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Human Urinary Exosomes as Innate Immune Effectors

† † ‡ Thomas F. Hiemstra,* Philip D. Charles, Tannia Gracia, Svenja S. Hester,§ † ‡ | Laurent Gatto, Rafia Al-Lamki,* R. Andres Floto,* Ya Su, Jeremy N. Skepper, † ‡ Kathryn S. Lilley, and Fiona E. Karet Frankl

*Department of Medicine, †Cambridge Centre for Proteome Research and Cambridge Systems Biology Centre, Department of Biochemistry, ‡Department of Medical Genetics, and |Multi-Imaging Centre, Department of Anatomy, University of Cambridge, Cambridge, United Kingdom; and §Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

ABSTRACT Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, presentation, modulation of , and shedding of obsolete . Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune that include antimicrobial proteins and and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm–Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.

J Am Soc Nephrol 25: 2017–2027, 2014. doi: 10.1681/ASN.2013101066

Exosomes form as intraluminal vesicles of multi- every cell type along the nephron.13,14 The array of vesicular bodies (MVBs), contain membrane and functions ascribed to exosomes in other tissues has cytoplasmic proteins, have a cytoplasmic-side in- kindled recent interest in the functional significance ward membrane orientation, and are released intact of urinary exosomes. Hogan et al.13 suggested inter- into the extracellular space (Figure 1A). First de- action of exosome-like vesicles with primary cilia of scribed in maturing ovine reticulocytes,1 exosomes renal epithelial cells, and Street and coworkers15 are released by many cell types2 and have been con- showed in vitro uptake of exosomes by a renal cortical ventionally regarded as a vehicle for shedding ob- solete protein. However, emerging evidence has revealed a variety of exosomal functions, including Received October 11, 2013. Accepted January 6, 2014. 3 the intercellular transfer of membrane receptors Published online ahead of print. Publication date available at 4–6 7 and RNA, induction of immunity, antigen pre- www.jasn.org. sentation,8 modulation of bone mineralization,9 Correspondence: Prof. Fiona E. Karet Frankl, Department of 10 and antiapoptotic responses. Medical Genetics, University of Cambridge, Cambridge Institute Nanovesicles were first shown in human urine by for Medical Research, Cambridge Biomedical Campus Box 139, Kanno and colleagues11 and subsequently, were Hills Road, Cambridge CB2 0XY, UK, or Prof. Kathryn S. Lilley, Cambridge Centre for Proteomics and Cambridge Systems Bi- 12 shown to represent exosomes. Consistent with a ology Centre, Department of Biochemistry, University of Cambridge, renal tubular epithelial origin, renal tubular epithelial Cambridge CB2 1QR, UK. Email: [email protected] or fek1000@ cells contain MVBs at the apical surface, and urine cam.ac.uk exosomes contain apical membrane proteins from Copyright © 2014 by the American Society of Nephrology

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Published reports of the urinary exoso- mal proteome have limited value in illumi- nating the potential functions of urinary exosomes for several reasons. First, protein identification by mass spectrometry (MS) has, until recently, yielded results with unacceptably low reproducibility and high false-positive rates,22,23 and previous re- ports are not free of these limitations. Sec- ond, studies have often aimed to maximize the number of protein identifications and hence, biomarker candidates rather than applying or reporting rigorous protein identification thresholds. Third, most have relied on pooled samples from up to six donors and have not reported interin- dividual variability or reproducibility. Here, we sought, for the first time, to ascribe functionality to human urinary exosomes. We initially performed rigorous, conservative tandem MS analysis of sepa- rate human urinary exosomal samples to allow enrichment scoring24 to elucidation of urine exosomal function.

RESULTS

Exosomal samples were obtained from 10 healthy volunteers (five men and five women) ages 23–36 years. Nine subjects were Caucasian, and one man was Mauri- Figure 1. Vesicles isolated from human urine are consistent with exosomes. (A) tian. Samples prepared from 455628 ml sec- – Exosomes are derived from the endocytic pathway (1 4) forming through invagination ond morning void contained 0.5460.18 mg of the limiting membrane of the MVB (3). They are released into the urinary space from protein/ml urine. Exosomal integrity was renal tubular epithelial cells through fusion of the MVB with the apical plasma mem- fi fi fi con rmed by the electron microscopy brane (4). (B and C) Exosomal isolation was con rmed by the identi cation by negative 6 stain EM of nanovesicles (black arrows; characteristic mean 50-nm size distribution. (D) (EM) demonstration of 54.5 14-nm ves- fi Uromodulin (white streaks in B and dark in C; open arrows in B and C) cofractionated icles (Figure 1, B and D), the identi ca- with exosomes but was confirmed to be extraexosomal (5 nm gold-labeled; white tion of known exosomal markers TSG101, arrows in C). (E) Western blot confirmed the presence of known exosomal constituents enolase-1, CD63, podocin, and aquaporin-2 in vesicle preparations but did not confirm them in precipitated protein from exosome- by Western blot and immunogold EM depleted urine. (F) Immuno-EM with 5 (TSG101 and CD63) or 15 nm (AQP2) gold (Figure 1, E and F), and the confirmation particle-labeled antibodies showed vesicular residency of known exosomal con- of a cytoplasmic side inward membrane ori- stituents (arrows). Vesicles were nonpermeabilized; thus, positive staining with an anti- entation (Figure 1F). CD63 antibody directed against an extracellular indicated the cytoplasmic We applied a number of methodological side inward membrane orientation characteristic of exosomes. EDUP, exosome-depleted approaches to overcome limitations of pre- urine protein; HKM, human membrane; MW, molecular weight; TSG101, tumour susceptibility 101. vious studies. (1) Samples were not pooled but analyzed separately. (2)MSwasper- formed without and with a uromodulin ex- collecting duct cell line, leading to speculation that exosomes clusion list25 on a high-sensitivity instrument. (3) Data were may provide intrarenal proximal-to-distal transapical renal tu- analyzed using stringent and novel bioinformatics ap- bular epithelial signaling through RNA transfer. However, most proaches26 (Supplemental Material). (4) All ambiguous pep- studies on urine exosomes to date have focused on biomarker tides were excluded unless matched only to products of a discovery, resulting in the publication of several urine exosome single gene. From 50 mg exosomal protein per subject, we protein compendia.12,13,16–21 identified 601 unique proteins by MS, with a median

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2 2 (interquartile range [IQR]) P value of 1.17310 13 (4.08310 4) suggesting a potential role in the targeting of RNA to exo- (Supplemental Table 1). Importantly, the use of a fixed uromodulin somes,4 or involved in innate immunity and the response to exclusion list25 and a posterior error-derived protein type I infection. Uromodulin was present in all 10 samples, consis- error estimator termed espresso (Supplemental Material) un- tent with previous reports, but confirmed as extraexosomal by masked proteins that would not otherwise have been evident immunogold EM (Figure 1D). All MS data have been depos- (Figure 2A). The complete proteome included the known exo- ited with the ProteomeXchange consortium repository somal markers TSG101, CD14, and CD59. However, 307 (51%) (http://proteomecentral.proteomexchange.org; reference had not previously been identified in exosomes from any source PXD000117). compared with the EXOCARTAdatabase.27 There was only min- Using enrichment scoring (ES),24 we found, in addition to imal overlap with the soluble urinary proteome (Supplemental the expected enrichment for cytoskeletal proteins (ES=10.45) Figure 1). and proteins involved in the endocytic pathway and vesicle As previously reported for exosomes,12 the cellular origin of formation (ES=8.06), a very significant enrichment for pro- the majority of identified proteins was in vesicles or the endo- teins involved in immunity and host defense (ES=3.27, cytic vesicular pathway, cytoplasm, plasma membrane, and P,0.001). The MS-derived type I error estimates for proteins nucleus (Figure 2B). Apical membrane proteins from all neph- falling into this grouping were consistently small (Figure 3A), ron segments were detected, including aminopeptidase N, car- indicating high significance. These 29 proteins (Figure 3B, bonic anhydrase II and IV, chloride intracellular channel 1, Table 1) included archetypal antimicrobial proteins and pep- Cubilin, Dipeptidase 1, -D28k,andVacuolarATPase tides and fell into two categories: those proteins with known 6V0 subunit C. Most proteins were identified from fewer than bacteriostatic (such as -1, fibronectin, and CD14) or one half of samples; 47 (8%) proteins were identified in all 10 bactericidal (e.g., lysozyme C, calprotectin [S100A8/A9], and samples (Figure 2, C and D), and 97 (16%) proteins were dermcidin) roles and those proteins that function as microbial identified in at least 8 of 10 samples. Each of the shared pro- receptors or bind to bacterial surface molecules.28–33 Impor- teins was identified from 6 (IQR=2–24) peptides, indicating a tantly, 28 of 29 immune proteins have known expression in set of consistently identifiable urinary exosomal constituents kidney (Table 1). We confirmed the presence and exosomal (Figure 2E). This core exosomal proteome included a large residency of a representative group of these innate immune number of proteins involved in the endocytic pathway, MVB exosomal proteins, including lysozyme C, dermcidin, mucin- formation, and exosomal biogenesis and cytoskeletal proteins 1, calprotectin, and myeloperoxidase, by Western blot and required for exosomal structural integrity. A considerable immunogold EM (Figure 3C, Supplemental Figure 9). The number was involved in and RNA processing, EM appearances differ markedly from the considerably larger

Figure 2. The human urinary exosomal proteome. (A) Using conventional methods (Mascot scoring and no uromodulin exclusion), only 237 proteins would have been evident (green). The application of a uromodulin exclusion list (purple) to conventional methods and the use of espresso (cyan) greatly increased protein identifications, which was further amplified by their combination. (B) Cellular locali- zation of 601 proteins identified from exosomal pellets by tandem MS. A significant proportion consisted of cytoplasmic and mem- brane proteins or constituents of the endocytic pathway or vesicles. (C) Heat map showing the overlap between subjects for all proteins identified from two or more peptides (yellow) or one (red) or not seen (black). (D) Most proteins were identified in the minority of subjects, consistent with the stochastic nature of MS. The x axis represents the number of subjects in whom each protein was observed; 47 proteins were observed in all 10 subjects, showing the value of analyzing multiple samples separately. (E) Principal protein groupings were consistent with previous reports of exosomes but also included those exosomes with an innate immune role.

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been proposed as an antimicrobial urinary defense protein38 and it cofractionates with exosomes during ultracentrifugation.25,39 Initial colony counting40 showed significant reduction in bacteria after incubation with exosomes (with or without uromodulin) but not uromodulin alone. However, this method is unable to assess real-time growth. To achieve this result, we trans- formed BL21 with the luxCDABE op- eron41 (BL21-lux) to constitutively express luciferase; transfected organisms sponta- neously omit 490-nm . Compared with buffer alone, exosomes from each of four healthy volunteers significantly in- hibited growth of BL21-lux,whereas growth was not altered by uromodulin (Figure 4). There was only minor interin- dividual variation (Supplemental Figure 2). The effect of exosomes on bacterial growth occurred rapidly, becoming appar- ent within 30 minutes of coincubation (Supplemental Figure 3). Complete inhibi- tion of bacterial growth was dependent on exosomal structural integrity (Figure 4A, Supplemental Figure 4). To assess clinical relevance, we first transformed a uropathogenic E. coli (UPEC) strain, obtained from an infected patient, to express the luxCDABE cluster (UPEC-lux). Despite more vigorous growth, UPEC-lux growth was still almost arrested by exosomes from healthy volun- teers (Figure 4A). We repeated these ex- periments with both BL21-lux and UPEC-lux using exosome-depleted urine Figure 3. Exosomes are enriched for innate immune proteins. (A) The exosomal as growth medium. Exosomes were equally proteome was significantly enriched for proteins with a known role in host defense. Proteins falling into this category (red) were identified with a high degree of statistical effective against both organisms, although confidence compared with all nonimmune proteins called. (B) The immune group as expected, their overall growth was slower included bactericidal and bacteriostatic proteins as well as those proteins known to in urine. We next sought to determine function as bacterial or viral receptors. (C) Western blot confirmed the presence of whether exosomes were effective against a representative group of proteins identified by MS, including myeloperoxidase (MPO), well characterized, luxCDABE-transformed, mucin-1 (MUC1), dermcidin (DCD), calprotectin (S100A8/A9 heterodimer), and lysozyme commensal (Nissle) or standard uropatho- C (LYZ). Positive controls represent human kidney membrane, except DCD (purified genic (UTI89 and CFT073) model strains of DCD) and calprotectin (neutrophil lysate). Immunogold EM shows the decoration of E. coli. In both exosome-depleted donor vesicles with 5-nm gold particles. urine and Luria-Bertani (LB) media, we ob- served consistent, significant growth inhi- and more heterogeneous neutrophil granule,34 where some of bition of all three organisms by exosomes but not by uromodulin these proteins are also found. alone (Figure 4A). Colony counting at peak bacterial growth Because of this enrichment, we next tested the hypothesis confirmed that the observed differences in luminescence were that exosomes may inhibit growth of or kill Escherichia coli,the attributable to significant differences in the number of viable organism responsible for up to 90% of urinary tract infections organisms (Figure 4B). (UTIs) in humans.35–37 First, BL21, a laboratory strain of The effect of coincubation with exosomes on bacterial E. coli, was incubated with exosomes, suspension buffer alone, or integrity was next evaluated by scanning EM. UPEC-lux in- purified human uromodulin, because uromodulin has itself cubated with exosomes or control and freeze-dried after 5 or

2020 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2017–2027, 2014 mScNephrol Soc Am J Table 1. Identified exosomal proteins with a known innate immune role No. of Known Present in P Peptide IPI Identifier Gene Symbol Protein Putative Immune Role Subjects Disambiguated Expression Exocarta Value Count with Protein in Kidney IPI00221224 ANPEP Aminopeptidase-N E. coli viral sensing Yes 0 10 292 Yes 25: IPI00022463 TF Transferrin Bactericidal Yes 0 10 23 ✔ Yes 2017 IPI00023673 LGALS3BP -3-binding protein Possibly bacteriostatic; Yes 0 10 31 ✔ Yes

– —

07 2014 2027, secreted in breast milk prevents diarrheal illness IPI00394972 IPI00394975 TRIM5 Tripartite motif Viral sensing; antiviral responses No ,0.001 10 10 ✔ Yes IPI00852949 IPI00939323 containing protein 5 IPI00909886 CARD9 Caspase recruitment Antifungal responses; deficiency No ,0.001 10 3 Yes domain-containing 9 results in mucosal and systemic candidiasis IPI00024254 IFIT3 IFN-induced protein Viral sensing No ,0.001 10 2 Yes with tetratricopeptide repeats 3 IPI00004573 PIGR Polymeric Ig receptor Expression of IgA and IgM at Yes 0 10 135 ✔ Yes apical surface IPI00247063 MME Unknown Yes 0 10 86 ✔ Yes IPI00797452 KRT10 10 Cell surface receptor for Yes 0 10 416 ✔ Yes Staphylococcus aureus IPI00027462 S100A9 S100A8/A9 (Calprotectin; Bactericidal Yes 0 (A9) 9 11 Yes IPI00007047 S100A8 occur as heterodiner) Yes ,0.001 (A8) 6 8 Yes IPI00019038 LYZ Lysozyme C Bactericidal Yes 0 8 102 Yes IPI00294713, IPI00306378 MASP2 Mannan-binding lectin Bactericidal enterobacterial Yes 0 8 35 ✔ Yes peptidase 2 receptor IPI00013955, IPI00218163, MUC1 Mucin-1 Unknown bacterial receptor Yes ,0.001 7 8 ✔ Yes IPI00218164, IPI00218165, IPI00218166, IPI00218168, IPI00218169, IPI00607673,

mueRl o rn Exosomes Urine for Role Immune IPI00902840, IPI00978078 IPI00007244, IPI00236554, MPO Myeloperoxidase Bactericidal fungicidal Yes ,0.001 4 5 ✔ Yes

IPI00236556 www.jasn.org IPI00029260 CD14 differentiation Binds bacterial cell walls Yes ,0.001 4 5 Yes antigen CD14 IPI00219806 S100A7 Psoriasin Bactericidal No ,0.001 3 19 ✔ Yes IPI00218733 SOD1 Superoxide dismutase 1 Protects against schistosomiasis; Yes ,0.001 3 4 ✔ Yes mechanism unknown AI RESEARCH BASIC IPI00027547 DCD Dermcidin Bactericidal Yes ,0.001 2 177 ✔ Yes IPI00032328, IPI00215894 KNG1 Kininogen-1 Bactericidal Yes ,0.001 2 95 ✔ Yes IPI00009276 PROCR receptor Unknown; protective against No ,0.001 2 4 Yes E. coli bacteremia 2021 BASIC RESEARCH www.jasn.org

15 minutes showed clear evidence of exosome-induced bacterial lysis (Figure 5). Organisms incubated with exo- No Yes Yes Yes No Yes Yes Yes

Known somes for 5 minutes showed an increased proportion of in Kidney Expression lysed (12%) compared with control samples (1.8%, P=0.003), increasing to 56% versus 2% after 15 minutes (Figure 5, A and C), consistent with the rapid in- ✔ ✔ ✔ ✔ ✔ hibition of BL21-lux growth observed earlier (Supplemen- tal Figure 3).

Disambiguated All these bacterial growth experiments were performed at pH 5.5–6.0, typical of omnivorous human urinary pH; exosomes were, however, also effective at pH 6.5 and pH Count Peptide 7.0 (Supplemental Figures 5 and 6). Furthermore, we noted that 0.5 mg/ml exosomes were effective against a 2 4 2 4 2 4 1 2 1111 1 1 1 starting 1 UPEC-lux concentration (representing 500 rela-

No. of .

Subjects tive light units) of 100 CFU/ml (Supplemental Figure

with Protein 8), confirming our observations to be highly physiologi- cally relevant. P Value 0.001 0.001 0.001 0.001 0.001 0.001 0.001 , , , , , , , DISCUSSION

Urinary exosomes released by renal tubular epithelia are No 0 No Yes No No Yes Exocarta

Present in present in human urine, where their function is unknown. Here, we examined the normal human urinary exosomal proteome through in-depth MS and showed significant enrichment for known innate immune proteins. EM studies confirmed the exosomal residency of antibacterial proteins and peptides. Urinary exosomes from healthy volunteers inhibited the growth of different uropathogenic and commensal E. coli strains and induced bacterial lysis, and these effects were dependent on the structural integrity of urinary exosomes. These findings suggest that urinary receptor exosomes function within the renal tract as innate immune Bactericidal bacteriostatic Bactericidal and bacteriostatic Yes Bactericidal Microbial receptor Bacteriostatic (binds iron) Bacteriostatic (binds iron) Bactericidal fungicidal Bactericidal and bacteriostatic Yes effectors. Although many of the innate immune proteins identi- fiedinouranalysis had beenpreviously identified inurinary exosomal analyses (Supplemental Table 1),12,13,16–21 en- richment for this protein grouping has not previously been reported. However, enrichment analysis relies on the reliability of the background proteome. Our analysis associated protein 1

-Defensin 9 of separate urinary exosomal preparations, along with key b methodological advances, has revealed a number of pro- teins identified from urinary exosomes for the first time. Furthermore, rather than place emphasis on maximizing the number of protein identifications, we made every at- FN1 Fibronectin DEFB109 PLUNC Palate lung and nasal LCN1LCN2 Lipocalin 1 Lipocalin 2 PGLYRP1 recognition HTN1 Histatin-1 LTF tempt to ensure the robustness of those proteins ultimately included in this proteome. These key aspects of our study enabled the clear identification of enrichment for innate immune proteins (confirmed by direct methods) as well as er Gene Symbol Protein Putative Immune Role

fi the expected enrichment for those proteins with a cyto- skeletal role, involved in MVB or exosomal biogenesis, or Continued resident on the luminal surface of the nephron. IPI Identi UTIs represent the most common bacterial infection in humans, with an estimated 150 million UTIs per annum IPI00339224, IPI00339225, IPI00339226, IPI00339227, IPI00339228, IPI00339319, IPI00411462, IPI00414283, IPI00479723, IPI00556632, IPI00855777, IPI00855785, IPI00867588 42,43 35,36,44 IPI00935308 IPI00009856 Table 1. IPI00021085 IPI00022418, IPI00339223, IPI00012024 Group: LCN1 Group: LCN2 IPI00298860 globally. Most of these UTIs are caused by E. coli.

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The anatomic location of the distal urethra results in its continuous exposure to large numbers of bacteria. Nevertheless, the uri- nary tract is usually sterile above the ure- thral meatus, indicating the existence of highlyeffectiveinnateimmunemecha- nisms within the renal tract. To date, these mechanisms remain incompletely under- stood. For infection to occur, uropatho- genic organisms must overcome a variety of physical and immune obstacles and as- cend the urinary tract. First, organisms must adhere to uroplakin-covered urothe- lium, which is limited by the shear flow of urine and the uromodulin, which impairs the ability of bacteria to ad- here and facilitates their expulsion during voiding.45 Second, invading organisms are exposed to free radicals and soluble antimi- crobial proteins and peptides present within urine, although the soluble concen- trations of these proteins and peptides seem too low to be directly bacteri- cidal.46,47 To evade these defenses, organ- isms typically invade epithelial cells, where they proliferate intracellularly before egressing back into the urinary space, a process that may, in turn, trigger acute in- flammatory responses through Toll-like re- ceptor activation. Our findings indicate that, in addition, this defensive array in- cludes exosomes, which must be evaded or overwhelmed for UTI to occur. Consti- tutively released by renal tubular epithelia, a continuous stream of exosomes acts as an

(P=0.04), bacterial growth did not significantly differ with uromodulin compared with control in any experiment. Intact exosomes inhibited growth of BL21 compared with lysed exosomes (shown in green; P,0.001); lysed exosomes in- duced some growth inhibition compared with buffer only (P,0.001). As expected, given the protein- and carbohydrate-rich nature of LB me- dium compared with urine, all organisms grew Figure 4. Exosomes inhibit growth of E. coli. (A) All exosomal additions were made at more vigorously in LB. For Nissle, UTI89, and 25 mg/ml. All curves represent mean6SEM; y axes show luminescence (relative light E. coli O6:H1 (CFT073) grown in urine, the num- units3106), and x axes show time in hours. Growth curves are left-censored at the ber of viable organisms at peak bacterial growth onset of detectable bacterial growth and arranged by growth medium (columns) and (dashed lines labeled A–C) was additionally as- organism (rows). All data represent at least three replicates per condition. Data for sessed by conventional colony counting (corre- BL21 represent triplicates for each of four biologic replicates. For all strains of E. coli sponding bar graphs in B). Addition of exosomes tested, highly significant inhibition of growth in either medium occurred with the to donor urine resulted in significant reductions addition of exosomes compared with controls (P values shown). With the exception of in the number of CFU for Nissle (P=0.009), UTI89 UPEC in LB, where uromodulin achieved some growth inhibition compared with control (P=0.05), and CFT073 (P=0.003). Colony counts are shown as CFU per milliliter3106.

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even with contemporary high-sensitivity mass spectrometers, mitigation against the masking effect of abundant proteins is incomplete. Lower abundance proteins may, therefore, have gone undetected or may not have been consistently seen. In contrast to the situation in vivo, where exosomes would be continually replenished, our in vitro experiments used a single exosomal dose; however, the strength of the observed effect further supports its physiologic relevance. Finally, uromodulin cofractionates with urinary exosomes during isolation; there- fore, we have not directly assessed the effects of uromodulin- free exosomal preparations, because the exosomal population would be highly compromised. Our findings add to an expanding body of evidence that exosomes are biologically active in a wide variety of tis- sues.4,10,48–50 They are consistent with a report of protection against viral respiratory pathogens by respiratory epithelium- derived exosomes,51 in which Kesimer and colleagues51 showed inhibition of influenza A virus infection of Madin– Darby canine kidney cells by respiratory epithelium-derived exosomes in vitro. The demonstration that urinary exosomes are able to induce bacterial lysis and inhibit growth at bacterial concentrations that are highly physiologically relevant makes Figure 5. Exosomes induce lysis of E. coli. (A) Incubation of UPEC identifying factors that modulate exosomal release and con- with exosomes (Exos) showed rapid induction of bacterial lysis. stitution key priorities for future work and may reveal poten- An increase in lytic phenotypes was detected as early as 5 mi- tial therapeutic targets for the treatment of UTIs. nutes after incubation (P=0.003); after 15 minutes, more than 50% of UPEC had undergone lysis (P,0.001). (B–D) Intact or- ganisms (B and D, white arrow) and whole organisms that had lost CONCISE METHODS integrity (C and D, black arrows) were counted, but lysed bac- terial fragments (D, open arrows) were not. Exosomal Isolation Exosomes were isolated from 10 healthy volunteer urine samples as innate immune sentinel within the urinary tract. Because exo- previously described.25 The five men and five women ages 23–36 somes contain molecules at once attractive and lethal to bac- years were on no regular medication and had not consumed anti- teria, they provide vehicles for the efficient, targeted distal biotics or other medication within the previous 1 month. Current delivery of antimicrobial molecules and confer on renal epithelia UTI was excluded. Briefly, subjects urinated directly into a container the ability to effect distant bacterial killing. Furthermore, they with protease inhibitors, including PMSF (500 ml 0.5 M solution), may function as decoys in limiting interaction of bacterial leupeptin (450 mg), and sodium azide (15 ml 100 mM solution). adhesion molecules with epithelial surfaces. Urine was centrifuged within 30 minutes of collection (Beckman Our study has several strengths. First, this study is the first AVANTI J26-XP centrifuge; JA-17 fixed angle rotor; polyallomer comprehensive report of in-depth proteomic analysis of 50-ml centrifuge bottles) for 20 minutes at 17,0003g. The superna- individual, rather than pooled, urinary exosomal samples. tant was passed through a sterile 0.22-mm filter and ultracentrifuged Second, we report that a urinary exosomal proteome was (Beckman Optim L-100 XP VAC Ultracentrifuge; Ti45 fixed-angle constituted by statistically robust and reproducibly identifiable titanium rotor; Beckman 70-ml polycarbonate ultracentrifuge bot- protein identifications. Third, we show a clinically relevant tles) for 135 minutes at 235,0003g and 4°C. Each ultracentrifugation effect of urinary exosomes on the most common human pellet was suspended in 50 ml suspension buffer (250 mM sucrose and urinary pathogen E. coli using several methodologies. How- 10 mM triethanolamine [pH 7.6]) and pooled with the other pellets ever, our findings should be interpreted within the limitations from the same urine sample. To avoid confounding effects on bacte- of the study. It is not possible to infer from our data which rial growth, urine samples for these experiments were collected with- exosomal proteins represent the key effectors, because targeted out protease inhibitors or sodium azide, triethanolamine was removed deletion of individual exosomal proteins has not yet been pos- from the suspension buffer, and exosomal pellets were washed one sible. More than one third of innate immune proteins were time by resuspension and ultracentrifugation. consistently identified ($8 of 10 samples), and this subgroup may be central. However, we cannot exclude the possibility that Exosome-Depleted Urinary Protein other less consistently identified members of this proteome or Samples were prepared as described for exosomal isolation above. The indeed, unidentified molecules are important. Furthermore, supernatant from the 235,0003g centrifugation was retained after

2024 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2017–2027, 2014 www.jasn.org BASIC RESEARCH harvesting exosomal pellets. This supernatant contained the nonexo- scoring was performed using the DAVID bioinformatics tool.24 This somal soluble urinary protein. For MS, protein was precipitated from method provides a measure (by tests of proportions) of whether these supernatants (typically 360 ml per subject) by the addition of functional categories are overrepresented within a gene list compared ammonium acetate and acetone (Supplemental Material). Pellets with what is expected from stochastic sampling of the entire human were resuspended with Laemmli sample buffer. gene set.

Sample Preparation for MS Protein Confirmation and Antibodies Protein from resuspended exosomal pellets was concentrated by Proteins identified by MS were confirmed by Western blotting precipitation and quantified using a Bradford protein binding according to standard methods and immuno-EM (described below). colorimetric assay (Bio-Rad). Protein pellets were stored at 280°C For each protein, the same antibodies (Supplemental Material) were until use. used for both techniques. For MS, protein pellets were suspended in Laemmli sample buffer and incubated at 95°C; 50 mg protein from each sample was fragmented Bacterial Transformation on a 4%/12% SDS–polyacrylamide gel. After staining, each gel track Bacteria were transformed by electroporation to express the luxCDABE was separated into 28 equal sections, which were processed indi- operon (Bioware plasmid pXEN13), which results in ampicillin re- vidually for the remainder of the workflow. After destaining, pro- sistance and constitutive expression of luciferase. teins were reduced and alkylated in gel, washed with NH4HCO3,and dehydrated in acetonitrile, and proteins were digested with modified Bacterial Growth Assays . Exosomal samples were collected from the same 10 healthy volunteers Liquid chromatography–MS/MS was performed using an Eksigent described earlier, but in contrast to samples prepared for MS, these NanoLC-1D Plus (Eksigent Technologies) HPLC system and an LTQ samples were handled without protease inhibitors, triethanolamine, Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Peptides or sodium azide. were separated by reverse-phase chromatography (Dionex). Peptides were loaded onto a 5-cm C18 precolumn (300 mm inner diameter; Bacterial Colony Counting LC Packings) from the autosampler. Peptides were eluted onto the Colony counting was performed essentially as in Miles and Misra.40 analytical column using gradients for solvent A (water+0.1% formic Briefly, serial dilutions of overnight bacterial cultures were spotted acid) and B (acetonitrile+0.1% formic acid) of 5%–50% B over onto ampicillin-impregnated agar plates and incubated at 37°C for 18 40 minutes. A New-Objective nanospray source was used for electro- hours. Colony counts were made from the highest bacterial concen- spray ionization. m/z Values of eluting ions were measured in the tration that yielded distinct colonies. Orbitrap mass analyzer with a mass range of 350–1600, and the res- olution was set at 7500. Bacterial Growth Curves For each growth assay, negative controls comprised suspension buffer MS Data Processing only or purified uromodulin (P135–1; SCIPAC) diluted in suspension Peptides from each gel segment were run two times. All segments were buffer. Overnight bacterial cultures were diluted in LB broth to lu- runwith dynamicexclusion. From these runs, a fixed exclusion list was minescence of 100–500 relative light units. At 4°C, 10 mlbacterial generated for the abundant protein uromodulin and superimposed culture was placed in each well of a 96-well plate; 5 mg exosomal on a dynamic exclusion list as described elsewhere.25 Data from these protein, uromodulin, or an equal volume of suspension buffer was two sets of runs were combined. MS data were processed using the added to each well. Volumes in all wells were standardized to 200 ml SEQUEST Bioworks Browser (version 3.3.1 SP1; Thermo Fisher Sci- with LB or exosome-depleted urine as appropriate (Supplemental entific) to generate MS/MS peak lists. Combined peak list files were Figure 7). All samples were evaluated at least in triplicate. Plates submitted to the MASCOT search algorithm (version 2.2.1; Matrix were incubated at 37°C, and luminescence readings were obtained Science) and searched against the IPI-Human Database, version 4.3. hourly until growth ceased. Exosomes were chemically lysed by se- Spectra were rescored using MASCOT-Percolator, a machine learning quential addition of ammonium acetate in methanol and acetone as tool that minimizes false discoveries and incorporates target decoy described for protein precipitation above. searching.26 Protein identification required two or more unique pep- tides, with a false discovery rate of 0.1. Single peptide identifications EM were included if the MASCOT-percolator posterior error probability Negative staining transmission EM with neutralized phosphotunstic was ,0.01. In addition, we included proteins based on an in-house acid or uranyl acetate on carbon film grids was performed with an FEI protein type I error estimator termed espresso (Supplemental Mate- Tecnai G2 electron microscope operated at 120 kV using an AMT rial). The cellular location of proteins was evaluated by searching the XR30B digital camera. For immunolabeling, samples were placed Ensembl gene identifier for each protein against the Human Protein on glow-discharged nickel grids for 30 seconds. Primary antibodies Atlas Database (http://www.proteinatlas.org). Renal expression was were applied for 15 minutes at room temperature. Secondary anti- evaluated using the bioGPS Gene Portal System (http://www.biogps. bodies labeled with 5-, 10-, or 15-nm gold particles were applied for org). Comparison of exosomal proteins with previous reports was 15 minutes. Grids were blocked with PBS. For scanning EM, bacterial made using the Exocarta exosomal protein database.27 Enrichment cultures were incubated with exosomes, and 10 mlwerespottedonto

J Am Soc Nephrol 25: 2017–2027, 2014 Immune Role for Urine Exosomes 2025 BASIC RESEARCH www.jasn.org glass coverslips, quench-frozen in propane-cooled liquid nitrogen, 8. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, freeze-dried (Edwards Auto 306 Turbo), and gold-coated. Scanning Melief CJ, Geuze HJ: B lymphocytes secrete antigen-presenting vesi- – EM was carried out with a Philips XL-30 FEG–scanning EM, and cles. JExpMed183: 1161 1172, 1996 9. Boyan BD, Wong KL, Fang M, Schwartz Z: 1alpha,25(OH)2D3 is an images were captured with an accelerating voltage of 5 kV. autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases. JSteroid Statistical Analyses Biochem Mol Biol 103: 467–472, 2007 Data were analyzed with Stata SE, version 12.1. Data are presented as 10. Sirois I, Raymond MA, Brassard N, Cailhier JF, Fedjaev M, Hamelin K, means6SDs or median (IQR) as appropriate. Parametric continuous Londono I, Bendayan M, Pshezhetsky AV, Hébert MJ: Caspase-3- dependent export of TCTP: A novel pathway for antiapoptotic in- variables were compared with the t test, and nonparametric contin- tercellular communication. Cell Death Differ 18: 549–562, 2011 uous variables were compared with the Wilcoxon sign test. Bac- 11. Kanno K, Sasaki S, Hirata Y, Ishikawa S, Fushimi K, Nakanishi S, Bichet terial growth was compared using response feature analysis with the DG, Marumo F: Urinary excretion of aquaporin-2 in patients with di- area under the curve as the response feature, and comparisons were abetes insipidus. NEnglJMed332: 1540–1545, 1995 fi made by one-way ANOVA. Proportions were compared with Pearson’s 12. Pisitkun T, Shen RF, Knepper MA: Identi cation and proteomic pro- filing of exosomes in human urine. Proc Natl Acad Sci U S A 101: 13368– chi-squared test of proportions. 13373, 2004 13. Hogan MC, Manganelli L, Woollard JR, Masyuk AI, Masyuk TV, Tammachote R, Huang BQ, Leontovich AA, Beito TG, Madden BJ, Charlesworth MC, ACKNOWLEDGMENTS Torres VE, LaRusso NF, Harris PC, Ward CJ: Characterization of PKD protein- positive exosome-like vesicles. J Am Soc Nephrol 20: 278–288, 2009 14. Dear JW, Street JM, Bailey MA: Urinary exosomes: A reservoir for We thank M. Clatworthy and M. Berry for the generous donation of biomarker discovery and potential mediators of intrarenal signalling. UTI89 E. coli and Ardeypharm GmbH for Nissle E. coli (Mutaflor). Proteomics 13: 1572–1580, 2013 This work was supported by an Action Medical Research Training 15. Street JM, Birkhoff W, Menzies RI, Webb DJ, Bailey MA, Dear JW: Exosomal transmission of functional aquaporin 2 in kidney cortical Fellowship (to T.F.H.), Wellcome Trust Grant 088489 (to F.E.K.F.)and collecting duct cells. J Physiol 589: 6119–6127, 2011 Strategic Award 079895 (to Cambridge Institute for Medical Research), 16. Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, the National Institute for Health Research Cambridge Biomedical Wang NS, Knepper MA: Large-scale proteomics and phosphoproteomics Research Centre (CBRC), and the Biotechnology and Biological Sci- of urinary exosomes. J Am Soc Nephrol 20: 363–379, 2009 ences Research Council (BBSRC). T.F.H. received a Raymond and 17. Zhou H, Pisitkun T, Aponte A, Yuen PS, Hoffert JD, Yasuda H, Hu X, Chawla L, Shen RF, Knepper MA, Star RA: Exosomal Fetuin-A identified Beverley Sackler Research Studentship and is currently supported by by proteomics: A novel urinary biomarker for detecting acute kidney the CBRC. P.D.C. was supported by BBSRC Research Studentship BB/ injury. Kidney Int 70: 1847–1857, 2006 D526088/1. L.G. is supported by a 7th Framework Programme of the 18. Nilsson J, Skog J, Nordstrand A, Baranov V, Mincheva-Nilsson L, European Union (262067-PRIME-XS). Breakefield XO, Widmark A: Prostate cancer-derived urine exosomes: A novel approach to biomarkers for prostate cancer. Br J Cancer 100: 1603– 1607, 2009 DISCLOSURES 19. Rood IM, Deegens JK, Merchant ML, Tamboer WP, Wilkey DW, Wetzels JF, Klein JB: Comparison of three methods for isolation of None. urinary microvesicles to identify biomarkers of nephrotic syndrome. Kidney Int 78: 810–816, 2010 20. Merchant ML, Powell DW, Wilkey DW, Cummins TD, Deegens JK, fi REFERENCES Rood IM, McAfee KJ, Fleischer C, Klein E, Klein JB: Micro ltration isolation of human urinary exosomes for characterization by MS. Pro- teomics Clin Appl 4: 84–96, 2010 1. Pan BT, Teng K, Wu C, Adam M, Johnstone RM: Electron microscopic 21. Moon PG, Lee JE, You S, Kim TK, Cho JH, Kim IS, Kwon TH, Kim CD, evidence for externalization of the in vesicular form Park SH, Hwang D, Kim YL, Baek MC: Proteomic analysis of urinary in sheep reticulocytes. 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Mittelbrunn M, Gutiérrez-Vázquez C, Villarroya-Beltri C, González S, in antimicrobial resistance among urinary tract infection isolates of Sánchez-Cabo F, González MA, Bernad A, Sánchez-Madrid F: Unidi- Escherichia coli from female outpatients in the United States. Antimicrob rectional transfer of microRNA-loaded exosomes from T cells to anti- Agents Chemother 46: 2540–2545, 2002 gen-presenting cells. Nat Commun 2: 282, 2011 37. Zhanel GG, Hisanaga TL, Laing NM, DeCorby MR, Nichol KA, Palatnik 50.SheldonH,HeikampE,TurleyH,DragovicR,ThomasP,OonCE,Leek LP, Johnson J, Noreddin A, Harding GK, Nicolle LE, Hoban DJ; NAUTICA R, Edelmann M, Kessler B, Sainson RC, Sargent I, Li JL, Harris AL: New Group: Antibiotic resistance in outpatient urinary isolates: Final results mechanism for Notch signaling to endothelium at a distance by Delta- from the North American Urinary Tract Infection Collaborative Alliance like 4 incorporation into exosomes. Blood 116: 2385–2394, 2010 (NAUTICA). 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J Am Soc Nephrol 25: 2017–2027, 2014 Immune Role for Urine Exosomes 2027

Supplementary Material

Contents

Immune modulating proteins identified from exosomal samples...... 2 Figure S1: Overlap between exosomal and soluble proteomes...... 4

Bacterial strains:...... 4 Figure S2: Variability between subjects of effects of exosomes on BL21-lux growth...... 5 Figure S3: Early effects of exosomes on growth of BL21 E. coli ...... 5 Figure S4: Exosomal Lysis...... 6 Figure S5: Effect of pH on exosomal action...... 7 Figure S6: Effect of exosomes on growth of UPEC (pH = 6.5) suspended in exosome-depleted urine supernatant ...... 8

Effective exosomal concentration...... 8 Figure S7: Sample constitution for luminometry experiments...... 8 Figure S8: Determining effective concentration ...... 9 Figure S9: Independent confirmation of immune proteins by Western blot ...... 10 Figure S10: Effect of serial washes on exosomal action against E coli ...... 11

Supplementary methods ...... 12 1. Protein precipitation for mass spectrometry ...... 12 2. Espresso protein type-I error estimating algorithm ...... 12 3. Antibodies ...... 13

Supplementary references...... 14

Supplementary Table S1. Proteins identified from human urinary exosomal samples by mass spectrometry...... 17

1

Immune modulating proteins identified from exosomal samples (see Table 1)

Aminopeptidase-N (ANPEP) serves as an E. coli fimbrial receptor,1 and functions as a viral receptor.2

Transferrin similarly sequesters iron and destabilises bacterial membranes,3 though not at as acidic a pH as

lactoferrin.4

LGALS3BP is present in breast milk and protects neonates from diarrhoeal illness, may protect against viral

invasion, and is implicated in NK cell-mediated immunity.5

TRIM5 is involved in viral sensing and restricts retroviral infection.6, 7

CARD9 is indispensible for fungal innate and acquired fungal immunity, and its deficiency results in mucosal

and systemic candidiasis.8

IFIT5 is highly conserved across species and reduces viral replication and mediates host antiviral defense,9

likely through direct binding of viral RNA.10

PIGR is a key mediator of mucosal immunity. Synthesised in epithelia, it binds basolateral IgA, is

transcytosed to the apical surface where it is either expressed with bound IgA, or cleaved as "secretory

component" which served to mediate IgA action in host defense.11

Neprilysin (also known as CD10 or CALLA) is an endopeptidase expressed on neutrophils and lymphocytes,

and is also highly expressed in kidney. It regulates levels of inflammatory peptides induced during infection

with E. coli.12 The homologue in Manduca sexta (Q86RS4) is implicated in the immune response to bacterial

infection.13

Keratin 10 is a cytoskeletal protein that is widely expressed. In epithelia, Staphylococcus aureus adheres to

the cell surface by binding to surface-expressed through the S aureus clumping factor B (ClfB).14

Calprotectin (S100A8/A9) is a heterotetramer consisting of S100A8 and S100A9. It inhibits growth and kills

gram positive and gram-negative bacteria, and Candida albicans.15, 16

Lysozyme C is microbicidal against gram-positive and -negative bacteria and Candida albicans, inducing lysis

of gram-negative bacteria by destabilising the outer membrane and forming pores in the inner membrane.17,

18

2

MASP2 is a mannan-binding lectin associated serine protease that is important for activation of the complement pathway. Deficiency in MASP2 is permissive to pneumococcal infection.19 Polymorphisms in the MASP2 gene are associated with HTLV-1 infection.20

Mucin-1 is widely expressed in epithelia. It binds to the surfaces of Pseudomonas aeruginosa and E. coli.21, 22

Furthermore, female mucin-1 null mice develop chronic genito-urinary infections.23

Myeloperoxidase (MPO) selectively binds and kills many gram-positive and -negative bacteria,24 and also has candidicidal and antiviral properties.25, 26 Its activity is enhanced at acidic pH.27

CD14 is the principal mammalian bacterial lipopolysaccharide receptor,28, 29 binds gram positive and gram negative bacterial surfaces,30 and may also function as a viral receptor.31

S100A7 (Psoriasin) is present in urine,32 kills E. coli by pore formation, and is maximally effective at pH 6.33, 34

Superoxide dismutase (SOD1) may inhibit bacterial growth by sequestering copper and zinc, and is implicated in host defence against Schistosoma infection.35

Dermcidin adheres to bacterial cell walls and inhibits growth of gram positive and negative bacteria, and

Candida albicans,36 by inhibiting bacterial RNA and protein synthesis.37

The D3 fragment of kininogen-1 (KNG-1) is potently bactericidal with activity against gram-positive and - negative bacteria.38

Protein C Receptor (PROCR) contributes to host defence against E. coli, although the mechanism is not clear.39

Fibronectin is a glycoprotein that is expressed at cell surfaces as a dimer, or secreted. It is the principle human receptor for Treponoema pallidum40 and Trichomonas vaginalis.41

PLUNC binds bacterial lipopolysaccharide, destabilises membranes and inhibits growth of Pseudomonas aeruginosa,42, 43 while overexpression of PLUNC in bronchial epithelium protects against mycoplasma infection.44

PGLYRP1 is a peptidoglycan recognition protein that demonstrates zinc-dependent killing of gram positive and gram negative bacteria.45

Lactoferrin is a cationic protein that inhibits bacterial growth by sequestering iron, and by adhering to and destabilising bacterial membranes.46, 47

3

Histatin-1 is a candidicidal protein.48

Lipocalin 1 and Lipocalin 2 are bacteriostatic proteins that inhibit bacterial growth by their ability to bind

iron.49, 50

Figure S1: Overlap between exosomal and soluble proteomes Exosomal Non-exosomal

476 125 239

Of the exosomal proteome, 21% were shared with soluble urinary proteins from simultaneously prepared samples.

Bacterial strains:

Strain Source

BL21 Commercially available, e.g. New England Biolabs, Ipswich MA, USA. Catalogue number C2530H

UPEC Clinical isolate from a patient with urinary tract infection, Addenbrooke's Hospital, Cambridge.

Organism characterised using the VITEK2 system version 04.02, bioMerieux

Nissle Mutaflor®, sourced from Ardeypharm GmBH, Hagen, Germany

UTI89 Gift of Dr Menna Clatworthy, Deparment of Medicine, University of Cambridge

CFT073 ATCC®, Manassas VA, USA

Catalogue number 700928™

4

Figure S2: Variability between subjects of effects of exosomes on BL-21-lux growth

Each of 4 volunteer samples, displayed in aggregate form in Figure 4, significantly inhibited growth of BL21 E. coli.

Figure S3: Early effects of exosomes on growth of BL21 E. coli

Addition of exosomes to BL21 (red arrow/curve) resulted in arrest of growth after approximately 30 min, with complete abolition of growth after 45 min. Uromodulin alone was not inhibitory; combination of exosomes with uromodulin after 45 min (pink arrow/curve) again abolished growth. BL21 was grown in LB medium, pH = 5.5.

5

Figure S4: Exosomal Lysis

To determine whether exosomal structural integrity was required for the observed bacterial growth inhibition, exosomal preparations were divided; one part was retained intact, whereas the other was subjected to chemical lysis.

Lysis was achieved with ammonium acetate and acetone precipitation as described under supplementary methods. Briefly, samples were incubated with 5 volumes 100% ammonium acetate in methanol overnight at 4°C, and centrifuged for 10 minutes at 3,000 x g. the resulting pellets were washed with 80% ammonium acetate at 4°C, and centrifuged for 10 minutes at 3,000 x g. The resulting pellets were washed with 80% acetone and centrifuged for 10 minutes at 3,000 x g. After removal of acetone, the final pellet was resuspended in 100 µl of a 250 mM sucrose solution, and equilibrated to a protein concentration matching that of the untreated exosomal aliquot.

To confirm that this protocol achieved exosomal lysis, samples were evaluated by transmission electron microscopy as described under supplementary methods below. Briefly, 10 µl of the treated or untreated samples were placed (in triplicate) on glow-discharged formvar grids and negatively stained with 2% uranyl acetate. All samples evaluated by EM had not been subjected to freezing at any point during the workflow.

A) Electron micrographs revealed extensive morphological differences between treated and untreated samples. In contrast to treated samples, where many characteristic vesicles with minimal luminal staining were evident, treated samples showed a granular appearance with only a small number of discernable vesicles present. Vesicles from treated samples demonstrated luminal staining with uranyl acetate, consistent with compromised membrane integrity. Uromodulin polymerisation was also disrupted in the treated samples. B) Given that intact vesicles were evident in the treated samples, vesicles were manually counted from 5 randomly selected 4.2 x 3.4 µm areas per grid. Areas were selected by an independent observer. Treatment reduced the number of intact vesicles per field by approximately 80%, from 537 ± 38 to 113 ± 8 vesicles per field (p < 0.0001).

6

We observed that exosomes of which the integrity had been disrupted as described above were less effective at inhibiting growth of E coli (Figure 4). Despite this, these preparations retained some degree of efficacy. Since the treated samples contain the innate immune proteins present in the exosomes of the parent sample in soluble form, and given that many of these proteins are known to induce bacterial lysis, it is not surprising that treated samples retain some activity against E coli. Furthermore, it is possible that the small number of vesicles that survive the lysis procedure intact may contribute to this residual activity.

Figure S5: Effect of pH on exosomal action

UPEC-lux growth did not differ at differing pH. Exosomes inhibited UPEC-lux growth at pH 5.5 (P = 0.001), pH 6.0 (P < 0.0001), pH 6.5 (P < 0.0001) and pH 7.0 (P = 0.0001). However, the inhibitory effect of exosomes on UPEC-lux appeared to decrease with increasing pH. Uromodulin limited bacterial growth to some extent across the pH range, although this was not statistically significant.

7

Figure S6: Effect of exosomes on growth of UPEC (pH = 6.5) suspended in exosome-depleted urine supernatant

UPEC-lux growth was greater at higher pH 6.5 (above) than pH 5.5 (Figure 4F). As shown here, provision of exosomes inhibited UPEC-lux growth at pH 6.5 (p = 0.0003). Uromodulin inhibited growth of UPEC-lux at pH 6.5 to a lesser extent (P = 0.0025).

Effective exosomal concentration

Growth curve experiments were performed in 96-well plates, with a well volume of 400μl. Wells were filled to a total of 200μl, as shown in the example below:

Figure S7: Sample constitution for luminometry experiments

20μl (5μg) 20μl (5μg) 20μl 250mM exosomes* uromodulin* sucrose buffer

170μl LB/ exosome- 170μl LB/ exosome- 170μl LB/ exosome- depleted urine depleted urine depleted urine

10μl bacterial culture 10μl bacterial culture 10μl bacterial culture

Sample A Sample B Sample C

*suspended in 250mM sucrose buffer

8

Within this system, we found 5μg of exosomal material to be effective against a starting culture with an rlu of 500 or less. To assess the clinical relevance of this inhibition, we determined the equivalent amount of bacteria by colony counting. Using a set of serially diluted cultures of UPEC-lux, we determined the rlu and corresponding bacterial concentration by colony counting.

Figure S8: Determining effective concentration

The maximum bacterial concentration at which 25μg/ml of exosomes prevented bacterial growth was approximately 500rlu, and this was equivalent to 5369 cfu/ml. Given the mean concentration of urinary exosomes (0.54μg/ml) of a large volume second morning void urine sample, we could infer that a physiological amount of exosomes should inhibit growth of approximately 116 cfu/ml (5369/24.3). Our observations are likely to underestimate the effects of exosomes in vivo, since this experimental model relies on the inoculation of established bacterial cultures with a single dose of exosomal material in contrast to the situation in vivo where exosomes are continuously shed into the renal tract and some invading organisms are expelled during micturition.

9

Figure S9: Independent confirmation of immune proteins by Western blot

Figure S9: Western blot was carried out on exosomal preparations from an independent cohort of healthy adult volunteers. For all proteins shown, 12 µg of exosomal material was used. The positive control (HKM) represents 50 µg of human kidney membrane in every case. Blots for all five proteins were carried out on the same exosomal preparations. There was insufficient material from subject 10 to allow blotting for dermcidin, and this subject is unfortunately no longer available. All proteins tested were clearly present in all samples. Sample ten/MUC1 required longer exposure. Bands shown for calprotectin represent S100A8/S100A9-heterotetramer; bands for lysozyme C represent dimer. All blots were carried out without deglycosylation.

10

Figure S10: Effect of serial washes on exosomal action against E coli

To determine whether extra-exosomal components of exosomal isolates were contributing to the inhibition of bacterial growth by exosomes, we performed serial washes on an exosomal sample from a healthy male volunteer. An exosomal sample was prepared from a second morning void by ultracentrifugation as previously. The exosomal sample was divided into three equal volumes. The three aliquots were resuspended in 10 ml 250 mM sucrose and ultracentrifuged for 2 hours at 56,800 x g using a Beckman Ti90 fixed angle rotor. This process was carried out once, twice or three times for the three aliquots respectively.

To assess the effect of serial washes on bacterial growth inhibition, 10 µg of exosomal material from each of the three pellets were incubated with BL21-lux. Uromodulin 10 µg was included as a control.

A) All three washed exosomal preparations significantly inhibited growth of BL21 E. coli. Increasing the number of washes augmented the inhibition of bacterial growth. B) Western blot demonstrated a reduction in the principal extra-exosomal protein uromodulin with serial washes.

The apparent increase in efficacy after serial washes is explained by the use of a similar protein load for each condition, such that increased washes would reduce the amount of extra-exosomal contamination but increase the amount of exosomal material.

11

Supplementary methods

1. Protein precipitation for mass spectrometry For mass spectrometry, protein was extracted by chemical precipitation. Briefly, 5 volumes of 1% ammonium acetate were added, and samples incubated at -20°C overnight, then centrifuged at 3,000 x g for 10 minutes at 4°C. The pellets were washed with 80% ammonium acetate, centrifuged at 3,000 x g for 10 minutes at 4°C, re-suspended in 80% acetone, and centrifuged at 3,000 x g for 15 minutes at 4°C. After removal of acetone, pellets were desiccated under a Speedvac for 3 minutes.

2. Espresso protein type-I error estimating algorithm Proteins were regarded as "seen" by MS if 1) there were two or more spectrum-peptide matches (SPMs) with a false discovery rate < 0.1, 2) A single SPM with a MASCOT-percolator posterior error probability of < 0.01 was present, or 3) if the product of posterior error probabilities of all unique peptides attributed to a protein was < 0.01. This latter approach is termed espresso, and is briefly described here.

Espresso excludes all SPMs that are ambiguous (not attributable to a single gene product). Since MASCOT-percolator assigns a posterior error probability (PEP) to each identified PSM in a dataset, and since the PEP is the probability of a peptide called correct to be false, the probability of a protein identification being false can be computed from the PEP of each contributing PSM. We let the minimum PEP of a PSM in fraction i for the jth unique peptide sequence u, deriving from protein P, be denoted E(uij). It follows that, for a protein with m unique peptides identified from n fractions, the protein error E(P) can be calculated as

The code for the espresso algorithm is freely available.

12

3. Antibodies Antibody Source, catalogue Type Clone Stock Usage Dilution (name/target) concentration (here)

Uromodulin (Tamm Cedarlane CL1032A Mouse mAb 10.32 57.2 mg/ml WB 1:1000 Horsfall Protein) EM 1:5

TSG101 Abcam 0083 Mouse mAb 4A10 1mg/ml WB 1:1000

EM 1:5

Enolase-1 Biogenesis 0100-0261 Mouse mAb 8G8 0.5mg/ml WB 1:500

Podocin Gift Mouse mAb Unknown 1mg/ml WB 1:1000

CD63 Abcam 8219 Mouse mAb MEM-259 1mg/ml WB 1:1000

EM 1:5

Mucin-1 Abcam 70475 Mouse mAb HMFG1 1.14mg/ml WB 1:250

EM 1:2

Lysozyme C Abcam 36362 Mouse mAb BGN/06/961 1mg/ml WB 1:1000

EM 1:5

Dermcidin Santa-Cruz sc-33656 Mouse mAb Unknown 0.2mg/ml WB 1:500

EM 1:5

Calprotectin Acris BM4025X Mouse mAb 27E10 1mg/ml WB 1:500 (MRP8/14) EM 1:5

Myeloperoxidase Abcam 25989 Mouse mAb 2C7 1mg/ml WB 1:1000

EM 1:5

mAb - Monoclonal antibody; WB - Western blot; EM - electron microscopy. For Western blot, positive controls were human kidney membrane, except for dermcidin (AnanSpec Dermcidin DCD-1L, catalogue 67265), calprotectin and myeloperoxidase (neutrophil lysate).

13

Supplementary references

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14

21. Kato K, Lillehoj EP, Kai H, Kim KC: MUC1 expression by human airway epithelial cells mediates Pseudomonas aeruginosa adhesion. Front Biosci, 2: 68-77, 2010. 22. Sando L, Pearson R, Gray C, Parker P, Hawken R, Thomson PC, Meadows JR, Kongsuwan K, Smith S, Tellam RL: Bovine Muc1 is a highly polymorphic gene encoding an extensively glycosylated mucin that binds bacteria. J Dairy Sci, 92: 5276-5291, 2009. 23. DeSouza MM, Surveyor GA, Price RE, Julian J, Kardon R, Zhou X, Gendler S, Hilkens J, Carson DD: MUC1/episialin: a critical barrier in the female reproductive tract. J Reprod Immunol, 45: 127-158, 1999. 24. Allen RC, Stephens JT Jr.: Myeloperoxidase selectively binds and selectively kills microbes. Infect Immun, 79: 474- 485, 2011. 25. Aratani Y, Koyama H, Nyui S, Suzuki K, Kura F, Maeda N: Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase. Infect Immun, 67: 1828-1836, 1999. 26. Yamamoto K, Miyoshi-Koshio T, Utsuki Y, Mizuno S, Suzuki K: Virucidal activity and viral protein modification by myeloperoxidase: a candidate for defense factor of human polymorphonuclear leukocytes against influenza virus infection. J Infect Dis, 164: 8-14, 1991. 27. Zgliczynski JM, Selvaraj RJ, Paul BB, Stelmaszynska T, Poskitt PK, Sbarra AJ: Chlorination by the myeloperoxidase- H2O2-Cl- antimicrobial system at acid and neutral pH. Proc Soc Exp Biol Med, 154: 418-422, 1977. 28. Wright SD: CD14 and innate recognition of bacteria. J Immunol, 155: 6-8, 1995. 29. Kusunoki T, Hailman E, Juan TS, Lichenstein HS, Wright SD: Molecules from Staphylococcus aureus that bind CD14 and stimulate innate immune responses. J Exp Med, 182: 1673-1682, 1995. 30. Pugin J, Heumann ID, Tomasz A, Kravchenko VV, Akamatsu Y, Nishijima M, Glauser MP, Tobias PS, Ulevitch RJ: CD14 is a pattern recognition receptor. Immunity, 1: 509-516, 1994. 31. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, Tripp RA, Walsh EE, Freeman MW, Golenbock DT, Anderson LJ, Finberg RW: Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol, 1: 398-401, 2000. 32. Heine G, Raida M, Forssmann WG: Mapping of peptides and protein fragments in human urine using liquid chromatography-mass spectrometry. J Chromatogr, 776: 117-124, 1997. 33. Glaser R, Harder J, Lange H, Bartels J, Christophers E, Schroder JM: Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection. Nat Immunol, 6: 57-64, 2005. 34. Michalek M, Gelhaus C, Hecht O, Podschun R, Schroder JM, Leippe M, Grotzinger J: The human antimicrobial protein psoriasin acts by permeabilization of bacterial membranes. Dev Comp Immunol, 33: 740-746, 2009. 35. Bender RC, Goodall CP, Blouin MS, Bayne CJ: Variation in expression of Biomphalaria glabrata SOD1: a potential controlling factor in susceptibility/resistance to Schistosoma mansoni. Dev Comp Immunol, 31: 874-878, 2007. 36. Harder J, Schroder JM: RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin. J Biol Chem, 277: 46779-46784, 2002. 37. Senyurek I, Paulmann M, Sinnberg T, Kalbacher H, Deeg M, Gutsmann T, Hermes M, Kohler T, Gotz F, Wolz C, Peschel A, Schittek B: Dermcidin-derived peptides show a different mode of action than the cathelicidin LL- 37 against Staphylococcus aureus. Antimicrob Agents Chemother, 53: 2499-2509, 2009. 38. Frick IM, Akesson P, Herwald H, Morgelin M, Malmsten M, Nagler DK, Bjorck L: The contact system--a novel branch of innate immunity generating antibacterial peptides. EMBO J, 25: 5569-5578, 2006. 39. Taylor FB Jr., Stearns-Kurosawa DJ, Kurosawa S, Ferrell G, Chang AC, Laszik Z, Kosanke S, Peer G, Esmon CT: The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis. Blood, 95: 1680-1686, 2000. 40. Lee JH, Choi HJ, Jung J, Lee MG, Lee JB, Lee KH: Receptors for Treponema pallidum attachment to the surface and matrix proteins of cultured human dermal microvascular endothelial cells. Yonsei Med J, 44: 371-378, 2003. 41. Crouch ML, Benchimol M, Alderete JF: Binding of fibronectin by Trichomonas vaginalis is influenced by iron and calcium. Microb Pathog, 31: 131-144, 2001. 42. Zhou HD, Li XL, Li GY, Zhou M, Liu HY, Yang YX, Deng, T, Ma, J, Sheng, SR: Effect of SPLUNC1 protein on the Pseudomonas aeruginosa and Epstein-Barr virus. Mol Cell Biochem, 309: 191-197, 2008. 43. Lukinskiene L, Liu Y, Reynolds SD, Steele C, Stripp BR, Leikauf GD, Kolls JK, Di YP: Antimicrobial Activity of PLUNC Protects against Pseudomonas aeruginosa Infection. J Immunol, 187: 382-390, 2011.

15

44. Chu HW, Thaikoottathil J, Rino JG, Zhang G, Wu Q, Moss T, Refaeli Y, Bowler R, Wenzel SE, Chen Z, Zdunek J, Breed R, Young R, Allaire E, Martin RJ: Function and regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflammation. J Immunol, 179: 3995-4002, 2007. 45. Wang M, Liu LH, Wang S, Li X, Lu X, Gupta D, Dziarski R: Human peptidoglycan recognition proteins require zinc to kill both gram-positive and gram-negative bacteria and are synergistic with antibacterial peptides. J Immunol, 178: 3116-3125, 2007. 46. Jenssen H, Hancock RE: Antimicrobial properties of lactoferrin. Biochimie, 91: 19-29, 2009. 47. Abrink M, Larsson E, Gobl A, Hellman L: Expression of lactoferrin in the kidney: implications for innate immunity and iron metabolism. Kidney Int, 57: 2004-2010, 2000. 48. Oppenheim FG, Xu T, McMillian FM, Levitz SM, Diamond RD, Offner GD, Troxler RF: Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans. J Biol Chem, 263: 7472-7477, 1988. 49. Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond KN, Strong RK: The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell, 10: 1033-1043, 2002. 50. Zasloff M: Antimicrobial peptides, innate immunity, and the normally sterile urinary tract. J Am Soc Nephrol, 18: 2810-2816, 2007.

16

Supplementary Table S1. Proteins identified from human urinary exosomal samples by mass spectrometry Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:A1BG Yes A1BG Alpha-1-B glycoprotein Both 6 1.20E-31 Both methods U

GROUP:A2ML1 Yes A2ML1 Alpha-2-macroglobulin-like 1 Exclusion 1 4.84E-14 Espresso only S, U

IPI00926669 ACAA1 Acetyl-Coenzyme A acyltransferase 1 Exclusion 1 1.92E-09 Espresso only - (peroxisomal 3-oxoacyl-Coenzyme A thiolase)

GROUP:ACLY Yes ACLY ATP citrate Exclusion 1 0.0000145 Espresso only Me, U

GROUP:ACTL6A Yes ACTL6A -like 6A Non-exclusion 1 4.55E-09 Espresso only -

IPI00032220 AGT Angiotensinogen (serpin peptidase inhibitor, Exclusion 1 4.84E-16 Espresso only P clade A, member 8)

GROUP:AKR1A1 Yes AKR1A1 Aldo-keto reductase family 1, member A1 Both 7 0 Both methods S, U (aldehyde reductase)

GROUP:AKR1B1 Yes AKR1B1 Aldo-keto reductase family 1, member B1 Both 3 0.000024 Both methods Me, U (aldose reductase)

IPI00293721 AKR7A3 Aldo-keto reductase family 7, member A3 Exclusion 1 0 Espresso only U (aflatoxin aldehyde reductase)

GROUP:ALB Yes ALB Albumin Both 53 0 Both methods CCC, MPE, P, S, U

IPI00916990 ALDH7A1 Aldehyde dehydrogenase 7 family, member A1 Non-exclusion 1 1.90E-06 Espresso only H

GROUP:ALDOA Yes ALDOA Aldolase A, fructose-bisphosphate Both 14 0 Both methods BC, B, CCC, S, U

17

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:ALDOB Yes ALDOB Aldolase B, fructose-bisphosphate Both 14 0 Both methods U

GROUP:ALDOC Yes ALDOC Aldolase C, fructose-bisphosphate Exclusion 1 1.23E-19 Espresso only U

IPI00419916 ALPL Alkaline , /bone/kidney Non-exclusion 3 0 Both methods U

IPI00022426 AMBP Alpha-1-microglobulin/bikunin precursor Both 7 0 Both methods U

IPI00238262 AMIGO2 Adhesion molecule with Ig-like domain 2 Non-exclusion 1 0.00302 Espresso only -

IPI00790622 AMOTL2 Angiomotin like 2 Exclusion 1 1.38E-11 Espresso only -

GROUP:AMY2B Yes AMY2B Amylase, alpha 2B (pancreatic) Non-exclusion 1 0.00716 Espresso only S

GROUP:ANGPTL2 Yes ANGPTL2 Angiopoietin-like 2 Both 11 0 Both methods U

GROUP:ANKRD13C Yes ANKRD13C repeat domain 13C Non-exclusion 1 2.03E-06 Espresso only -

GROUP:ANKRD18B Yes ANKRD18B Ankyrin repeat domain 18B Exclusion 1 0.0001665 Espresso only -

GROUP:ANP32B Yes ANP32B Acidic (-rich) nuclear phosphoprotein 32 Non-exclusion 1 0.00826 Espresso only - family, member B

IPI00221224 ANPEP Alanyl (membrane) aminopeptidase Both 292 0 Both methods S, U (aminopeptidase N, aminopeptidase M, microsomal aminopeptidase, CD13, p150)

GROUP:ANXA1 Yes ANXA1 Both 9 0 Both methods A, CCC, M, S, TB, U

GROUP:ANXA4 Yes ANXA4 Annexin A4 Both 48 0 Both methods CCC, S, U

18

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:ANXA6 Yes ANXA6 Annexin A6 Exclusion 1 3.79E-16 Espresso only B, CCC, Me, M, S, U

GROUP:AP3D1 Yes AP3D1 Adaptor-related protein complex 3, delta 1 Non-exclusion 1 0.000029 Espresso only - subunit

IPI00022391 APCS Amyloid P component, serum Both 19 0 Both methods U

GROUP:APLP1 Yes APLP1 Amyloid beta (A4) precursor-like protein 1 Exclusion 1 0.0006056 Espresso only -

GROUP:APOA1 Yes APOA1 Apolipoprotein A-I Non-exclusion 1 1.03E-07 Espresso only CCC, S, U

IPI00021854 APOA2 Apolipoprotein A-II Both 5 4.10E-31 Both methods P, S, U

GROUP:APOA4 Yes APOA4 Apolipoprotein A-IV Both 6 5.33E-33 Both methods U

GROUP:APOE Yes APOE Both 28 0 Both methods P, U

IPI00910625 APOH (beta-2-glycoprotein I) Non-exclusion 1 1.76E-15 Espresso only -

IPI00910333 AQP3 Non-exclusion 2 2.48E-20 Both methods A, U

GROUP:ARF6 Yes ARF6 ADP-ribosylation factor 6 Both 6 0 Both methods CCC, U

IPI00374285 ARHGEF37 Rho guanine exchange factor (GEF) Exclusion 1 3.22E-06 Espresso only - 37

IPI00003327 ARL3 ADP-ribosylation factor-like 3 Both 6 0 Both methods U

IPI00008405 ARSF F Both 4 0 Both methods U

19

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00940046 ASAH1 N-acylsphingosine amidohydrolase (acid Both 6 0 Both methods U ceramidase) 1

IPI00102281 ASPRV1 Aspartic peptidase, retroviral-like 1 Exclusion 1 3.26E-16 Espresso only -

IPI00872410 ATG2B ATG2 autophagy related 2 homolog B (S. Exclusion 1 4.43E-14 Espresso only - cerevisiae)

IPI00852619 ATP10B ATPase, class V, type 10B Non-exclusion 2 3.10E-26 Both methods -

IPI00927118 ATP13A4 ATPase type 13A4 Exclusion 2 1.62E-06 Both methods -

GROUP:ATP5B Yes ATP5B ATP synthase, H+ transporting, mitochondrial Both 4 7.09E-23 Both methods CCC, S, U F1 complex, beta polypeptide

GROUP:ATP6V0C Yes ATP6V0C ATPase, H+ transporting, lysosomal 16kDa, V0 Exclusion 1 2.93E-11 Espresso only U subunit c

GROUP:ATP6V1A Yes ATP6V1A ATPase, H+ transporting, lysosomal 70kDa, V1 Both 12 0 Both methods S, U subunit A

GROUP:ATP6V1D Yes ATP6V1D ATPase, H+ transporting, lysosomal 34kDa, V1 Non-exclusion 2 3.62E-17 Both methods S, U subunit D

GROUP:ATP6V1E1 Yes ATP6V1E1 ATPase, H+ transporting, lysosomal 31kDa, V1 Both 5 0 Both methods S, U subunit E1

GROUP:ATXN3 Yes ATXN3 Ataxin 3 Non-exclusion 2 5.07E-08 Both methods -

IPI00179326 BAIAP2L1 BAI1-associated protein 2-like 1 Both 2 6.53E-19 Both methods CCC, I, U

20

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00299024 BASP1 Brain abundant, membrane attached signal Exclusion 2 0.0000101 Both methods TB, U protein 1

IPI00027416 BBOX1 Butyrobetaine (gamma), 2-oxoglutarate Non-exclusion 1 0.0000401 Espresso only U dioxygenase (gamma-butyrobetaine hydroxylase) 1

GROUP:BCAM Yes BCAM Basal cell adhesion molecule (Lutheran blood Exclusion 1 2.41E-06 Espresso only U group)

IPI00004101 BHMT Betaine-homocysteine methyltransferase Exclusion 3 0 Both methods U

GROUP:BRDT Yes BRDT Bromodomain, testis-specific Exclusion 2 6.38E-07 Both methods -

IPI00166533 C10orf140 10 140 Exclusion 1 0.00155 Espresso only -

GROUP:C11orf49 Yes C11orf49 open reading frame 49 Non-exclusion 1 0.00264 Espresso only -

IPI00059185 C11orf52 Chromosome 11 open reading frame 52 Both 9 0 Both methods U

GROUP:C14orf106 Yes C14orf106 Chromosome 14 open reading frame 106 Non-exclusion 3 1.78E-11 Both methods -

GROUP:C17orf49 Yes C17orf49 open reading frame 49 Non-exclusion 1 0.0000363 Espresso only - (BAP18)

GROUP:C19orf20 Yes C19orf20 Chromosome 19 open reading frame 20 Non-exclusion 0 0.0000509 Espresso only -

IPI00024925 C19orf77 Chromosome 19 open reading frame 77 Both 8 0 Both methods -

IPI00145260 C1orf69 open reading frame 69 Non-exclusion 1 0.0000116 Espresso only -

21

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:C20orf114 Yes C20orf114 Chromosome 20 open reading frame 114 Both 12 0 Both methods S, U

IPI00382821 C5orf32 Chromosome 5 open reading frame 32 Both 9 0 Both methods CCC, U

IPI00945363 C6orf1 Chromosome 6 open reading frame 1 Both 2 2.12E-12 Both methods -

IPI00396439 C6orf168 Chromosome 6 open reading frame 168 Exclusion 1 6.44E-26 Espresso only -

GROUP:CA2 Yes CA2 Carbonic anhydrase II Both 14 0 Both methods U

GROUP:CA4 Yes CA4 Carbonic anhydrase IV Exclusion 1 1.76E-26 Espresso only U

GROUP:CAB39 Yes CAB39 Calcium binding protein 39 Both 4 0 Both methods S, U

IPI00220361 CALB1 Calbindin 1, 28kDa Both 10 0 Both methods U

GROUP:CALCRL Yes CALCRL Calcitonin receptor-like Non-exclusion 1 5.07E-06 Espresso only -

IPI00216984 CALML3 -like 3 Exclusion 2 2.65E-31 Both methods U

IPI00021536 CALML5 Calmodulin-like 5 Exclusion 3 2.13E-36 Both methods -

IPI00909886 CARD9 Caspase recruitment domain family, member 9 Both 3 2.89E-13 Both methods -

IPI00925214 CASP12 Caspase 12 Both 16 0 Both methods -

GROUP:CBR1 Yes CBR1 Carbonyl reductase 1 Both 7 0 Both methods CCC, S, U

GROUP:CCDC6 Yes CCDC6 Coiled-coil domain containing 6 Non-exclusion 1 0.00477 Espresso only -

22

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00893019 CCDC88A Coiled-coil domain containing 88A Non-exclusion 1 1.46E-11 Espresso only -

GROUP:CCT2 Yes CCT2 Chaperonin containing TCP1, subunit 2 (beta) Non-exclusion 1 0.0001455 Espresso only BCCC, CCC, U

IPI00029260 CD14 CD14 molecule Both 3 5.68E-28 Both methods S, U

GROUP:CD2AP Yes CD2AP CD2-associated protein Exclusion 2 2.79E-34 Both methods U

IPI00011302 CD59 CD59 molecule, complement regulatory protein Both 13 0 Both methods CCC, S, TB, U

GROUP:CEBPA Yes CEBPA CCAAT/enhancer binding protein (C/EBP), alpha Exclusion 1 5.37E-16 Espresso only -

IPI00386504 CELF4 Elav-like family member 4 Exclusion 1 0.0000834 Espresso only -

IPI00074020 CENPK Centromere protein K Exclusion 0 0.000722 Espresso only -

GROUP:CENTD1 Yes CENTD1 Centaurin, delta 1 Exclusion 1 0.0015115 Espresso only -

IPI00165972 CFD Complement factor D (adipsin) Non-exclusion 1 0.0000549 Espresso only U

GROUP:CFL1 Yes CFL1 Cofilin 1 (non-muscle) Both 2 7.96E-19 Both methods B, CCC, S, TB, U

GROUP:CFL2 Yes CFL2 Cofilin 2 (muscle) Exclusion 1 4.62E-16 Espresso only CCC

GROUP:CHMP1A Yes CHMP1A Chromatin modifying protein 1A Both 5 3.26E-19 Both methods CCC, U

IPI00156984 CHMP1B Chromatin modifying protein 1B Both 13 0 Both methods U

GROUP:CHMP2A Yes CHMP2A Chromatin modifying protein 2A Both 22 0 Both methods CCC, U

23

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:CHMP4B Yes CHMP4B Chromatin modifying protein 4B Both 18 0 Both methods CCC, U

IPI00060414 CHMP4C Chromatin modifying protein 4C Both 2 6.51E-17 Both methods -

GROUP:CHMP5 Yes CHMP5 Chromatin modifying protein 5 Both 27 0 Both methods U

GROUP:CLIC1 Yes CLIC1 Chloride intracellular channel 1 Both 9 0 Both methods BCCC, CCC, TB, U

IPI00383046 CMBL Carboxymethylenebutenolidase homolog Non-exclusion 3 0 Both methods U (Pseudomonas)

GROUP:CNP Yes CNP 2',3'-cyclic nucleotide 3' phospho-diesterase Both 3 0 Both methods M, S, U

GROUP:CNTN3 Yes CNTN3 Contactin 3 ( associated) Both 2 2.12E-07 Both methods -

GROUP:CNTN6 Yes CNTN6 Contactin 6 Non-exclusion 1 1.20E-06 Espresso only MC

GROUP:COBRA1 Yes COBRA1 of BRCA1 Non-exclusion 3 0 Both methods -

GROUP:COL15A1 Yes COL15A1 Collagen, type XV, alpha 1 Both 8 0 Both methods U

GROUP:COL18A1 Yes COL18A1 Collagen, type XVIII, alpha 1 Both 2 1.87E-20 Both methods S, U

IPI00297646 COL1A1 Collagen, type I, alpha 1 Non-exclusion 1 0.0013002 Espresso only Fe

GROUP:COL27A1 Yes COL27A1 Collagen, type XXVII, alpha 1 Exclusion 1 7.96E-06 Espresso only -

IPI00291136 COL6A1 Collagen, type VI, alpha 1 Exclusion 7 0 Both methods S, U

IPI00514903 COQ3 Coenzyme Q3 homolog, methyltransferase (S. Non-exclusion 1 0.0000432 Espresso only -

24

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

cerevisiae)

IPI00464968 COX1 Cytochrome c oxidase subunit I Non-exclusion 1 0.0052906 Espresso only -

IPI00737939 COX8C Cytochrome c oxidase subunit 8C Exclusion 1 4.98E-06 Espresso only -

GROUP:CPM Yes CPM Carboxypeptidase M Exclusion 1 0.000892 Espresso only -

IPI00024403 CPNE3 Copine III Exclusion 1 2.46E-06 Espresso only S, U

GROUP:CPT1C Yes CPT1C Carnitine palmitoyltransferase 1C Exclusion 1 0.0025859 Espresso only -

IPI00428057 CREB3L2 CAMP responsive element binding protein 3- Exclusion 1 0.000291 Espresso only - like 2

IPI00916918 CREB5 CAMP responsive element binding protein 5 Non-exclusion 2 1.54E-21 Both methods U

GROUP:CREBBP Yes CREBBP CREB binding protein (Rubinstein-Taybi Exclusion 3 0.0000457 Two peptide rule - syndrome) only

IPI00297056 CRNN Cornulin Both 6 0 Both methods S, U

GROUP:CRYZ Yes CRYZ Crystallin, zeta (quinone reductase) Non-exclusion 1 5.60E-14 Espresso only CCC, U

GROUP:CSGALNACT1 Yes CSGALNACT1 N- Non-exclusion 2 0 Both methods - acetylgalactosaminyltransferase 1

IPI00019157 CSPG4 Chondroitin sulfate 4 Exclusion 1 0.0005923 Espresso only CCC

IPI00032293 CST3 Cystatin C Exclusion 1 0.0035644 Espresso only CCC, S, U

25

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:CSTA Yes CSTA Cystatin A (stefin A) Exclusion 2 4.50E-31 Both methods -

IPI00021828 CSTB Cystatin B (stefin B) Exclusion 2 0 Both methods S, U

IPI00607841 CSTF2 Cleavage stimulation factor, 3' pre-RNA, subunit Exclusion 1 0.0002918 Espresso only - 2, 64kDa

IPI00909622 CSTF2 Cleavage stimulation factor, 3' pre-RNA, subunit Exclusion 1 7.25E-10 Espresso only - 2, 64kDa

GROUP:CTDP1 Yes CTDP1 CTD (carboxy-terminal domain, RNA Both 2 0.00005 Both methods - polymerase II, polypeptide A) phosphatase, subunit 1

GROUP:CTSA Yes CTSA Cathepsin A Both 10 0 Both methods U

GROUP:CTSC Yes CTSC Cathepsin C Both 2 0 Both methods S, U

GROUP:CUBN Yes CUBN Cubilin (-cobalamin receptor) Both 37 0 Both methods U

IPI00911104 CYB5R1 Cytochrome b5 reductase 1 Non-exclusion 1 0.00709 Espresso only S

IPI00916645 CYBRD1 Cytochrome b reductase 1 Non-exclusion 2 1.08E-06 Both methods U

IPI00032876 CYTL1 Cytokine-like 1 Non-exclusion 1 4.01E-23 Espresso only -

IPI00936290 DACH1 Dachshund homolog 1 (Drosophila) Both 3 1.55E-07 Both methods -

GROUP:DAK Yes DAK Dihydroxyacetone kinase 2 homolog (S. Exclusion 2 0 Both methods U cerevisiae)

26

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:DCD Yes DCD Dermcidin Exclusion 1 1.99E-35 Espresso only CCC, U

IPI00478916 DCLRE1A DNA cross-link repair 1A (PSO2 homolog, S. Non-exclusion 1 0.0039046 Espresso only - cerevisiae)

IPI00220342 DDAH1 Dimethylarginine dimethylaminohydrolase 1 Non-exclusion 2 2.13E-08 Both methods U

GROUP:DDAH2 Yes DDAH2 Dimethylarginine dimethylaminohydrolase 2 Both 2 1.40E-16 Both methods U

GROUP:DDX49 Yes DDX49 DEAD (Asp-Glu-Ala-Asp) box polypeptide 49 Exclusion 3 0 Both methods -

IPI00513969 DDX59 DEAD (Asp-Glu-Ala-Asp) box polypeptide 59 Non-exclusion 1 1.65E-06 Espresso only -

IPI00935308 DEFB109 Defensin, beta 109 Both 2 0.0001019 Both methods -

GROUP:DHX9 Yes DHX9 DEAH (Asp-Glu-Ala-His) box polypeptide 9 Exclusion 1 0.0000111 Espresso only -

GROUP:DNAH5 Yes DNAH5 , axonemal, heavy chain 5 Exclusion 1 5.31E-10 Espresso only -

IPI00031065 DNASE1 I Non-exclusion 3 9.70E-12 Both methods -

IPI00941939 DNM1 1 Exclusion 1 5.88E-16 Espresso only -

IPI00059476 DPEP1 Dipeptidase 1 (renal) Both 74 0 Both methods CCC, I, U

IPI00018953 DPP4 Dipeptidyl-peptidase 4 (CD26, adenosine Both 18 0 Both methods CCC, I, S, U deaminase complexing protein 2)

IPI00025753 DSG1 Desmoglein 1 Exclusion 10 0 Both methods -

27

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00515022 DSG4 Desmoglein 4 Exclusion 1 0.0007645 Espresso only -

GROUP:DSP Yes DSP Both 101 0 Both methods M, S, U

GROUP:DSPP Yes DSPP Dentin sialophosphoprotein Exclusion 1 0.001841 Espresso only -

IPI00944963 DUSP7 Dual specificity phosphatase 7 Exclusion 2 1.23E-24 Both methods -

GROUP:ECE1 Yes ECE1 Endothelin converting 1 Non-exclusion 1 6.11E-16 Espresso only U

GROUP:EEF2 Yes EEF2 Eukaryotic translation elongation factor 2 Exclusion 1 0.0011582 Espresso only CCC, Me, S, U

IPI00019501 EFNB3 Ephrin-B3 Exclusion 1 0.00472 Espresso only -

IPI00000073 EGF Epidermal growth factor (beta-urogastrone) Both 104 0 Both methods U

GROUP:EHD1 Yes EHD1 EH-domain containing 1 Exclusion 1 0.0007326 Espresso only CCC, S, U

GROUP:EHD4 Yes EHD4 EH-domain containing 4 Both 7 0 Both methods B, CCC, U

GROUP:EIF4A1 Yes EIF4A1 Eukaryotic translation initiation factor 4A, Non-exclusion 1 1.34E-13 Espresso only CCC isoform 1

GROUP:ENO1 Yes ENO1 Enolase 1, (alpha) Both 51 0 Both methods BC, CCC, I, Me, M, S, U

IPI00014375 ENPEP (aminopeptidase A) Both 7 0 Both methods U

IPI00157414 ENPP6 Ectonucleotide Exclusion 1 7.89E-27 Espresso only U pyrophosphatase/ 6

28

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00169058 ENSG0000018 Non-exclusion 1 0.00954 Espresso only - 1943

GROUP:EP300 Yes EP300 E1A binding protein p300 Both 3 8.23E-14 Both methods -

GROUP:EPPK1 Yes EPPK1 Epiplakin 1 Both 9 0 Both methods CCC, U

GROUP:EPS8 Yes EPS8 Epidermal growth factor receptor pathway Exclusion 1 1.12E-12 Espresso only I, U substrate 8

GROUP:EPS8L1 Yes EPS8L1 EPS8-like 1 Both 2 9.64E-25 Both methods U

GROUP:EPS8L2 Yes EPS8L2 EPS8-like 2 Exclusion 3 0 Both methods S, TB, U

GROUP:ERBB3 Yes ERBB3 V-erb-b2 erythroblastic leukemia viral oncogene Exclusion 1 0.0000408 Espresso only - homolog 3 (avian)

GROUP:ESCO2 Yes ESCO2 Establishment of cohesion 1 homolog 2 (S. Non-exclusion 1 0.0006284 Espresso only - cerevisiae)

IPI00024167 ESF1 ESF1, nucleolar pre-rRNA processing protein, Exclusion 2 4.50E-24 Both methods - homolog (S. cerevisiae)

GROUP:ESX1 Yes ESX1 ESX homeobox 1 Exclusion 2 1.45E-07 Both methods -

GROUP:EXOC2 Yes EXOC2 Exocyst complex component 2 Exclusion 1 0.0016665 Espresso only -

GROUP:EZR Yes EZR Ezrin Both 67 0 Both methods CCC, Me, M, TB, U

IPI00219684 FABP3 Fatty acid binding protein 3, muscle and heart Exclusion 1 0.0001728 Espresso only B, U (mammary-derived growth inhibitor)

29

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:FABP5 Yes FABP5 Fatty acid binding protein 5 (psoriasis- Exclusion 3 0 Both methods MC associated)

GROUP:FAM125A Yes FAM125A Family with sequence similarity 125, member A Exclusion 1 1.92E-28 Espresso only CCC, U

GROUP:FBP1 Yes FBP1 Fructose-1,6-bisphosphatase 1 Both 7 0 Both methods U

GROUP:FGA Yes FGA Both 19 0 Both methods P, U

GROUP:FGB Yes FGB Fibrinogen beta chain Exclusion 14 0 Both methods MPE, P, SF

GROUP:FGL2 Yes FGL2 Fibrinogen-like 2 Exclusion 3 2.05E-11 Both methods U

GROUP:FLG Yes FLG Both 30 0 Both methods -

GROUP:FLG2 Yes FLG2 Filaggrin family member 2 Exclusion 20 0 Both methods -

IPI00744322 FLJ39582 THAP7 antisense RNA 1 Exclusion 1 0.00684 Espresso only -

GROUP:FLJ43950 Yes FLJ43950 FLJ43950 protein Non-exclusion 1 6.84E-15 Espresso only -

GROUP:FLT1 Yes FLT1 Fms-related tyrosine kinase 1 (vascular Exclusion 1 2.89E-08 Espresso only - endothelial growth factor/vascular permeability factor receptor)

GROUP:FN1 Yes FN1 Fibronectin 1 Exclusion 2 5.31E-13 Both methods M, P, SF, U

GROUP:FUT10 Yes FUT10 Fucosyltransferase 10 (alpha (1,3) Exclusion 1 0.00306 Espresso only - fucosyltransferase)

30

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:FXYD2 Yes FXYD2 FXYD domain containing ion transport regulator Both 3 4.15E-18 Both methods U 2

IPI00911090 GANC Glucosidase, alpha; neutral C Exclusion 0 0.0001682 Espresso only -

GROUP:GAPDH Yes GAPDH Glyceraldehyde-3-phosphate dehydrogenase Both 43 0 Both methods BC, BCCC, CCC, I, Me, M, S, TB, U

IPI00008832 GAS1 Growth arrest-specific 1 Exclusion 1 0.0008886 Espresso only -

IPI00215768 GCLC Glutamate-cysteine , catalytic subunit Non-exclusion 1 0.0040525 Espresso only -

GROUP:GFAP Yes GFAP Glial fibrillary acidic protein Non-exclusion 6 1.55E-26 Both methods -

IPI00023728 GGH Gamma-glutamyl (conjugase, Both 7 0 Both methods U folylpolygammaglutamyl hydrolase)

GROUP:GGT1 Yes GGT1 Gamma-glutamyltransferase 1 Exclusion 2 0.0000765 Both methods U

GROUP:GIPC2 Yes GIPC2 GIPC PDZ domain containing family, member 2 Exclusion 1 6.34E-06 Espresso only U

IPI00220766 GLO1 Glyoxalase I Exclusion 2 9.60E-16 Both methods U

IPI00018236 GM2A GM2 ganglioside activator Non-exclusion 1 1.13E-17 Espresso only U

GROUP:GNA11 Yes GNA11 Guanine nucleotide binding protein (G protein), Both 3 7.09E-25 Both methods Me, S, U alpha 11 (Gq class)

IPI00027243 GNA15 Guanine nucleotide binding protein (G protein), Non-exclusion 1 6.78E-07 Espresso only - alpha 15 (Gq class)

31

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:GNAI3 Yes GNAI3 Guanine nucleotide binding protein (G protein), Exclusion 2 5.96E-35 Both methods CCC, U alpha inhibiting activity polypeptide 3

GROUP:GNB1 Yes GNB1 Guanine nucleotide binding protein (G protein), Exclusion 5 0 Both methods CCC, Me, S, U beta polypeptide 1

IPI00221232 GNG12 Guanine nucleotide binding protein (G protein), Both 6 0 Both methods CCC, U gamma 12

IPI00232571 GPC4 4 Non-exclusion 1 4.90E-18 Espresso only S

IPI00019180 GPC5 Glypican 5 Non-exclusion 1 6.45E-08 Espresso only -

GROUP:GPX3 Yes GPX3 Glutathione peroxidase 3 (plasma) Exclusion 1 1.24E-31 Espresso only S

GROUP:GRIN3A Yes GRIN3A Glutamate receptor, ionotropic, N-methyl-D- Non-exclusion 0 9.11E-09 Espresso only - aspartate 3A

GROUP:GSK3A Yes GSK3A Glycogen synthase kinase 3 alpha Non-exclusion 1 6.97E-18 Espresso only -

GROUP:GSTP1 Yes GSTP1 Glutathione S- pi Exclusion 1 0.0000351 Espresso only CCC, Me, S, TB, U

GROUP:GYPC Yes GYPC C (Gerbich blood group) Non-exclusion 1 1.33E-08 Espresso only -

IPI00913960 HAS1 Hyaluronan synthase 1 Exclusion 2 1.36E-08 Both methods -

GROUP:HBA1; HBA2 Yes HBA1; HBA2 Haemogolbin alpha1, alpha 2 Both 3 0 Both methods P

GROUP:HBB Yes HBB Haemoglobin beta Both 2 0 Both methods P, U

32

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00045223 HEJ1 DNAJA1P5 DnaJ (Hsp40) homolog, subfamily A, Both 3 3.84E-23 Both methods - member 1 pseudogene 5

GROUP:HERC2 Yes HERC2 Hect domain and RLD 2 Exclusion 2 0.0004256 Two peptide rule - only

GROUP:HES2 Yes HES2 Hairy and enhancer of split 2 (Drosophila) Exclusion 1 5.41E-10 Espresso only -

GROUP:HIST1H1D Yes HIST1H1D Histone cluster 1, H1d Both 2 3.28E-24 Both methods MC

IPI00014165 HIST1H2APS4 Histone cluster 1, H2a, pseudogene 4 Exclusion 1 1.37E-07 Espresso only -

IPI00453473 HIST2H4B Histone cluster 1, Histone cluster 2 Both 19 0 Both methods CC, Pa, MC, O, HIST2H4A HIST1H4A HIST1H4D HIST1H4F HIST1H4C HIST1H4H HIST1H4B HIST1H4E HIST1H4I HIST1H4K HIST1H4J HIST1H4L HIST4H4

GROUP:HLA-DQB1; Yes HLA-DQB1; Major histocompatibility complex, class II Both 7 0 Both methods BC, T cells HLA-DQB2; HLA-DRB1; HLA-DQB2; HLA-DRB2; HLA-DRB3; HLA-DRB1; HLA-DRB4; HLA-DRB5 HLA-DRB2; HLA-DRB3; HLA-DRB4; HLA-DRB5

33

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00007339 HMG20B High-mobility group 20B Both 2 4.88E-07 Two peptide rule - only

IPI00514742 HMGCL 3-hydroxymethyl-3-methylglutaryl-Coenzyme A Exclusion 1 0.00265 Espresso only - lyase (hydroxymethylglutaricaciduria)

GROUP:HNRNPA2B1 Yes HNRNPA2B1 Heterogeneous nuclear ribonucleoprotein Non-exclusion 1 7.11E-10 Espresso only BCCC, CCC A2/B1

IPI00927894 HNRNPA3 Heterogeneous nuclear ribonucleoprotein A3 Non-exclusion 1 4.82E-16 Espresso only MC

GROUP:HNRNPF Yes HNRNPF Heterogeneous nuclear ribonucleoprotein F Non-exclusion 1 1.17E-08 Espresso only MC

IPI00022488 HPX Both 3 5.99E-19 Both methods U

IPI00022371 HRG Histidine-rich glycoprotein Non-exclusion 1 9.51E-17 Espresso only U

GROUP:HRH2 Yes HRH2 Histamine receptor H2 Both 2 0.0003105 Two peptide rule - only

GROUP:HRNR Yes HRNR Hornerin Both 85 0 Both methods P

GROUP:HRSP12 Yes HRSP12 Heat-responsive protein 12 Exclusion 2 1.53E-27 Both methods U

GROUP:HSP90AA1 Yes HSP90AA1 Heat shock protein 90kDa alpha (cytosolic), Non-exclusion 3 0 Both methods BC, CCC, I, MPE, M, S, class A member 1 U

GROUP:HSP90AB1 Yes HSP90AB1 Heat shock protein 90kDa alpha (cytosolic), Non-exclusion 4 0 Both methods CCC, M, U class B member 1

IPI00007765 HSPA9 Heat shock 70kDa protein 9 (mortalin) Exclusion 1 1.15E-08 Espresso only BCCC, CCC

34

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:HTN1 Yes HTN1 Histatin 1 Exclusion 1 1.55E-30 Espresso only -

GROUP:IDS Yes IDS Iduronate 2- (Hunter syndrome) Non-exclusion 1 0.00471 Espresso only -

IPI00024254 IFIT3 Interferon-induced protein with Both 2 3.35E-16 Both methods - tetratricopeptide repeats 3

IPI00016915 IGFBP7 Insulin-like growth factor binding protein 7 Both 18 0 Both methods -

GROUP:IGHA1; Yes IGHA1; Immunoglobulin heavy constant alpha 1 Both 21 0 Both methods B, S IGHV3OR16-13 IGHV3OR16-13

GROUP:IGHG1 Yes IGHG1 Immunoglobulin heavy constant gamma 1 (G1m Non-exclusion 1 0.0020099 Espresso only CCC, MPE marker)

IPI00930442 IGHG4 Immunoglobulin heavy constant gamma 4 Non-exclusion 1 0.0031042 Espresso only CCC

IPI00382488 IGHV4-59 Immunoglobulin heavy variable 4-59 Non-exclusion 1 0.0000642 Espresso only -

IPI00178926 IGJ Immunoglobulin J polypeptide, linker protein Both 3 6.65E-34 Both methods P, S for immunoglobulin alpha and mu polypeptides

IPI00830057 IGK Immunoglobulin kappa Exclusion 1 1.21E-27 Espresso only -

IPI00387113 IGK@ Immunoglobulin kappa locus Both 3 3.26E-10 Both methods CCC

IPI00909649 IGKC Immunoglobulin Kappa constant Both 6 0 Both methods CCC, S

GROUP:IGL@ Yes IGL@ Immunoglobulin lambda locus Both 2 3.27E-08 Both methods S

35

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00382440 IGLV3-25 Immunoglobulin lambda variable 3-25 Exclusion 4 0 Both methods -

IPI00152189 IGLV9-49 Immunoglobulin lambda variable 9-49 Non-exclusion 1 0.000353 Espresso only -

IPI00930108 IQCC IQ motif containing C Exclusion 1 6.17E-07 Espresso only -

IPI00023410 ITGA8 Integrin, alpha 8 Both 2 0.0017499 Two peptide rule - only

GROUP:ITGB1 Yes ITGB1 Integrin, beta 1 (fibronectin receptor, beta Both 3 1.79E-20 Both methods CCC, U polypeptide, antigen CD29 includes MDF2, MSK12)

IPI00555554 ITGB8 Integrin, beta 8 Non-exclusion 1 0.00155 Espresso only U

GROUP:ITPR2 Yes ITPR2 Inositol 1,4,5-triphosphate receptor, type 2 Non-exclusion 1 0.000119 Espresso only -

IPI00011692 IVL Involucrin Exclusion 4 0 Both methods S

GROUP:JUP Yes JUP Junction Both 32 0 Both methods CCC, S, U

IPI00020003 KCNJ8 Potassium inwardly-rectifying channel, Non-exclusion 1 0.00121 Espresso only - subfamily J, member 8

IPI00060715 KCTD12 Potassium channel tetramerisation domain Both 4 0 Both methods - containing 12

GROUP:KHSRP Yes KHSRP KH-type splicing regulatory protein (FUSE Exclusion 1 9.45E-28 Espresso only - binding protein 2)

IPI00643007 KIAA0174 KIAA0174 Exclusion 1 0.004036 Espresso only CCC, S, U

36

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00844000 KIAA0776 KIAA0776 Non-exclusion 1 6.61E-17 Espresso only -

IPI00852954 KIAA1045 KIAA1045 Non-exclusion 1 0.00429 Espresso only -

GROUP:KIAA1529 Yes KIAA1529 KIAA1529 Both 20 0 Both methods U

GROUP:KIAA2022 Yes KIAA2022 KIAA2022 Exclusion 1 0.0072328 Espresso only -

GROUP:KIF12 Yes KIF12 family member 12 Exclusion 1 4.65E-08 Espresso only U

GROUP:KIR2DL3 Yes KIR2DL3 Killer cell immunoglobulin-like receptor, two Exclusion 1 0.000151 Espresso only - domains, long cytoplasmic tail, 3

GROUP:KLK15 Yes KLK15 Kallikrein-related peptidase 15 Exclusion 1 1.88E-10 Espresso only -

GROUP:KNG1 Yes KNG1 Kininogen 1 Both 3 0 Both methods U

IPI00514908 KPRP Keratinocyte proline-rich protein Exclusion 6 2.72E-32 Both methods S, U

IPI00186166 KPTN Kaptin (actin binding protein) Non-exclusion 1 6.78E-07 Espresso only -

GROUP:KRT1 Yes KRT1 (epidermolytic hyperkeratosis) Both 647 0 Both methods CCC, I, S

GROUP:KRT10 Yes KRT10 Keratin 10 (epidermolytic hyperkeratosis; Both 416 0 Both methods CCC, I, Me, S keratosis palmaris et plantaris)

GROUP:KRT13 Yes KRT13 Both 24 0 Both methods CCC, S

GROUP:KRT13 Yes KRT13 Keratin 13 Both 10 0 Both methods CCC, S

37

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:KRT14 Yes KRT14 (epidermolysis bullosa simplex, Both 18 0 Both methods CCC, S Dowling-Meara, Koebner)

GROUP:KRT16 Yes KRT16 (focal non-epidermolytic Both 69 0 Both methods CCC, S palmoplantar keratoderma)

GROUP:KRT17 Yes KRT17 Non-exclusion 1 1.41E-07 Espresso only CCC, S

GROUP:KRT18 Yes KRT18 Exclusion 4 1.56E-32 Both methods CCC, S

GROUP:KRT2 Yes KRT2 Keratin 2 (epidermal ichthyosis bullosa of Both 325 0 Both methods CCC, S Siemens)

GROUP:KRT20 Yes KRT20 Exclusion 1 0.0000383 Espresso only CCC

GROUP:KRT31 Yes KRT31 Keratin 31 Exclusion 1 0.00102 Espresso only -

GROUP:KRT4 Yes KRT4 Both 66 0 Both methods CCC, S

GROUP:KRT5 Yes KRT5 (epidermolysis bullosa simplex, Both 161 0 Both methods CCC, S Dowling-Meara/Kobner/Weber-Cockayne types)

GROUP:KRT73 Yes KRT73 Keratin 73 Both 4 6.08E-16 Both methods S

GROUP:KRT76 Yes KRT76 Keratin 76 Exclusion 3 0 Both methods CCC

GROUP:KRT78 Yes KRT78 Keratin 78 Exclusion 7 0 Both methods S

GROUP:KRT79 Yes KRT79 Keratin 79 Non-exclusion 2 1.54E-18 Both methods CCC, S

38

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:KRT82 Yes KRT82 Keratin 82 Both 6 0 Both methods -

GROUP:KRT84 Yes KRT84 Keratin 84 Exclusion 1 0.00123 Espresso only O

GROUP:KRT9 Yes KRT9 (epidermolytic palmoplantar Both 521 0 Both methods CCC, I, S keratoderma)

IPI00020487 LACRT Lacritin Both 4 2.65E-36 Both methods -

GROUP:LCN1 Yes LCN1 Lipocalin 1 (tear prealbumin) Exclusion 1 3.46E-12 Espresso only -

GROUP:LCN2 Yes LCN2 Lipocalin 2 (oncogene 24p3) Exclusion 1 3.79E-16 Espresso only U

IPI00297169 LCP2 Lymphocyte cytosolic protein 2 (SH2 domain Exclusion 2 4.48E-08 Both methods - containing leukocyte protein of 76kDa)

GROUP:LDHB Yes LDHB Lactate dehydrogenase B Both 32 0 Both methods B, CCC, Me, U

IPI00465431 LGALS3 Lectin, galactoside-binding, soluble, 3 Non-exclusion 1 0.0000814 Espresso only CCC, S, U

GROUP:LGALS3BP Yes LGALS3BP Lectin, galactoside-binding, soluble, 3 binding Both 31 0 Both methods CCC, P, S, U protein

IPI00219221 LGALS7 Lectin, galactose binding, soluble 7 Both 21 0 Both methods S, Pa LGALS7B

GROUP:LGMN Yes LGMN Legumain Non-exclusion 1 1.40E-16 Espresso only -

IPI00009950 LMAN2 Lectin, mannose-binding 2 Both 25 0 Both methods U

39

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00796067 LMBR1L Limb region 1 homolog (mouse)-like Non-exclusion 1 3.88E-07 Espresso only -

IPI00103672 LOC100129503 Hypothetical protein LOC100129503 Exclusion 1 0.0037422 Espresso only -

IPI00307761 LOC100132178 Hypothetical protein LOC100132178 Exclusion 1 6.98E-07 Espresso only -

IPI00888515 LOC100287547 Hypothetical protein LOC100287547 Non-exclusion 1 0.00538 Espresso only -

GROUP:LOC124220 Yes LOC124220 Similar to common salivary protein 1 Exclusion 1 2.84E-17 Espresso only Pa, U

GROUP:LOC198437 Yes LOC198437 BA299N6.3 Non-exclusion 0 0.000013 Espresso only -

GROUP:LOC259308 Yes LOC259308 Hypothetical LOC259308 Exclusion 1 1.96E-07 Espresso only -

IPI00293276 MIF Macrophage migration inhibitory factor Exclusion 1 3.05E-38 Espresso only B, CCC, TB, U (-inhibiting factor)

IPI00937151 LOC440335 Hypothetical gene supported by BC022385; Both 3 2.43E-38 Both methods U BC035868; BC048326

GROUP:LOC643951 Yes LOC643951 Similar to Transmembrane protein 162 Exclusion 1 9.89E-08 Espresso only -

IPI00787576 LOC644070 Similar to germ cell associated 1 isoform 2 Non-exclusion 3 1.03E-16 Both methods -

IPI00740701 LOC644656 Hypothetical protein LOC644656 Exclusion 1 0.00829 Espresso only -

IPI00888865 LOC728637 Similar to acyl-CoA synthetase long-chain family Both 7 0 Both methods - member 6

GROUP:LOC93349 Yes LOC93349 Hypothetical protein BC004921 Non-exclusion 1 0.000639 Espresso only -

40

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00008107 LRFN2 Leucine rich repeat and fibronectin type III Exclusion 1 0.0009778 Espresso only - domain containing 2

IPI00022417 LRG1 Leucine-rich alpha-2-glycoprotein 1 Exclusion 1 5.94E-06 Espresso only -

IPI00917696 LRP1B Low density lipoprotein-related protein 1B Exclusion 1 0.0003092 Espresso only P (deleted in tumors)

IPI00024292 LRP2 Low density lipoprotein-related protein 2 Both 12 0 Both methods U

GROUP:LTF Yes LTF Lactotransferrin Exclusion 1 4.84E-16 Espresso only B, S

IPI00019038 LYZ Lysozyme (renal amyloidosis) Both 16 0 Both methods CCC, S, U

IPI00152487 MAGEE2 antigen family E, 2 Both 5 2.23E-21 Both methods -

IPI00844511 MAN1A1 Mannosidase, alpha, class 1A, member 1 Both 8 0 Both methods CCC, U

GROUP:MAPK4 Yes MAPK4 Mitogen-activated protein kinase 4 Exclusion 1 0.0049631 Espresso only -

GROUP:MASP2 Yes MASP2 Mannan-binding lectin serine peptidase 2 Both 35 0 Both methods P, U

IPI00946481 MBNL1 Muscleblind-like (Drosophila) Both 1 1.14E-12 Espresso only -

IPI00946481 MBNL1 Muscleblind-like (Drosophila) Both 2 5.49E-23 Both methods -

GROUP:MCAT Yes MCAT Malonyl CoA:ACP acyltransferase Exclusion 1 1.39E-06 Espresso only - (mitochondrial)

IPI00916431 MCF2L MCF.2 cell line derived transforming sequence- Exclusion 1 0.0001862 Espresso only -

41

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

like

GROUP:MGAT5 Yes MGAT5 Mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N- Exclusion 1 0.0047913 Espresso only - acetyl-glucosaminyltransferase

IPI00103065 MITD1 MIT, interacting and transport, Both 3 1.21E-18 Both methods CCC, U domain containing 1

GROUP:MLX Yes MLX MAX-like protein X Non-exclusion 2 1.90E-28 Both methods -

IPI00910964 MMD Monocyte to macrophage differentiation- Non-exclusion 1 3.17E-17 Espresso only - associated

GROUP:MME Yes MME Membrane metallo-endopeptidase Both 86 0 Both methods U

IPI00004489 MOCS3 Molybdenum cofactor synthesis 3 Non-exclusion 1 0.00891 Espresso only -

GROUP:MPO Yes MPO Myeloperoxidase Exclusion 3 2.02E-18 Both methods U

IPI00877839 MRPS17 Mitochondrial ribosomal protein S17 Non-exclusion 1 0.0030474 Espresso only -

IPI00942799 MS4A4E Membrane-spanning 4-domains, subfamily A, Exclusion 1 0.0006638 Espresso only - member 4E

GROUP:MTSS1 Yes MTSS1 suppressor 1 Exclusion 1 0.0000589 Espresso only -

GROUP:MUC1 Yes MUC1 Mucin 1, cell surface associated Both 2 1.23E-09 Both methods S, TB

IPI00918002 MUC5B , oligomeric /gel-forming Both 2 6.64E-06 Both methods -

42

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:MYH1 Yes MYH1 , heavy chain 1, non-muscle Exclusion 3 4.69E-35 Both methods -

GROUP:MYH10 Yes MYH10 Myosin, heavy chain 10, non-muscle Both 2 7.65E-07 Both methods CCC

GROUP:MYH2 Yes MYH2 Myosin, heavy chain 2, non-muscle Exclusion 2 2.87E-06 Both methods -

GROUP:MYH4 Yes MYH4 Myosin, heavy chain 4, non-muscle Exclusion 3 7.10E-38 Both methods -

GROUP:MYH9 Yes MYH9 Myosin, heavy chain 9, non-muscle Both 3 2.04E-18 Both methods CCC, M, P, S, U

IPI00002352 MYLPF Fast skeletal 2 Exclusion 2 7.23E-23 Both methods -

GROUP:NAALADL1 Yes NAALADL1 N-acetylated alpha-linked acidic dipeptidase- Exclusion 1 0.0005712 Espresso only - like 1

IPI00007471 NACA2 Nascent polypeptide-associated complex alpha Non-exclusion 1 0.00426 Espresso only - subunit 2

GROUP:NAGLU Yes NAGLU N-acetylglucosaminidase, alpha- (Sanfilippo Exclusion 4 0 Both methods U disease IIIB)

GROUP:NALCN Yes NALCN Sodium leak channel, non-selective Exclusion 1 0.0020017 Espresso only -

GROUP:NAPSA Yes NAPSA Napsin A aspartic peptidase Both 10 0 Both methods U

IPI00748534 NCRNA00107 PPP2R3B antisense RNA 1 Non-exclusion 1 1.29E-07 Espresso only -

IPI00642734 NEK6 NIMA (never in mitosis gene a)-related kinase 6 Exclusion 1 2.73E-17 Espresso only -

IPI00470875 NIPAL1 NIPA-like domain containing 1 Exclusion 1 0.000923 Espresso only -

43

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:NKX2-1 Yes NKX2-1 NK2 homeobox 1 Exclusion 1 0.000157 Espresso only -

IPI00412982 NOTCH1 Notch homolog 1, translocation-associated Exclusion 1 0.0066901 Espresso only CCC (Drosophila)

GROUP:NPM1 Yes NPM1 Nucleophosmin (nucleolar phosphoprotein B23, Non-exclusion 1 0.000193 Espresso only U numatrin)

IPI00306369 NSUN2 NOL1/NOP2/Sun domain family, member 2 Exclusion 1 1.69E-15 Espresso only -

GROUP:NT5C Yes NT5C 5', 3'-, cytosolic Both 2 6.89E-06 Both methods U

GROUP:NT5DC3 Yes NT5DC3 5'-nucleotidase domain containing 3 Exclusion 1 5.86E-07 Espresso only -

GROUP:NUAK1 Yes NUAK1 NUAK family, SNF1-like kinase, 1 Non-exclusion 4 0 Both methods -

IPI00103142 NUDCD2 NudC domain containing 2 Non-exclusion 2 0.0000113 Both methods -

GROUP:NUTF2 Yes NUTF2 Nuclear transport factor 2 Both 5 0 Both methods U

IPI00218116 OASL 2'-5'-oligoadenylate synthetase-like Non-exclusion 1 0.0003353 Espresso only -

IPI00554443 OBFC1 Oligonucleotide/-binding fold Exclusion 1 6.17E-13 Espresso only - containing 1

IPI00022255 OLFM4 Olfactomedin 4 Both 77 0 Both methods S, U

IPI00556491 ONECUT3 One cut homeobox 3 Exclusion 1 0.0038407 Espresso only -

IPI00169254 OR14C36 Olfactory receptor, family 14, subfamily C, Exclusion 1 1.06E-13 Espresso only -

44

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

member 36

IPI00073066 OR1F2P Olfactory receptor, family 1, subfamily F, Exclusion 2 3.45E-07 Both methods - member 2

IPI00183993 OR2M5 Olfactory receptor, family 2, subfamily M, Exclusion 1 0.00154 Espresso only - member 5

IPI00375736 OR5B2 Olfactory receptor, family 5, subfamily B, Exclusion 1 6.72E-07 Espresso only - member 2

IPI00171603 OR7E91P Olfactory receptor, family 7, subfamily E, Non-exclusion 1 3.13E-06 Espresso only - member 91 pseudogene

IPI00169151 OR8K5 Olfactory receptor, family 8, subfamily K, Exclusion 1 0.00769 Espresso only - member 5

IPI00923610 OTOF Otoferlin Exclusion 1 3.85E-06 Espresso only -

GROUP:OTOL1 Yes OTOL1 Otolin 1 Exclusion 1 0.00706 Espresso only -

IPI00329572 PACSIN3 Protein kinase C and casein kinase substrate in Both 2 4.08E-38 Both methods U neurons 3

IPI00418630 PAPD7 PAP associated domain containing 7 Exclusion 1 0.00263 Espresso only -

IPI00910435 PBRM1 Polybromo 1 Exclusion 1 0.0000978 Espresso only -

IPI00299402 PC Pyruvate carboxylase Exclusion 1 1.06E-08 Espresso only CCC, S

GROUP:PCCA Yes PCCA Propionyl Coenzyme A carboxylase, alpha Both 2 1.00E-11 Both methods -

45

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

polypeptide

IPI00647500 PCCA Propionyl Coenzyme A carboxylase, alpha Both 2 1.67E-20 Both methods - polypeptide

IPI00168563 PCGF3 Polycomb group ring finger 3 Non-exclusion 1 0.000648 Espresso only -

GROUP:PDDC1 Yes PDDC1 Parkinson disease 7 domain containing 1 Both 2 4.82E-15 Both methods U

GROUP:PDZD4 Yes PDZD4 PDZ domain containing 4 Exclusion 1 0.00681 Espresso only -

IPI00011858 PDZK1IP1 PDZK1 interacting protein 1 Both 5 0 Both methods U

GROUP:PEBP1 Yes PEBP1 Phosphatidylethanolamine binding protein 1 Exclusion 1 1.90E-17 Espresso only CCC, S, U

IPI00018235 PEF1 Penta-EF-hand domain containing 1 Both 18 0 Both methods U

IPI00216691 PFN1 1 Both 4 0 Both methods B, CCC, U

GROUP:PGK1 Yes PGK1 Phosphoglycerate kinase 1 Non-exclusion 2 0.0000751 Both methods CCC, I, S, U

GROUP:PGLS Yes PGLS 6-phosphogluconolactonase Both 4 0 Both methods U

IPI00021085 PGLYRP1 Peptidoglycan recognition protein 1 Both 2 4.19E-23 Both methods U

GROUP:PGM5P1 Yes PGM5P1 Phosphoglucomutase 5 pseudogene 1 Non-exclusion 1 0.0000154 Espresso only -

IPI00792870 PHF1 PHD finger protein 1 Exclusion 2 1.23E-14 Both methods -

GROUP:PIB5PA Yes PIB5PA Phosphatidylinositol (4,5) bisphosphate 5- Non-exclusion 1 7.98E-07 Espresso only -

46

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

phosphatase, A

GROUP:PIGR Yes PIGR Polymeric immunoglobulin receptor Both 135 0 Both methods P, S, U

GROUP:PKP1 Yes PKP1 1 (ectodermal dysplasia/skin Exclusion 2 2.03E-09 Both methods - fragility syndrome)

GROUP:PKP3 Yes PKP3 Plakophilin 3 Non-exclusion 1 0.0011305 Espresso only -

IPI00925427 PLA2G4B A2, group IVB (cytosolic) Non-exclusion 1 3.51E-26 Espresso only -

IPI00925632 PLCD1 , delta 1 Non-exclusion 1 0.0015779 Espresso only U

IPI00009856 PLUNC Palate, lung and nasal epithelium Exclusion 4 0 Both methods - associated

GROUP:POF1B Yes POF1B Premature ovarian failure, 1B Non-exclusion 2 2.34E-13 Both methods -

GROUP:POU2F1 Yes POU2F1 POU class 2 homeobox 1 Non-exclusion 1 1.10E-06 Espresso only -

GROUP:PPIB Yes PPIB Peptidylprolyl B (cyclophilin B) Non-exclusion 1 2.46E-20 Espresso only CCC, S, U

IPI00024129 PPIC Peptidylprolyl isomerase C (cyclophilin C) Non-exclusion 1 0.000019 Espresso only N

GROUP:PPL Yes PPL Exclusion 3 9.11E-27 Both methods S, U

GROUP:PRAMEF21; Yes PRAMEF21; PRAME family member 21 Non-exclusion 1 0.0089765 Espresso only - PRAMEF20 PRAMEF20

IPI00023038 PRB1 Proline-rich protein BstNI subfamily 1 Exclusion 1 1.45E-06 Espresso only -

47

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00797380 PRCD Progressive rod-cone degeneration Exclusion 1 1.19E-06 Espresso only -

GROUP:PRDX1 Yes PRDX1 Peroxiredoxin 1 Both 3 0 Both methods BCCC, B, CCC, S

GROUP:PRDX5 Yes PRDX5 Peroxiredoxin 5 Both 4 0 Both methods CCC, S, U

IPI00220301 PRDX6 Peroxiredoxin 6 Both 9 0 Both methods CCC, S, U

GROUP:PRIC285 Yes PRIC285 Peroxisomal proliferator-activated receptor A Non-exclusion 1 0.0042504 Espresso only - interacting complex 285

IPI00009276 PROCR Protein C receptor, endothelial (EPCR) Both 4 9.99E-25 Both methods -

IPI00909774 PRPF40A PRP40 pre-mRNA processing factor 40 homolog Both 2 2.76E-07 Both methods - A (S. cerevisiae)

IPI00909774 PRPF40A PRP40 pre-mRNA processing factor 40 homolog Both 3 2.45E-15 Both methods - A (S. cerevisiae)

IPI00915929 PRPF40A PRP40 pre-mRNA processing factor 40 homolog Both 3 1.38E-13 Both methods - A (S. cerevisiae)

IPI00915929 PRPF40A PRP40 pre-mRNA processing factor 40 homolog Both 2 0.0000155 Both methods - A (S. cerevisiae)

IPI00027019 PRR4 Proline rich 4 Both 2 0 Both methods -

GROUP:PRSS7 Yes PRSS7 Protease, serine, 7 (enterokinase) Non-exclusion 1 0.0053887 Espresso only -

IPI00329538 PRSS8 Protease, serine, 8 Exclusion 2 0 Both methods S, U

48

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:PSCA Yes PSCA Prostate stem cell antigen Both 11 0 Both methods U

GROUP:PSPC1 Yes PSPC1 Paraspeckle component 1 Exclusion 1 0.0021192 Espresso only -

GROUP:PTBP1 Yes PTBP1 Polypyrimidine tract binding protein 1 Exclusion 6 0 Both methods U

GROUP:PTGER3 Yes PTGER3 Prostaglandin E receptor 3 (subtype EP3) Exclusion 1 0.000339 Espresso only -

IPI00000940 PTH Parathyroid hormone Non-exclusion 1 2.21E-08 Espresso only -

GROUP:PTPRJ Yes PTPRJ Protein tyrosine phosphatase, receptor type, J Exclusion 2 6.00E-09 Both methods U

IPI00747534 PYCR2 Pyrroline-5-carboxylate reductase family, Exclusion 1 0.0008554 Espresso only - member 2

GROUP:PYGB Yes PYGB Phosphorylase, glycogen; brain Both 2 0.0011184 Two peptide rule CCC only

GROUP:PYGM Yes PYGM Phosphorylase, glycogen; muscle (McArdle Both 16 0 Both methods - syndrome, glycogen storage disease type V)

GROUP:RAB10 Yes RAB10 RAB10, member RAS oncogene family Both 4 0 Both methods CCC, U

GROUP:RAB13 Yes RAB13 RAB13, member RAS oncogene family Non-exclusion 1 8.25E-08 Espresso only CCC, U

GROUP:RAB14 Yes RAB14 RAB14, member RAS oncogene family Both 12 0 Both methods CCC, Fe, U

GROUP:RAB15 Yes RAB15 RAB15, member RAS onocogene family Both 6 0 Both methods CCC, U

GROUP:RAB21 Yes RAB21 RAB21, member RAS oncogene family Both 2 1.21E-26 Both methods U

49

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:RAB35 Yes RAB35 RAB35, member RAS oncogene family Both 3 0 Both methods CCC, U

GROUP:RAB5B Yes RAB5B RAB5B, member RAS oncogene family Non-exclusion 1 4.54E-16 Espresso only CCC, Me, P, U

GROUP:RAB5C Yes RAB5C RAB5C, member RAS oncogene family Both 12 0 Both methods CCC, S, U

GROUP:RAB8A Yes RAB8A RAB8A, member RAS oncogene family Non-exclusion 1 7.25E-06 Espresso only CCC, U

GROUP:RAB8B Yes RAB8B RAB8B, member RAS oncogene family Exclusion 1 9.98E-16 Espresso only CCC, U

GROUP:RAC1 Yes RAC1 Ras-related C3 botulinum toxin substrate 1 (rho Both 2 0 Both methods CCC, U family, small GTP binding protein Rac1)

GROUP:RAP1A Yes RAP1A RAP1A, member of RAS oncogene family Exclusion 1 0.0000754 Espresso only CCC

GROUP:RAP1B Yes RAP1B RAP1B, member of RAS oncogene family Exclusion 1 5.34E-08 Espresso only CCC, S, U

GROUP:RAP2B Yes RAP2B RAP2B, member of RAS oncogene family Both 3 3.22E-37 Both methods CCC, U

IPI00893458 RAPGEF5 Rap guanine nucleotide exchange factor (GEF) 5 Exclusion 1 5.15E-08 Espresso only -

GROUP:RASGEF1A Yes RASGEF1A RasGEF domain family, member 1A Non-exclusion 1 5.88E-06 Espresso only -

GROUP:RAVER2 Yes RAVER2 Ribonucleoprotein, PTB-binding 2 Non-exclusion 2 1.68E-09 Both methods -

GROUP:RBMS3 Yes RBMS3 RNA binding motif, single stranded interacting Non-exclusion 1 0.0016 Espresso only MC protein

IPI00938339 RBMX RNA binding motif protein, X-linked Exclusion 2 1.51E-15 Both methods -

50

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00167369 RBMXL3 RNA binding motif protein, X-linked-like 3 Non-exclusion 1 0.0000196 Espresso only -

IPI00640136 RFX7 Regulatory factor X, 7 Exclusion 1 0.0003863 Espresso only MC

IPI00251559 RNF20 Ring finger protein 20 Non-exclusion 1 2.27E-07 Espresso only -

IPI00011044 RP1 Retinitis pigmentosa 1 (autosomal dominant) Exclusion 1 1.17E-06 Espresso only -

GROUP:GABPB2 Yes GABPB2 GA binding protein , beta Exclusion 1 0.0035783 Espresso only - subunit 2

GROUP:RP5-1022P6.2 Yes RP5-1022P6.2 Hypothetical protein KIAA1434 Exclusion 1 3.27E-08 Espresso only -

GROUP:RPS14 Yes RPS14 Ribosomal protein S14 Non-exclusion 1 2.01E-17 Espresso only CCC

IPI00008433 RPS5 Ribosomal protein S5 Non-exclusion 1 2.33E-09 Espresso only MC

IPI00221088 RPS9 Ribosomal protein S9 Non-exclusion 1 2.79E-30 Espresso only -

IPI00020418 RRAS Related RAS viral (r-ras) oncogene homolog Non-exclusion 2 1.70E-25 Both methods CCC, U

IPI00006541 RSPH6A Radial spoke head 6 homolog A Exclusion 1 0.0033614 Espresso only -

IPI00034010 RWDD1 RWD domain containing 1 Non-exclusion 1 0.000263 Espresso only -

GROUP:RYR2 Yes RYR2 2 (cardiac) Exclusion 1 0.0001449 Espresso only -

GROUP:S100A11 Yes S100A11 S100 calcium binding protein A11 Both 3 0 Both methods CCC, S, U

IPI00218131 S100A12 S100 calcium binding protein A12 Exclusion 1 0.00563 Espresso only -

51

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00027463 S100A6 S100 calcium binding protein A6 Non-exclusion 4 7.84E-21 Both methods CCC, S, U

GROUP:S100A7 Yes S100A7 S100 calcium binding protein A7 Both 5 0 Both methods -

IPI00007047 S100A8 S100 calcium binding protein A8 Both 11 0 Both methods S, U

IPI00027462 S100A9 S100 calcium binding protein A9 Both 8 0 Both methods S, U

IPI00017526 S100P S100 calcium binding protein P Both 2 4.46E-19 Both methods CCC, U

IPI00798360 SARNP SAP domain containing ribonucleoprotein Non-exclusion 1 2.59E-21 Espresso only -

IPI00947285 SBSN Suprabasin Exclusion 4 0 Both methods S

IPI00217766 SCARB2 Scavenger receptor class B, member 2 Non-exclusion 1 0.000167 Espresso only U

IPI00413194 SCFV Single-chain Fv fragment Non-exclusion 1 4.66E-16 Espresso only S

IPI00026126 SCGB2A1 Secretoglobin, family 2A, member 1 Both 3 0 Both methods -

IPI00300117 SCN7A Sodium channel, voltage-gated, type VII, alpha Both 2 5.65E-12 Both methods -

GROUP:SCPEP1 Yes SCPEP1 Serine carboxypeptidase 1 Exclusion 1 0.0002755 Espresso only S, U

IPI00011564 SDC4 Syndecan 4 Exclusion 3 5.18E-14 Both methods CCC, S

IPI00170635 SECTM1 Secreted and transmembrane 1 Both 2 0 Both methods U

GROUP:SEMG2 Yes SEMG2 Semenogelin II Exclusion 1 0 Espresso only -

52

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:SERPINA1 Yes SERPINA1 Serpin peptidase inhibitor, clade A (alpha-1 Both 4 0 Both methods CCC, P, U antiproteinase, antitrypsin), member 1

GROUP:SERPINA13 Yes SERPINA13 Serpin peptidase inhibitor, clade A (alpha-1 Non-exclusion 1 0.0009138 Espresso only - antiproteinase, antitrypsin), member 13 (pseudogene)

IPI00007221 SERPINA5 Serpin peptidase inhibitor, clade A (alpha-1 Both 6 0 Both methods U antiproteinase, antitrypsin), member 5

IPI00292946 SERPINA7 Serpin peptidase inhibitor, clade A (alpha-1 Exclusion 2 3.54E-15 Both methods U antiproteinase, antitrypsin), member 7

GROUP:SERPINB12 Yes SERPINB12 Serpin peptidase inhibitor, clade B (ovalbumin), Exclusion 2 6.88E-22 Both methods - member 12

GROUP:SERPINB4 Yes SERPINB4 Serpin peptidase inhibitor, clade B (ovalbumin), Both 3 2.56E-19 Both methods U member 4

GROUP:SERPING1 Yes SERPING1 Serpin peptidase inhibitor, clade G (C1 Both 10 0 Both methods P, U inhibitor), member 1, (angioedema, hereditary)

GROUP:SFN Yes SFN Stratifin Exclusion 14 0 Both methods CCC, K, U

IPI00795239 SGCZ Sarcoglycan zeta Non-exclusion 1 2.39E-06 Espresso only -

IPI00879791 SH2B2 SH2B adaptor protein 2 Exclusion 1 4.37E-10 Espresso only -

IPI00872618 SH3BGRL3 SH3 domain binding glutamic acid-rich protein Non-exclusion 1 0.00666 Espresso only U like 3

GROUP:SH3GL2 Yes SH3GL2 SH3-domain GRB2-like 2 Non-exclusion 2 0 Both methods -

53

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:SH3KBP1 Yes SH3KBP1 SH3-domain kinase binding protein 1 Non-exclusion 2 8.09E-08 Both methods -

GROUP:SHOX Yes SHOX Short stature homeobox Exclusion 1 0.00743 Espresso only -

IPI00946734 SLC12A8 Solute carrier family 12 (potassium/chloride Exclusion 1 0.0005679 Espresso only - transporters), member 8

IPI00807688 SLC22A12 Solute carrier family 22 (organic anion/cation Exclusion 1 0.00028 Espresso only U transporter), member 12

GROUP:SLC30A9 Yes SLC30A9 Solute carrier family 30 (zinc transporter), Exclusion 1 0.0001164 Espresso only - member 9

IPI00099939 SLC35C2 Solute carrier family 35, member C2 Exclusion 1 5.59E-09 Espresso only -

GROUP:SLC38A4 Yes SLC38A4 Solute carrier family 38, member 4 Exclusion 1 0.0041818 Espresso only -

IPI00094624 SLC38A8 Solute carrier family 38, member 8 Non-exclusion 1 0.0000984 Espresso only -

GROUP:SLC41A3 Yes SLC41A3 Solute carrier family 41, member 3 Non-exclusion 2 2.35E-20 Both methods -

IPI00218248 SLC5A4 Solute carrier family 5 (low affinity glucose Exclusion 1 0.0005436 Espresso only - cotransporter), member 4

GROUP:SLC7A4 Yes SLC7A4 Solute carrier family 7 (cationic Exclusion 1 0.00221 Espresso only - transporter, y+ system), member 4

IPI00043337 SLMO1 Slowmo homolog 1 (Drosophila) Exclusion 1 0.00933 Espresso only -

GROUP:SMARCC1 Yes SMARCC1 SWI/SNF related, matrix associated, actin Exclusion 0 0.0001018 Espresso only - dependent regulator of chromatin, subfamily c,

54

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

member 1

GROUP:SMN1; SMN2; Yes SMN1; SMN2; Survival of motor neuron 1, telomeric Exclusion 1 7.71E-09 Espresso only - LOC100133835 ; LOC100133836

IPI00550115 SMPDL3B Sphingomyelin phosphodiesterase, acid-like 3B Non-exclusion 1 4.86E-16 Espresso only CCC, U

GROUP:SMR3B Yes SMR3B Submaxillary gland androgen regulated protein Exclusion 2 2.62E-18 Both methods - 3 homolog B (mouse)

IPI00220528 SNRPF Small nuclear ribonucleoprotein polypeptide F Non-exclusion 1 0.0012027 Espresso only -

GROUP:SNRPN Yes SNRPN Small nuclear ribonucleoprotein polypeptide N Non-exclusion 1 4.83E-08 Espresso only -

IPI00001885 SNX8 Sorting nexin 8 Non-exclusion 1 0.0001285 Espresso only -

GROUP:SOBP Yes SOBP Sine oculis binding protein homolog Non-exclusion 1 7.78E-06 Espresso only - (Drosophila)

GROUP:SOD1 Yes SOD1 Superoxide dismutase 1, soluble (amyotrophic Exclusion 2 3.17E-06 Both methods CCC, U lateral sclerosis 1 (adult))

IPI00027827 SOD3 Superoxide dismutase 3, extracellular Both 5 0 Both methods -

GROUP:SORD Yes SORD Sorbitol dehydrogenase Exclusion 2 0 Both methods U

GROUP:SOX3 Yes SOX3 SRY (sex determining region Y)-box 3 Both 2 0.0000168 Both methods -

GROUP:SPATA7 Yes SPATA7 Spermatogenesis associated 7 Both 4 1.18E-25 Both methods -

55

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00915976 SPATS2L Spermatogenesis associated, serine-rich 2-like Exclusion 1 0.00168 Espresso only -

GROUP:SPEG Yes SPEG SPEG complex locus Exclusion 1 3.99E-16 Espresso only -

GROUP:SPTBN2 Yes SPTBN2 , beta, non-erythrocytic 2 Exclusion 1 3.71E-13 Espresso only -

IPI00241148 SSBP4 Single stranded DNA binding protein 4 Both 2 8.22E-08 Both methods -

IPI00241148 SSBP4 Single stranded DNA binding protein 4 Both 2 3.31E-06 Both methods -

IPI00793211 ST13 Suppression of tumorigenicity 13 (colon Exclusion 1 0.0000719 Espresso only U carcinoma) (Hsp70 interacting protein)

GROUP:STOM Yes STOM Stomatin Non-exclusion 3 9.07E-25 Both methods B, TB, U

GROUP:STT3B Yes STT3B STT3, subunit of the oligosaccharyltransferase Exclusion 1 0.0031163 Espresso only - complex, homolog B (S. cerevisiae)

IPI00021302 SUSD2 Sushi domain containing 2 Exclusion 2 2.09E-24 Both methods U

GROUP:SVOP Yes SVOP SV2 related protein homolog (rat) Non-exclusion 1 1.17E-13 Espresso only -

IPI00651630 TAAR2 Trace amine associated receptor 2 Exclusion 1 0.00683 Espresso only -

IPI00879057 TAF3 TAF3 RNA polymerase II, TATA box binding Both 8 0 Both methods - protein (TBP)-associated factor, 140kDa

IPI00643828 TDRD3 Tudor domain containing 3 Both 1 1.59E-21 Espresso only -

IPI00065229 TEKT5 5 Exclusion 1 5.38E-06 Espresso only -

56

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:TF Yes TF Transferrin Both 23 0 Both methods CCC, U

IPI00029174 TGIF2 TGFB-induced factor homeobox 2 Non-exclusion 1 4.44E-16 Espresso only -

IPI00300376 TGM3 Transglutaminase 3 (E polypeptide, protein- Exclusion 1 1.62E-10 Espresso only S glutamine-gamma-glutamyltransferase)

GROUP:THBS1 Yes THBS1 Thrombospondin 1 Exclusion 2 8.40E-13 Both methods CCC, P, S, U

GROUP:THRA Yes THRA Thyroid hormone receptor, alpha Non-exclusion 1 2.58E-08 Espresso only - (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian)

GROUP:TLE3 Yes TLE3 Transducin-like enhancer of split 3 (E(sp1) Exclusion 2 1.97E-07 Both methods - homolog, Drosophila)

IPI00791212 TM6SF1 Transmembrane 6 superfamily member 1 Non-exclusion 1 0.00199 Espresso only -

IPI00395888 TMEM123 Transmembrane protein 123 Exclusion 1 3.61E-07 Espresso only -

IPI00009441 TMEM216 Transmembrane protein 216 Both 11 0 Both methods -

IPI00375577 TMEM65 Transmembrane protein 65 Exclusion 1 0.0007594 Espresso only -

GROUP:TMTC1 Yes TMTC1 Transmembrane and tetratricopeptide repeat Both 9 0 Both methods - containing 1

IPI00100247 TMX4 thioredoxin-related transmembrane protein 4 Both 7 0 Both methods -

GROUP:TNNI3K Yes TNNI3K TNNI3 interacting kinase Exclusion 1 0.0026919 Espresso only -

57

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

IPI00940106 TOX2 TOX high mobility group box family member 2 Non-exclusion 1 0.00719 Espresso only -

GROUP:TPCN1 Yes TPCN1 Two pore segment channel 1 Exclusion 1 7.16E-07 Espresso only -

GROUP:TPM2 Yes TPM2 2 (beta) Exclusion 1 4.55E-06 Espresso only -

GROUP:TRAFD1 Yes TRAFD1 TRAF-type zinc finger domain containing 1 Non-exclusion 2 0.0000102 Both methods -

GROUP:TRIM5 Yes TRIM5 Tripartite motif-containing 5 Non-exclusion 1 2.19E-19 Espresso only -

IPI00926749 TRIP6 Thyroid hormone receptor interactor 6 Non-exclusion 1 0.0015232 Espresso only -

GROUP:TSC22D2 Yes TSC22D2 TSC22 domain family, member 2 Both 2 9.99E-08 Both methods -

GROUP:TSG101 Yes TSG101 Tumor susceptibility gene 101 Both 10 0 Both methods CCC, TB, U

IPI00877047 TSHR Thyroid stimulating hormone receptor Exclusion 1 0.0004033 Espresso only -

IPI00030936 TSPAN1 Tetraspanin 1 Both 2 4.15E-10 Both methods CCC, TB, U

IPI00296805 TSPAN10 Tetraspanin 10 Non-exclusion 1 0.0036006 Espresso only -

IPI00514593 TTC4 Tetratricopeptide repeat domain 4 Non-exclusion 0 1.88E-08 Espresso only -

IPI00023234 UBA2 SUMO1 activating enzyme subunit 2 Non-exclusion 1 0.0073359 Espresso only -

GROUP:UBR2 Yes UBR2 Ubiquitin protein ligase E3 component n- Non-exclusion 2 4.09E-20 Both methods MC recognin 2

GROUP:UMOD Yes UMOD Uromodulin (uromucoid, Tamm-Horsfall Exclusion 169 0 Both methods U

58

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

glycoprotein)

IPI00012113 UPK2 Uroplakin 2 Both 9 0 Both methods U

IPI00289920 UPK3A Uroplakin 3A Exclusion 1 3.94E-06 Espresso only U

GROUP:USH1C Yes USH1C Usher syndrome 1C (autosomal recessive, Exclusion 1 0.00363 Espresso only - severe)

GROUP:USP6 Yes USP6 Ubiquitin specific peptidase 6 (Tre-2 oncogene) Non-exclusion 1 1.64E-13 Espresso only -

GROUP:VAPA Yes VAPA VAMP (vesicle-associated membrane protein)- Non-exclusion 1 4.82E-16 Espresso only MC associated protein A, 33kDa

IPI00395488 VASN Vasorin Both 13 0 Both methods U

IPI00383676 VGLL1 Vestigial like 1 (Drosophila) Non-exclusion 1 0.0028287 Espresso only -

IPI00783024 VH87-2 immunoglobulin heavy variable 3-15 Non-exclusion 7 0 Both methods -

GROUP:VIM Yes VIM Exclusion 1 0.0010829 Espresso only N

IPI00166079 VKORC1L1 Vitamin K epoxide reductase complex, subunit Exclusion 1 0.0000769 Espresso only - 1-like 1

IPI00382436 VL4 Non-exclusion 1 2.29E-31 Espresso only -

GROUP:VMO1 Yes VMO1 Vitelline membrane outer layer 1 homolog Exclusion 1 4.62E-16 Espresso only U (chicken)

59

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:VPS28 Yes VPS28 Vacuolar protein sorting 28 homolog (S. Both 16 0 Both methods CCC, S, U cerevisiae)

GROUP:VPS37B Yes VPS37B Vacuolar protein sorting 37 homolog B (S. Both 17 0 Both methods CCC, U cerevisiae)

IPI00401773 VPS37C Vacuolar protein sorting 37 homolog C (S. Non-exclusion 1 6.32E-07 Espresso only CCC, U cerevisiae)

GROUP:VPS4A Yes VPS4A Vacuolar protein sorting 4 homolog A (S. Both 11 0 Both methods S, U cerevisiae)

GROUP:VPS4B Yes VPS4B Vacuolar protein sorting 4 homolog B (S. Both 13 0 Both methods U cerevisiae)

IPI00216313 VSNL1 Visinin-like 1 Non-exclusion 1 1.58E-06 Espresso only -

GROUP:VTA1 Yes VTA1 Vps20-associated 1 homolog (S. cerevisiae) Exclusion 2 2.85E-35 Both methods S, U

IPI00298971 VTN Both 12 0 Both methods P, S, U

IPI00797789 WBP2 WW domain binding protein 2 Exclusion 1 0.0000451 Espresso only -

GROUP:XPNPEP2 Yes XPNPEP2 X-prolyl aminopeptidase (aminopeptidase P) 2, Both 24 0 Both methods U membrane-bound

IPI00553187 YIPF6 Yip1 domain family, member 6 Exclusion 1 1.50E-06 Espresso only -

GROUP:YWHAB Yes YWHAB Tyrosine 3-monooxygenase/tryptophan 5- Both 13 0 Both methods CCC, S, U monooxygenase activation protein, beta polypeptide

60

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:YWHAE Yes YWHAE Tyrosine 3-monooxygenase/tryptophan 5- Both 13 0 Both methods CCC, Me, S, U monooxygenase activation protein, epsilon polypeptide

GROUP:YWHAG Yes YWHAG Tyrosine 3-monooxygenase/tryptophan 5- Non-exclusion 1 0.0000289 Espresso only CCC, S, U monooxygenase activation protein, gamma polypeptide

GROUP:YWHAQ Yes YWHAQ Tyrosine 3-monooxygenase/tryptophan 5- Exclusion 3 4.86E-36 Both methods CCC, S, U monooxygenase activation protein, theta polypeptide

GROUP:YWHAZ Yes YWHAZ Tyrosine 3-monooxygenase/tryptophan 5- Both 27 0 Both methods CCC, S, TB, U monooxygenase activation protein, zeta polypeptide

GROUP:ZBTB10 Yes ZBTB10 Zinc finger and BTB domain containing 10 Exclusion 1 0.000193 Espresso only -

IPI00455725 ZC3H11A Zinc finger CCCH-type containing 11A Both 2 6.16E-10 Both methods - LOC441155

IPI00307325 ZFP161 Zinc finger protein 161 homolog (mouse) Exclusion 1 1.08E-07 Espresso only -

IPI00293575 ZNF184 Zinc finger protein 184 Non-exclusion 1 0.0020634 Espresso only -

IPI00902668 ZNF268 Zinc finger protein 268 Exclusion 1 0.000443 Espresso only -

GROUP:ZNF282 Yes ZNF282 Zinc finger protein 282 Non-exclusion 2 2.95E-06 Both methods -

IPI00141925 ZNF296 Zinc finger protein 296 Non-exclusion 1 0.0006851 Espresso only -

IPI00879271 ZNF355P Zinc finger protein 355P Non-exclusion 1 0.0065168 Espresso only -

61

Protein Identifier Dis- Gene Name Description Exclusion List Peptide Type I Error Identification Previously Identified ambiguated Count Method in Exosomes*

GROUP:ZNF41 Yes ZNF41 Zinc finger protein 41 Exclusion 1 4.04E-06 Espresso only -

IPI00922712 ZNF418 Zinc finger protein 418 Non-exclusion 1 2.39E-11 Espresso only -

IPI00745436 ZNF451 Zinc finger protein 451 Non-exclusion 1 0.00135 Espresso only -

IPI00073580 ZNF689 Zinc finger protein 689 Exclusion 1 0.0002782 Espresso only -

GROUP:ZSWIM4 Yes ZSWIM4 Zinc finger, SWIM-type containing 4 Exclusion 1 0.00231 Espresso only -

* A Amniotic fluid M Mesothlioma cells B Breast milk MC Mast cells BC B cells Me Melanoma cells BCC cells N Neuroglial cells CC Colorectal cells O Oligodendrocytes CCC: cells P Plasma Fe Embryonic fibrobalsts Pa Parrotid gland H Hepatcytes S Saliva I Intestinal epithelial cells SF Synovial fluid K Keratinocytes TB Tracehobronchila cells MPE Malignant pleural effusions U Urine

62