2.31E-04 63 HV310_HUMAN P01771 Immunoglobulin heavy variable 3-33 IGHV3-33 -4.25 5.57E-04 Beta-galactoside alpha-2,6- 64 SIAT1_HUMAN P15907 ST6GAL1 -5.45 8.71E-04 sialyltransferase 1 65 APOA5_HUMAN Q6Q788 Apolipoprotein A-V APOA5 -5.74 1.06E-04

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Author Contributions

F. Gao and F. Jiao designed experiments and wrote the original draft; F. Gao, F. Jiao and C. Xia carried out experiments; Y. Zhao performed the data analyses; W. Ying and Y. Xie helped perform the data acquisition; X Guan and M Tao collected serum from patients; Y Zhang, W Qin and X. Qian reviewed and edited the manuscript. All authors read and approved the manuscript.