Purification and Identification of Membrane Proteins from Urinary

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Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning Shuiwang Hu, Luca Musante, Dorota Tataruch, Xiaomeng Xu, Oliver Kretz, Michael Henry, Paula Meleady, Haihua Luo, Hequn Zou, Yong Jiang, and Harry Holthofer J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00386 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 3, 2017

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Journal of Proteome Research

Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning Shuiwang Hu1,2 Luca Musante1, Dorota Tataruch1, Xiaomeng Xu7, Oliver Kretz 3,4,5, Michael Henry6, Paula Meleady6, Haihua Luo2, Hequn Zou7, Yong Jiang2*, Harry Holthofer3,8* 1

Centre for BioAnalytical Sciences (CBAS), Dublin City University, Dublin, Ireland

2

Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure

Research, Southern Medical University, Guangzhou, China 3

Department of Medicine IV, Medical Center, Faculty of Medicine, University of Freiburg,

Freiburg, Germany 4

Department of Neuroanatomy, University of Freiburg, Freiburg, Germany

5

III. Medical Clinic, University Hospital Hamburg-Eppendorf, Hamburg, Germany

6

National Institute for Cellular Biotechnology (NICB), Dublin City University, Dublin, Ireland

7

Institute of Nephrology and Urology, the Third Affiliated Hospital, Southern Medical University,

Guangzhou, China 8

Freiburg Institute for Advanced Studies (FRIAS), Albert-Ludwigs University, Freiburg, Germany

* Co-corresponding author

1

ACS Paragon Plus Environment


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Abstract Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport and signaling. Hence membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall Protein (THP), the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral trans-membrane proteins present in human urinary vesicles. Firstly, uEVs were enriched using a “hydrostatic filtration dialysis’’ appliance (HFDa), and then the enriched uEVs and the lysates were verified by transmission electron microscopy (TEM). After using Triton X-114 phase partitioning we generated an insoluble pellet fraction (PF), aqueous phase (AP) and detergent phase (DP) -fractions and analyzed these with LC-MS/MS. Both in-gel and off-gel protein digestion methods were used to reveal increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP were much higher than of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. 2


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In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs. Keywords Membrane protein; urine; extracellular vesicle; Triton X-114 partitioning

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INTRODUCTION Almost all cells release a variety of membrane-bound vesicles into their extracellular environment under physiological and pathological conditions(1). These secreted Extracellular Vesicles (EVs) are of endosomal and plasma membrane origin and include, for example, exosomes, microvesicles, exosome-like vesicles, retrovirus-like particles and apoptotic bodies(2-4). EVs reflect not only a novel mechanism of intercellular communication(5) by their surface coat and cargo, for example, Alvarez-Erviti L et al delivered siRNA to the mouse brain by systemic injection of targeted exosomes to treat Alzheimer's disease(6). However, EVs also mirror faithfully the physiological condition of their cells of origin(7). For instance, Baj-Krzyworzeka M et al found that tumour-derived microvesicles carry several surface determinants and mRNA of tumor cells and transfer some of these determinants to monocytes(8). Additionally, EVs carry a variety of functional membrane and cytosolic proteins, diversity of RNA species as well as distinct lipids known to participate in physiological and/or pathological processes(9). Within the past decade interest in EVs has increased due to their demonstrated participation in processes such as intercellular communication, drug and gene vector delivery and as a rich yet untapped reservoir of biomarkers(10, 11). Among different body fluids, urine is an easily accessible, abundant source for repetitive samples. uEVs originate from the entire epithelial lining of the nephrons and uroepithelium but most likely also from the circulation(12, 13). Accordingly, uEVs contain protein biomarkers that can be used to diagnose or monitor disease progress or response to therapy(14-16). The isolation of uEVs with the currently established methods is tedious and further complicated by the interference of, for example, Tamm-Horsfall protein (THP), which is the most abundant glycoprotein in urine. While its exact functions remain to be defined, THP easily polymerizes into a high molecular weight protein meshwork which co-precipitates in uEV isolation process and efficiently entraps uEVs(17). This complicates the downstream analysis of uEVs, requiring additional steps to reveal, for 4


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instance, low abundance proteins in mass spectrometry (MS) analysis. THP can also remarkably interfere with glycoconjugate glycan analysis(18-20). Interestingly, uEVs contain an abundance of membrane proteins with plausible roles such as in signal transduction(21,

22)

. Thus, their targeted analysis for particularly

membrane protein content may open invaluable insights into roles of protein complexity. Currently there are no such targeted methods available. In this study we developed a modification of Triton X-114 phase separation to isolate and purify membrane proteins from uEVs. Specifically, the non-ionic detergent Triton X-114 was used to separate the sample into two distinct phases. When above the clouding point (22oC) temperature of Triton X-114, a lower oily appearing detergent phase (DP) and an upper aqueous phase (AP) are formed(23, 24). Hydrophilic proteins are found entirely in the aqueous phase and integral membrane proteins are recovered in the detergent phase. Using this method, most of the membrane proteins of uEVs were shown to partition into the detergent fraction.

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MATERIALS AND METHODS Urine Samples Urine samples were collected from healthy volunteers among the laboratory staff, aged 28-44 years (N=4). First morning void urine was processed within 3 h without adding protease inhibitors. Urine was anonymously labeled and pooled together. Written informed consent was obtained from all participants. This study was approved by The Ethical Committee of Dublin City University (DCUREC/2014/222). All experiments were performed in accordance with the declaration of Helsinki. uEV Purification Urinary vesicles were enriched using a “hydrostatic filtration dialysis’’ appliance (HFDa)(25). Briefly, urine samples were centrifuged first at relative centrifugation force of 2,000 g in a benchtop Universal 320 centrifuge (Hettich Zentrifugen, Tuttingen, Germany), then the supernatant (SN) was collected and used to isolate uEVs by our proprietary system, with a separating funnel equipped with a dialysis membrane with molecular weight cut-off (MWCO) of 1,000 kDa (Spectra/Por, SpectrumLaboratories Inc, Rancho Dominguez, CA). The uEVs fraction recovered from the dialysis tube is referred to as HFDa(25). Pre-Condensation of Triton X-114 Triton X-114 (Sigma-Aldrich, St. Louis, USA) was pre-condensed as described in the original publication(24) in order to remove the more hydrophilic components from the commercial reagent, and the concentration of pre-condensed Triton X-114 was 11.2% (w/v). Briefly, 20 mL of Triton X-114 was dissolved in 980 mL of 10 mM Tris HCl buffer, pH 7.5 containing 150 mM NaCl (Buffer A) in a 1 L conical flask immersed in a bath of ice and water placed on top of a magnetic mixer. The solution was gently agitated (300 rpm) until a clear and transparent solution was observed. The conical flask was then placed in the water bath set at 30°C and incubated overnight for phase separation. The upper turbid aqueous phase mixed with detergent was discarded and 6


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replaced by the same volume of buffer A. The detergent enriched phase was clearly seen on the bottom of the flask as an oily clear solution. These steps were repeated for a total of 3 times. The final lower detergent phase was collected and stored at 4oC. Membrane Protein Isolation and Purification from uEVs Protein quantification of uEVs was done by Bradford protein assay(26). The equivalent volume of 1 mg protein was dried by vacuum concentration. Dried pellet was then dissolved in 1 mL Lysis Buffer A (1% SDS, 10 mM Hepes, pH 7.4, 150 mM NaCl and 1 mM PMSF) at room temperature (RT) for 1 h with end-over-end rotation. After transferring the lysate into a 15 mL centrifugal tube, 9 mL of Lysis Buffer B (10 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM PMSF and 2.2% pre-condensed Triton X-114) (23)

was added (equivalent to 200 mg of detergent per mg of protein) and incubated at

4oC overnight with end-over-end rotation. The lysate was then centrifuged at 8,800 g, at 4°C for 10 min in a Mikro 220R 320 microcentrifuge (Hettich Zentrifugen) to sediment the insoluble pellet fraction (PF). The PF then was washed with 1 mL of Washing Buffer (10 mM Hepes, pH 7.4, and 150 mM NaCl) for 5 min on ice, and centrifuged at 8,800 g at 4°C for 10 min, and spun repeatedly for 3 times. The supernatant was collected to a volume of 15 mL and warmed up at 30°C water bath for 10 min to achieve phase partitioning. The mixture was centrifuged at 3,000 g at 30°C for 10 min, and the upper aqueous phase (AP) and lower detergent phase (DP) were carefully collected. The AP was further purified by adjusting the Triton X-114 concentration to 2% (w/v) and repeating the phase partitioning, as above. Similarly, the DP was further purified by adding an equal volume of aqueous Washing Buffer and repeating the phase partitioning process(27). Detergent phase separation procedure for AP and DP as above was repeated at least 3 times. Finally, proteins in each of the AP, DP, and PF fractions extracted from uEVs were recovered by acetone precipitation (three volumes ice-cold acetone: one volume sample)(28) and stored at -80°C before use. The simplified schematic workflow of our method is shown in Figure 1.

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Transmission Electron Microscopy Vesicles in HFDa and the lysate of the above paragraph were centrifuged at 8,800 g, 4°C for 10 min in a Mikro 220R 320 microcentrifuge (Hettich Zentrifugen) to sediment vesicles and the insoluble pellet fraction. Vesicles in HFDa, HFDa-pellet (HFDa-P8,800g) and supernatant (HFDa-SN8,800g) were used as reference in the transmission electron microscopy analysis. HFDa, HFDa-P8,800g, HFDa-SN8,800g and DP were re-suspended in 0.1 M PBS. Drops of these suspensions were placed on parafilm. The carbon-coated copper meshed grids (Plano, Wetzlar, Germany) were placed on the drops for 5 min for probe adsorption. After 5 min of fixation on drops of 1% glutaraldehyde (Sigma-Aldrich, St. Louis, USA) grids were washed 4 times for 3 s and negative contrasted using 1% uranyl acetate. Grids were air-dried and analyzed using a Zeiss 906 transmission microscope (Zeiss, Oberkochen, Germany). SDS-PAGE and Gel Staining Detergent phase extracted fractions were first resolubilized in 1% SDS at RT for 1 h. After that the solution was diluted 10× with Milli-Q H2O, and protein concentration determined by Bradford protein assay(26). The equivalent of 15 µg of each detergent portioning fraction was then resuspended in 7 M urea, 2 M thiourea, 5% (w/v) SDS, 40 mM Tris-HCl, pH 6.8, 0.5 mM EDTA, 20% (v/v) glycerol and 50 mM DTT in a ratio of 0.25 mg of protein per ml of solution(29). Protein denaturation was obtained after an overnight incubation at RT. Proteins were separated by SDS-PAGE employing freshly cast T 6-18%, C 2.6% gradient resolving gel (80 mm×50 mm×1.5 mm)(30). The gel was then stained with colloidal Coomassie G-250(31). Off-Gel Trypsin Digestion Sample preparation for MS analysis was performed according to Liu et al(32) with minor modifications. Briefly, 50 µg of dried protein samples were resolubilized and reduced by 100 µL of solution made of 8 M urea, 10 mM tris carboxyethyl phosphine (TCEP), 0.2 mM EDTA, 1% (w/v) sodium deoxycholate (DOC) and 100 mM Tris buffer, pH 8.8, in the dark for 1 h at RT, followed by alkylation with 55 mM 8


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(iodoacetamide) IAA in the dark for 1.5 h at RT. Thereafter, 40 mM N-acetyl cysteine (NAC) was added to quench the excess of IAA. Samples were digested by sequencing grade modified trypsin (Promega, Madison, WI) in a ratio of 1 µg of trypsin for 50 µg of total protein, at 37°C overnight. Finally, the samples were acidified with 1% (v/v) formic acid (FA) to precipitate DOC(33) and then the supernatant was cleaned up by Sep-Pak C18 cartridge (Waters, Milford Massachusetts, USA) according to manufacturer’s instructions.

Enzymatic In-Gel Digestion The lane of DP from Coomassie stained gel was excised into 9 bands of equal width and destained with 50% acetonitrile (ACN) in 50 mM ammonium bicarbonate (NH4HCO3). Disulfide bonds were reduced with 10 mM DTT and incubated for 45 min at 55°C. Free cysteines were then alkylated with 55 mM IAA, and incubated for 30 min at RT in the dark. Gel pieces were washed for 3 times with 50% ACN in 50 mM NH4HCO3 to remove the excess of IAA and dehydrated in 100% ACN. Finally, gel pieces were rehydrated and digested with 0.13 µg/mL sequencing grade modified trypsin (Promega, Madison, WI) in 50 mM NH4HCO3 for 12 h at 37°C. Digested peptides were extracted from the gel and acidified with 1% (v/v) final concentration of formic acid. The extracts were then cleaned up using a Sep-Pak C18 cartridge (Waters, Milford Massachusetts, USA) according to manufacturer’s instructions.

Nano-LC-ESI-MS/MS Analysis and Database Search Cleaned up samples were dried and resuspended in 20 µL of 0.1% (v/v) FA, 2% (v/v) ACN solution and 1 µg of peptide mixture estimated at 280 nm by Nanodrop ND-1000 (Thermo Scientific, Waltham, USA) was analyzed by Ultimate 3000 nano LC-system (Dionex, Sunnyvale, USA) coupled to an Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, USA). Peptides were eluted with the following binary gradients: solvent A (2% ACN and 0.1% FA in LC-MS grade water) and 0-65% solvent B (80% ACN and 0.08% FA in LC-MS grade water) for 60 min using a 9



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nanoRPC column (PepMap C18, 75 mm id_250 mm, 3 mm particle and 100 A pore size (Dionex). The linear trap quadrupole was operated in a data dependent acquisition mode with Xcalibur software (Thermo Scientific). Full mass spectra were recorded in profile mode over a mass range of 300-2000 m/z and tandem mass spectra recorded in profile mode. Dynamic exclusion was enabled with an exclusion duration of 30 s. Protein identification searches were performed using the information in the tandem mass spectra by searching against the UniProtKB/Swissprot protein database (Homo sapiens) with MASCOT search engine (Version 2.3, Matrix Science). Searches were carried out with trypsin specificity (one missed cleavage allowed), 20 ppm for MS and 0.6 Da for MS/MS (oxidation of Met and carbamidomethylation of Cys were set as variable modifications).

Diagonal SDS-PAGE. Two-dimensional (2D) SDS-PAGE was performed according to a standard protocol(34) with some modifications. Briefly, for the first dimension the protein solubilization and separation was carried out under non-reducing (NR) conditions, 60 µg of protein was denatured in 60 µL of a buffer containing 7 M Urea, 2 M thiourea, 40 mM Tris-HCl, pH 6.8, 5% SDS, 0.4 mM EDTA, 20% glycerol, at 20°C overnight with agitation in a thermomixer (TherMixer C Eppendorf, Hamburg, Germany). Gels were run at 15 mA (constant) using a gradient gel (T 6-18%; C 2.6%), size 86 mm×68 mm×1 mm. At the end of the run strips of acrylamide gel of the size of the well were cut and they were reduced first in an equilibration buffer (6 M Urea, 100 mM Tris-HCl, pH 8.8, 5% SDS, 0.4 mM EDTA, 20% glycerol), containing 1% (w/v) DTT for 20 min and then alkylated with the above equilibration buffer containing 2.6% (w/v) IAA for 20 min under agitation. Strips were then placed on top of a T 6-18%, C 2.6% gradient gel with size of 86 mm×68 mm×1.5 mm and sealed with a 0.5% (w/v) agarose of low electroendosmosis and low melting point with running buffer. At the end of the separation carried out at 20 mA (constant), gels were stained either with silver staining(35) or transferred to 0.45 µm nitrocellulose (NC) membrane (Whatman, 10


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Clifton, UK). Bioinformatics Analysis Transmembrane-spanning alpha helices were predicted using the web-based prediction program Transmembrane Hidden Markov Model (TMHMM) v2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/)(36). Grand Average of Hydropathy (GRAVY) scores was calculated according to the Kyte and Doolittle hydropathy scoring system (http://www.gravy-calculator.de/)(37). Post-translational modification (PTM) of lipidation analysis was performed with Uniprot database on-line. KEGG pathway was predicted with DAVID Bioinformatics Resources 6.8 online (https://david.ncifcrf.gov/home.jsp).

Western Blot After SDS-PAGE electrophoresis, the gels were transferred to 0.45 µm NC membrane (Whatman)(38). The NC membranes were then saturated with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, USA) and incubated with specific antibodies in Odyssey blocking buffer diluted 1 to 1 with PBS and 0.1% (v/v) Tween-20 according to manufacturer’s instructions: 1 µg/mL mouse anti-CD9 (R&D Biosystems, Minneapolis, Mn), 1 µg/mL mouse anti-CD63 (R&D Biosystems), 1 µg/mL rabbit anti-CD81 (R&D Biosystems), 1 µg/mL mouse anti-NEP (R&D Biosystems), 1 µg/mL rat anti-DPP4 (R&D Biosystems) and 1 µg/mL rabbit anti-TSG101 (R&D Biosystems). After 6 washes in PBS-Tween (0.1%, v/v), membranes were incubated with red (Excitation 680 nm; Emission 700 nm) and/or infrared (Excitation 780 nm; Emission 800 nm) dye-coupled secondary antibodies (LI-COR Biosciences) at a ratio of 1:5000 at RT for 1 h. Acquisition of the fluorescent signal was performed by Odyssey infrared imaging system (LI-COR Biosciences).

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RESULTS Vesicle Isolation, Triton X-114 Partitioning and Protein Pattern of Different Fractions Urinary extracellular vesicles were isolated by hydrostatic filtration dialysis(25). The membrane protein extraction protocol was performed using a multiple washing technique instead of a sucrose cushion to improve protein partitioning based on their solubility in the aqueous and detergent phase (Figure 1)(27). Modification of the original protocol included solubilization of uEVs with 1% (w/v) SDS and then a buffer containing Triton X-114 was added to extend the solubilization of membrane proteins while decreasing the SDS concentration ≤0.1% (w/v). The presence of SDS at a value less than 0.1% did not affect the cloudy point of Triton X-114 and the phase separation was achieved when samples were warmed up to 30°C (Figure 2 A and B). The interface line was clearly visible and it lasted long enough to allow a careful separation of the phases with pipette. The pre-solubilization step with SDS along with an overall increase of the protein/Triton X-114 ratio from 1 to 10 mg to 1 to 200 mg increased considerably the solubilization of the phospholipidic bilayer and membrane protein with a smaller insoluble pellet fraction (PF) (Figure 2 C and D). This procedure provides a better yield of the detergent phase (DP). Solubilization of the membrane proteins with SDS diluted with Triton X-114 was performed at 4oC and end-over-end agitation overnight. Multi-wash technique was chosen because it provided an efficient separation of the two layers while sucrose cushion separation repeatedly failed to separate the two phases sufficiently. Colloidal Coomassie brilliant blue stained gel showed a clear protein pattern of the different fractions of uEVs (Figure 2 D). More than 30 protein bands were detected in DP while many protein bands were found to be enriched preferentially (arrows) in DP compared to other fractions.

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Transmission Electron Microscopy (TEM) Transmission electron microscopy analysis (Figure 3 A) confirmed the presence and the heterogeneity in size and morphology of the vesicles (HFDa) recovered from the dialysis tube. In the centrifugation pellet HFDa-P8,800g (Figure 3 B) and supernatant HFDa-SN8,800g (Figure 3 C) showed the presence of clusters of vesicles while in PF (Figure 3 D) showed amorphous material with no evidence of remaining intact vesicles when compared with the starting material, HFDa, HFDa-P8,800g and HFDa-SN8,800g (Figure 3 A-C). Higher magnification of uEVs are shown in Figure 3 a,b,c,d. Additional TEM imaging can be found in Supporting Information, Figures S1. Proteomic Analysis of Detergent Phase Separation Fractions The Triton X-114 extraction generated an insoluble pellet fraction (PF), an aqueous phase (AP) and detergent phase (DP). 1µg of digested peptides from each fraction was injected online to a Thermo LTQ Orbitrap XL. A list of identified proteins was obtained according to the criteria presented in the materials and methods section. We identified 75 distinct proteins in the PF (Supporting Tables S 1.1), 134 in the AP (Supporting Tables S 1.2) and 221 proteins in the DP (Supporting Tables S 1.3) fractions, respectively. 40 proteins were common to all datasets while 42 and 13 proteins were shared between DP and AP and DP and FP, respectively. Interestingly, 126 proteins of DP were found to be unique proteins compared to other fractions (Supporting Figures S2). In order to reduce the complexity of the protein samples and to achieve the most complete and reproducible analysis(39), we used SDS-PAGE separation and in-gel digestion before LC-MS/MS in parallel. 30 µg of DP protein was separated on a 5 cm 4-20% gel and stained with colloidal Coomassie blue. The entire gel lane was sliced into 9 fractions (Figure 4 A), and each lane was manually destained first and then reduced and alkylated and finally digested with trypsin. We identified 275 proteins (including keratins) with at least 2 unique-sequence peptides (Supporting Tables S 2). After analyzing the data, we found abundance of proteins in the different gel-slice 13



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fractions. However, among these proteins, 168 were uniquely distributed in the different gel strips, and 11 proteins were found to be present in all the gel fractions (Supporting Tables S 2). The presence of ubiquitin could explain why many other proteins like TSG101 were detected at a molecular weight slightly higher than their theoretical ones.

Diagonal SDS gel electrophoresis was performed to check if

ubiquitins were present at any molecular weight higher than its monomeric form of 8kDa. Fifteen µg of AP (Figure 5 A) and DP proteins (Figure 5 B) were loaded for diagonal SDS gel electrophoresis, respectively, and then silver stained. WB analysis with monoclonal (Figure 5 C and D) or polyclonal (Figure 5 E and F) anti-UBQ antibodies confirmed their presence in the entire diagonal, evidently enriched at medium range molecular weight. This result is in line with previous reports(40-42). Detection of TSG101 in both AP and DP (Figure 5 E and F) revealed a higher organization suggesting that TSG101 can be engaged in protein complexes stabilized by disulfide bonds (Circle) and other type of covalent bonds (Rectangle) which seem to be abundant in the AP (Figure 5 G). Conversely, in the DP (Figure 5 H) the bulk of TSG101 signal appeared on the diagonal at a molecular weight higher than 46 kDa. Multiple monoubiquitination(43) and ISGylation(44) may explain these higher molecular weight isoforms. After comparing with the DP proteins from in-gel and off-gel analyses, 329 proteins were found in DP fraction in total (Supporting Figures S 2), and 167 proteins were common between DP in-gel and off-gel (Supporting Figures S 2). All these data showed that integrating both the in-gel and off-gel methods might be very helpful to achieve much more membrane proteins of uEVs. Moreover, after comparing with the identified proteins without phase separation in our earlier publication(32), we detected membrane proteins more selectively, especially the low abundance proteins (Supporting Figures S3). Thus, 199 different unique proteins were found specifically in DP. In addition, we carried out the GO analysis with the 199 different proteins, out of which 34 proteins (Supporting Tables S3) were integral components of plasma membrane. Interestingly, among these proteins, several typical membrane spanning proteins were detected including e.g. 14


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Tetraspanin-1,

Tetraspanin-4,

Tetraspanin-6,

Tetraspanin-8,

Neprilysin

and

Transmembrane protease serine 2.

Bioinformatic Analysis Analysis of transmembrane domains (TMDs) revealed that the percentage of transmembrane proteins was 9.3%, 23.9%, 30.8% and 25.5% in PF, AP, DP off-gel and DP in-gel, respectively (Figure 6 A). The average GRAVY score of these groups was -0.36, -0.36, -0.17 and -0.25, respectively, and more than 17.2% of DP off-gel and 10.2% of DP in-gel were hydrophobic proteins with the GRAVY score higher than 0.3 (Figure 6 B). 46 highly hydrophobic proteins from DP are shown in Table 1, and more than 91.3% of them had TMDs. Furthermore, the percentage of proteins with lipid anchor were 14.7% in PF off-gel, 11.2% in AP off-gel, 21.3% in DP off-gel and 20.0% in DP in-gel (Figure 6 C). The DP fraction had more lipidated proteins than the other groups. Additionally, after performing KEGG pathway analysis with the total of DP proteins, the results showed that 9.8% of the DP proteins played important roles in endocytosis pathway, and many of the proteins identified participate in signaling pathways (Figure 7). Notably, ADP-ribosylation factor 6 (ARF6) is a lipid-anchored membrane protein involved in protein trafficking to regulate endocytic recycling and cytoskeleton remodeling(45). Importantly, we only identified ARF6 in DP proteins instead of AP proteins.

This is taken as yet another

evidence to support validity of our method.

Western Blot Verification In order to verify whether the detergent-released proteins were membrane proteins truly, the study revealed 5 typical membrane proteins (CD9, CD63, CD81, NEP and DPP4) and a peripheral one (TSG101) as detected by shotgun LC-MS/MS. These were further verified by Western blot analysis. Surprisingly, the results showed that all of these typical membrane proteins (CD9, CD63, CD81, NEP and DPP4) were 15



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detectable in DP fraction, while TSG101 could be found in both AP and DP fractions (Figure 8). This provided further evidence that membrane proteins are successfully enriched in the DP -fraction.

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DISCUSSION Urinary extracellular vesicles express typical surface protein assembly reminiscent of their site of origin but in their “cargo” there is also a selected pattern of proteins, mRNA and microRNAs(16). After being secreted out form their site of origin, they can directly bind to distant sites with their surface proteins and finally deliver their biological cargo(46). Moreover, uEVs released from epithelial cells along the nephron may represent a valuable source of information for diagnostic purposes and future therapeutic applications(47). Despite continuous improvements in the uEV purification methods, THP is still a problem due to its interference with downstream analytics. To solve this problem, THP depolymerization by dithiothreitol (DTT) treatment or uEV isolation by sucrose gradient centrifugation have been used, but both approaches have drawbacks(48). Therefore, new strategies to simplify targeted protein harvesting are needed. The known involvement of membrane proteins in cell-cell communication, adhesion, and cell signaling(49) further call for improved methods for targeted protein harvesting. However, the hydrophobicity of membrane proteins often leads to aggregation and precipitation upon detergent removal, causing complications in downstream analytics(50). Among techniques successfully used, phase separation is a simple, efficient, and inexpensive method to purify and concentrate detergent-solubilized membrane proteins(51). Detergent molecules typically aggregate to form micelles above a critical concentration. Suspensions of these detergents separate into two phases when reaching a threshold temperature (cloud point)(52). Triton X-114 has a cloud point near room temperature (22°C). Accordingly, it has been successfully used to extract and separate hydrophobic and hydrophilic membrane proteins from each other. To our knowledge, Triton X-114 has not been used to characterize membrane proteins of uEVs. Here we used a modified serial Triton X-114 phase separation protocol to isolate and 17



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Page 18 of 44

purify membrane proteins of uEVs. The TEM analysis revealed that our method could increase the solubility of uEVs. According to the SDS-PAGE protein profile, more than 30 protein bands were detected in DP. Several protein bands were found to be enriched obviously in DP compared with the other phases. To provide further details of the membrane proteins of uEVs, we performed shotgun LC-MS/MS to identify the proteins of different Triton X-114 extracted fractions. As a result, 40 shared proteins were seen in all three fractions, while 126 unique proteins were detected in the DP fraction. In parallel, we performed in-gel trypsin digestion with the gel lane of DP, and then did the LC-MS/MS analysis. From the MS results, 108 more DP proteins were found with in-gel digestion. A comparison of the identified DP proteins from the in-gel and off-gel methods revealed that integration of digestion methods would increase the amount of unique membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier report(32), we detected almost 200 different proteins in the DP fraction, such as Tetraspanin-1,

Tetraspanin-4,

Tetraspanin-6,

Tetraspanin-8,

Neprilysin

and

Transmembrane protease serine 2. This is a strong evidence of validity and success of the present method in separating selectively membrane proteins. Interestingly, as some common proteins were found in each gel slice, diagonal SDS-PAGE was used to validate these, especially for ubiquitin. Ubiquitination is

a

key event in posttranslational protein modification(53), sorting and translocation inside the lumen of nascent exosomes(54) and protein degradation(55). Interestingly, a deubiquitination step is not required in the secretion of exosomes secreted in urine(56), and ubiquitin can still be detected in uEVs(42, 56) as seen in this study. As is well known, membrane proteins comprise three major protein categories: transmembrane proteins (TMDs), cytosolic membrane-associated proteins, and lipid-linked proteins(57).

Prediction of TMDs from the three protein fractions showed

that DP had more TMDs than the other groups. Afterwards, analysis of hydrophobicity also revealed that the GRAVY score of DP were much higher than other fractions. Furthermore, the analysis of proteins with lipid anchor also found that 18


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DP proteins had more lipid anchors than other fractions. Therefore, all these data above showed the usefulness of our method in separating different protein groups. Moreover, to further characterize the DP proteins we performed KEGG pathway analysis with the total of DP proteins found, the results showed that these appeared important especially in endocytosis and signaling pathways(58). This is well in line with the general biological functions of membrane proteins. Thus, the KEGG pathway informatics analysis results also provided positive evidence that the membrane proteins were enriched successfully with our method. Finally, in order to validate if the detergent rich proteins were membrane proteins, 5 specific transmembrane proteins (CD9, CD63, CD81, NEP and DPP4) and a peripheral one (TSG101) were analyzed by Western blot. CD9, CD63 and CD81 antibodies are against the tetraspanins that belong to membrane proteins(59), while NEP and DPP4 are single transmembrane proteins(60), so they could be good standards to prove whether the DP proteins are membrane proteins. Interestingly, the results showed the positive bands for the 5 proteins, and the membrane protein bands could be found in the DP fraction compared with AP. As a control, TSG101 could be detected in AP and DP fractions. The Western blot results supported the membrane purification method from uEVs. Overall, our modified Triton X-114 method for uEV studies as detailed here can be used to simplify profiles of the subcellular proteomes and hugely increase success to detect also the low abundance membrane proteins in uEVs.

CONCLUSIONS Our protocol of serial Triton X-114 phase separation is time-saving, efficient, and easy to perform in any laboratory to purify membrane proteins from uEVs. This will facilitate the detection of potential biomarkers and druggable targets in uEVs. SUPPROTING INFORMATION Supporting Figures S1: Additional transmission electron microscopy of vesicles in 19



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Page 20 of 44

HFDa, pellet, supernatant and detergent insoluble pellet.

Supporting Figures S2: Venn analysis of different fractionated proteins and DP proteins with different digestion method Supporting Figures S3 Venn analysis of identified proteins with None-phase separation and Phase separation method Supporting Tables S1: Full list of the protein identifications obtained from mass spectrometry off-gel analysis Supporting Tables S2: Full list of the protein identifications obtained from mass spectrometry in-gel analysis Supporting Tables S3: Full list of the integral component of plasma membrane proteins after GO analysis between the identified proteins with None-phase separation and Phase separation method AUTHOR INFORMATION Corresponding Author *

H.H.: E-mail: [email protected], Phone 0035840-557 4525 *

Y.J.: E-mail: [email protected], Phone number: 0086-20-61648231

Notes The authors declare no competing financial interests ACKNOWLEDGEMENTS This study was supported by ITN FP-7 Marie Curie European Union funded programmes

"UroSense"

(IAAP-GA-2011-286386),

FP7-Health

programme

"KidneyConnect" (Grant number 602422), the EU-Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115974 (BEAt-DKD) (“Biomarkers for diabetic nephropathy”), the FRIAS program of the Albert-Ludwigs University, Freiburg, the NSFC-Guangdong Joint Foundation of China (No. U1601225), the National Natural Science Foundation of China (No. 81372030 and No. 81671965), the Key Scientific and Technological Program of Guangzhou City (No. 201607020016) 20


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and the South Wisdom Valley Innovative Research Team Program (CXTD-001).

Author Contributions SH, LM, MH and OK performed experiments, LM and SH designed and developed the study. SH, LM, DT, XX, MH, OK, PM and HL analyzed the data. SH, LM, YJ, HZ and HH interpreted the results and wrote the manuscript. HH supervised the project.

21



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25



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Page 26 of 44

Table 1. The highly hydrophobic proteins that are unique in DP

No.

Accession No.a

Gene name

TMDsb

GRAVY scorec

Lipid anchor

Off-gel

In-gel

1

Q8IWA5

SLC44A2

11

0.407

—

√

√

2

Q53GD3

SLC44A4

10

0.488

—

√

√

3

Q495M3

SLC36A2

10

0.620

—

√

√

4

P31639

SLC5A2

14

0.585

—

√

√

5

O00322

UPK1A

4

0.527

—

√

√

6

Q53TN4

CYBRD1

6

0.571

—

√

√

7

P22732

SLC2A5

12

0.632

—

√

√

8

Q13183

SLC13A2

11

0.644

—

√

—

9

Q1EHB4

SLC5A12

13

0.526

—

√

—

10

O14817

TSPAN4

4

0.721

—

√

—

11

O43657

TSPAN6

4

0.359

—

√

√

12

P60033

CD81

4

0.689

—

√

√

13

P21926

CD9

4

0.487

√

√

√

14

Q8WWT9

SLC13A3

11

0.508

—

√

—

15

Q96FL8

SLC47A1

13

0.646

—

√

—

16

P41181

AQP2

6

0.514

—

√

—

17

P49447

CYB561

6

0.577

—

√

—

18

Q7Z3F1

GPR155

15

0.352

—

√

√

19

O43653

PSCA

0

0.476

√

√

√

20

Q8NFJ5

GPRC5A

7

0.310

—

√

—

21

Q9UBD6

RHCG

12

0.457

—

√

—

22

O15244

SLC22A2

11

0.314

—

√

—

23

Q8N357

SLC35F6

10

0.561

—

√

—

24

Q695T7

SLC6A19

12

0.449

—

√

—

25

P13866

SLC5A1

14

0.563

—

√

√

26

Q9NRX5

SERINC1

11

0.486

√

√

—

27

Q687X5

STEAP4

6

0.301

—

√

—

28

Q16563

SYPL1

3

0.346

—

√

—

29

Q9NS93

TM7SF3

7

0.319

—

√

√

30

O60635

TSPAN1

4

0.675

—

√

—

31

P19075

TSPAN8

4

0.729

—

√

—

32

O00526

UPK2

1

0.656

—

√

√

33

A0PJK1

SLC5A10

14

0.626

—

—

√

34

O95716

RAB3D

0

0.345

√

√

√

35

P04899

GNAI2

0

0.364

√

√

√

36

P08962

CD63

4

0.767

—

√

√

26


Page 27 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


No.

Accession No.a

Gene name

TMDsb

GRAVY scorec

Lipid anchor

Off-gel

In-gel

37

P29972

AQP1

6

0.495

—

√

√

38

P48509

CD151

4

0.33

√

—

√

39

P57739

CLDN2

4

0.558

√

—

√

40

Q53GD3

SLC44A4

10

0.488

—

√

√

41

Q71RC9

SMIM5

1

0.568

—

—

√

42

Q969X1

TMBIM1

7

0.387

—

—

√

43

Q9H3U5

MFSD1

11

0.708

—

—

√

44

Q9HD89

RETN

0

0.381

—

√

√

45

Q9NQ84

GPRC5C

7

0.362

—

√

√

46

Q9NZH0

GPRC5B

7

0.503

—

√

√

a

Uniprot ID, bTransmembrane domains, cGRAVY score>0.3 means that the protein is highly hydrophobic.

27



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Figure 1. Schematic workflow of purification of membrane proteins from uEVs in Triton X-114 solution. Serial Triton X-114 phase separation is used to purify membrane proteins from uEVs. When the temperature is above cloud point (22°C), pellet fraction (PF), aqueous phase (AP) and detergent phase (DP) are formed after centrifugation.

28


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Figure 2. Triton X-114 partitioning in presence of SDS and increased protein detergent ratio. Panel A, phase separation of Triton X-114 is still visible (arrows) in presence of SDS up to 0.25 % final concentration where the phase line starts to broaden as better represented in panel B where traces of blue bromophenol was added as visual aid. Panel C, protein pattern of the detergent partitioning in absence of SDS and with a protein detergent ratio of 1 to 200 mg. Panel D, protein pattern of the detergent partitioning in presence of 0.1 % SDS and with a protein detergent ratio of 1 to 200 mg, band 1-9 showed several enriched proteins in DP fraction.

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Figure 3 Transmission electron micrograph of vesicles in HFDa, pellet, supernatant and detergent insoluble pellet. 10 µg of protein was adsorbed on the grid and negative stained by uranyl acetate. Images were taken at ×40,000 to give a broad overview of the fractions. A, HFDa; B, HFDa-P8,800 g; C HFDa-SN8,800; D, insoluble pellet PF. a, b, c, d, the enlarged pictures of uEVs.

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Figure 4 Protein profile of different fractions of uEVs and the MS data analysis of the DP in-gel A, Colloidal Coomassie brilliant blue stained gel showed that many differentially expressed protein bands were enriched in DP compared to other groups. Slice 1-9 showed the picked slices for in-gel digestion. B, percentage of proteins respecting their theoretical molecular weight.

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Figure 5 Diagonal SDS page. Protein profiles of 15 µg AP (A) and DP (B) proteins after silver staining, monoclonal anti UBQ of AP (C) and DP (D); Polyclonal anti UBQ of AP (E) and DP (F); polyclonal anti TSG101 of AP (G) and DP (H). Molecular weight standard in kDa

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Percentage of transmembrane proteins (%) PF of f-g el A P of f-g el D P of f-g el

A

40

30.8%

30

25.5%

23.9%

20

in

of f-g el

P D

PF

-g el

0

P

............................................................-0.4 -0.5

5

D

............................................................0.3

0.0

11.2% 10

of f-g el

0.5

20.0%

14.7%

15

P

1.0

21.3% 20

A

B

25

of f-g el

D P

in

-g el

0

Percentage of proteins with lipid anchor

C

9.3%

10

GRAVY score

-1.0

in -g el D P

of f-g el D P

of f-g el A P

of f-g el

-1.5

PF

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Transmembrane domains, hydrophobicity distribution and lipid anchor of proteins identified in different fractionated groups A, proteins containing one or more transmembrane domains were predicted using the TMHMM algorithm for different fractionated proteins. The results indicated that DP had more TMDs than other groups. B, GRAVY scores of proteins identified in different fractions were calculated based on Kyte and Doolittle algorithms, and represented by dots. The two dotted lines indicate the thresholds required for hydrophobic (-0.4) and highly hydrophobic (0.3) determination, and the black line represents the average GRAVY score for the dataset. DP contained more hydrophobic proteins than other groups. C, ratios of proteins with lipid anchor in different fractions.

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Figure 7. KEGG pathway analysis of the total DP proteins The results of KEGG pathway analysis showed that the DP proteins played important roles in endocytosis pathway and signaling pathway

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A

St

TP

PF

AP

B

DP

St

TP

PF

AP

DP

250 150 100 75

250 150 100 75

DPP4

50 37

50 25 20

37 25 20 15 10

C

250 150 100 75

15 10

St

TP

PF

AP

D

DP

St

TP

PF

AP

DP

250 150 100 75

NEP

50 37

50 37 25 20

CD9

TSG101 CD63

25 20

CD81

15 15 10 10

Figure 8 Membrane protein verification by Western blot A, Coomassie stained gel; B, CD9 and DPP4; C, CD81 and NEP; D, CD63 and TSG101 The results of Western blot showed that 3 typical membrane proteins (CD9, CD63 and DPP4) could only be found in TP and DP compared with other protein groups, which supported that membrane proteins of uEVs were enriched successfully in DP.

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For TOC only

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250 kDa

A

kDa

250

10 kDa

250 150 100 75 50 37 25 20 15 10

10 C

D 250 150 100 75 50 37 25 20 15 10

250 150 100 75 50 37 25 20 15 10 E

F 250 150 100 75 50 37 25 20 15 10

250 150 100 75 50 37 25 20 15 10 G 250 150 100 75 50 37 25 20 15 10

B

250 150 100 75 50 37 25 20 15

H 250 150 100 75 50 37 25 20 15 ACS Paragon Plus 10 Environment

kDa

10


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Purification and Identification of Membrane Proteins from Urinary

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