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Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

Patricia A. Gonzales^*,, Trairak Pisitkun^*, Jason D. Hoffert^*, Dmitry Tchapyjnikov^*, Robert A. Star, Robert Kleta^,||,¶, Nam Sun Wang and Mark A. Knepper^*

^* Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Renal Diagnostics and Therapeutics Unit, National Institute of Diabetes and Digestive and Kidney Diseases, Section of Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, and ^|| Office of Rare Diseases, Office of the Director, National Institutes of Health, Bethesda, and Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland; and ^¶ London Epithelial Group, Centre for Nephrology, University College London, London, United Kingdom

Correspondence: Dr. Mark A. Knepper, 10 Center Drive, MSC-1603, National Institutes of Health, Bethesda, MD 20892-1603. Phone: 301-496-3064; Fax: 301-402-1443; E-mail: knep{at}helix.nih.gov

Received for publication April 21, 2008. Accepted for publication July 30, 2008.

	Abstract

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Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.

	Introduction

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Urinary exosomes are small extracellular vesicles (<100 nm in diameter) that originate from the internal vesicles of multivesicular bodies (MVB) in renal epithelial cells, including glomerular podocytes, renal tubule cells, and the cells lining the urinary drainage system.¹ Exosomes are released into the urine when the outer membrane of the MVB fuses with the apical plasma membrane of the epithelial cell.

Exosomes can be recovered from the urine by differential centrifugation as a low-density membrane fraction. Exosome isolation can result in marked enrichment of low-abundance urinary proteins that have potential pathophysiologic significance. As a consequence, we and others have been working to define optimal conditions for their isolation and purification as a prelude to their use in biomarker discovery studies.^1–3

In this study, we thoroughly expanded the known proteome of human urinary exosomes by using a highly sensitive LC-MS/MS system, improved software for identification of peptide ions and a more elaborate data analysis strategy than in our previous study. In addition, we used a neutral loss scanning approach⁴ to investigate the phosphoproteome of human urinary exosomes. The study identified 1412 proteins including 14 phosphoproteins in human urinary exosomes. Overall, there are 177 proteins that are associated with diseases as judged by their presence on the Online Mendelian Inheritance in Man (OMIM) database, 34 of which are known to be associated with renal diseases. The potential clinical usefulness of urinary exosomes was demonstrated using the well-defined renal tubulopathy, Bartter syndrome type I, as an example. The rich information from the proteomic analysis also provides further insight into the biogenesis of urinary exosomes.

	RESULTS

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Large-Scale Proteomic Profiling of Human Urinary Exosomes
In this study, we carried out proteomic profiling of a low-density membrane fraction from human urine consisting chiefly of exosomes, using a highly sensitive LC-MS/MS system, based on an ion trap mass spectrometer (LTQ; Thermo-Finnigan; Thermo Electron, San Jose, CA). We unambiguously identified 1132 proteins including 205 proteins seen in our previous study and 927 proteins not seen in our previous study of human urinary exosomes.¹ The full list (ambiguous and unambiguous identifications) contains 1412 proteins and can be viewed in Supplemental Table 1, and the list of proteins that were unambiguously identified in both studies can be viewed at http://dir.nhlbi.nih.gov/papers/lkem/exosome/. The expanded list of exosomal proteins includes 177 proteins that are disease related, on the basis of their presence in the OMIM database (Table 1).

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Disease-related protein: NKCC2 and Bartter syndrome type I. (A) Details of clinical phenotype for patients with Bartter syndrome type I, patient 1 and patient 2. (B) Ultrasound images showing calcium deposits (white arrowheads) in the kidneys of patients 1 and 2. (C) Immunoblot of urinary exosomes samples from patient 1, patient 2, control 1, and control 2 using polyclonal rabbit anti-NKCC2 and NCC antibodies.

Nineteen phosphorylation sites corresponding to 14 phosphoproteins were identified (Table 5). These included both newly identified phosphorylation sites and sites that had been previously identified. Two orphan G-protein–coupled receptors are included in the former group, viz. GPRC5B and GPRC5C. In GPRC5B, we identified one new phosphorylation site, T389, and, in GPRC5C, we identified three new phosphorylation sites, T435, S395, and Y426. These proteins are also known as retinoic acid–induced gene 2 (GPRC5B) and retinoic acid–induced gene 3 (GPRC5C).

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Novel phosphorylation site in the NCC. The serine-811 on the NCC protein is phosphorylated. (A) The phosphorylation site on the peptide is denoted by an asterisk (*). (B) The neutral loss peak (NL) from the +2 mass spectrum and the site-determining ions b5, b6, y7, and y8.

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Detection of AQP2-S256 phosphorylation in urinary exosomes. IMCD, rat inner medullary collecting duct treated with dDAVP (V2R-selective vasopressin analog) for 30 min; exo (10 µg), is human urinary exosomes, 10 µg; exo (72 µg), human urinary exosomes, 72 µg.

	DISCUSSION

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As illustrated in Figure 1, analysis of human urinary exosomes by mass spectrometry and immunoblotting can provide information with regard to genetic diseases involving apical proteins as shown by the qualitative assessment of urinary exosome samples from patients with Bartter syndrome type I. The urinary exosome patient samples showed a complete absence of NKCC2 protein bands. Mutations in the SLC12A1 gene cause Bartter syndrome type I.⁹ Many such mutations presumably result in misfolding of the NKCC2 protein, preventing the apical delivery of the protein and therefore preventing incorporation into urinary exosomes.

Barriers to Clinical Use of Human Urinary Exosome Analysis
We previously reviewed the potential of urinary exosome analysis as a route to biomarker discovery in renal diseases and delineated barriers to success with the approach.^{2, 21, 22} One important barrier is the lack of standard protocols for collection, processing, and storage of urine samples to allow reproducible measurements to be made in any clinical laboratory. We have proposed a set of procedures that can serve as a beginning point in the development of such techniques (http://intramural.niddk.nih.gov/research/uroprot/). Our current approach includes an ultracentrifugation step, which requires expensive instrumentation and long processing times. Filtration methods have been proposed to replace the ultracentrifugation step.³ A particular knotty problem is removal of Tamm-Horsfall protein, an extraordinary abundant urinary protein that interferes with successful mass spectrometry and immunoblotting.²³ In the long run, the most important technical challenge may be to develop quantification approaches that allow detection of changes in excretion rates of particular biomarker candidates. Both labeling and nonlabeling methods have been developed to make protein mass spectrometry quantitative²⁴; however, the biggest barrier to quantification lies in development of adequate normalization techniques providing surrogates for timed collections of urine, which are notoriously inaccurate.²¹ Use of creatinine as a normalizing variable may be inadequate because of high subject-to-subject variability in its rate of excretion.²¹ Even without quantification, urinary exosome analysis can be valuable in situations such as genetic diseases (e.g., Bartter syndrome type I [Figure 1]), where a protein may be entirely absent from urinary exosomes.

Relevance to Renal Biology
Several of the proteins newly identified in urinary exosomes in this study may have considerable relevance to renal biology and the mechanism of renal disease. Our previous study¹ identified proteins that were characteristic of most of the cell types facing the urinary space from podocytes through transitional epithelial cells of the urinary drainage system. In this study, we identified markers of two additional cell types, type A and B intercalated cells. Specifically, the B1 subunit of the H⁺-ATPase is apically located in type A intercalated cells,²⁵ and the anion transporter pendrin is present in type B intercalated cells.²⁶ Previously, we showed that urinary exosomes are derived from the apical endosomal pathway so that, although the B1 subunit of the H⁺-ATPase is also expressed in type B intercalated cells, its basolateral location probably precludes delivery to urinary exosomes. Overall, we identified 17 different vacuolar H⁺-ATPase subunits in urinary exosomes, 78% of the whole V₀–V₁ complex.⁸

We identified all of the subunits of the four ESCRT complexes (ESCRT-0 through ESCRT-III) in urinary exosomes in this study. The ESCRT complexes play a central role in the formation of MVB and the secretion of exosomes.⁵

Four different orphan G-protein–coupled receptors were identified in urinary exosomes in this study, namely GPR98, GPRC5A, GPRC5B, and GPRC5C. These receptors are presumably apically located in one or more renal tubule cells. GPR98 (also known as very large G-protein–coupled receptor 1 or Neurepin) has more than 6000 amino acids. The three GPRC5 proteins are members of the metabotropic glutamate family, but their natural ligands are unknown. It will be of interest in future studies to discover the role of these proteins in renal development and regulation.

Phosphoproteomic Analysis of Human Urinary Exosomes
Posttranslational modifications (PTM) of proteins play an important role in protein function. Among the most important PTM is phosphorylation. Protein phosphorylation regulates cellular signaling processes and may determine protein structure, function, and subcellular localization.¹² The ability to detect PTM, such as phosphorylation, in urinary exosomes may provide an additional level of information that could aid in diagnosis and treatment of a variety of renal disorders. Furthermore, discovery of PTM in urinary exosomes can provide clues about physiologic and pathophysiologic mechanism. In this study, we identified 14 phosphoproteins. The specific phosphorylation sites identified included six that were previously identified and eight that had not been previously identified. Among the novel sites was serine-811 in the NCC protein. This amino acid is conserved in humans, chimpanzees, rhesus monkeys, and horses but not in mice and rats. The amino acid sequence surrounding this site is absent in rodents owing to a difference in exon splicing.¹⁵ Finally, AQP2 phosphorylated at serine-256 was readily detectable in urinary exosomes. Because this phosphorylation event is increased by vasopressin-stimulated activation of adenylyl cyclase,¹⁶ measurements of the amount of serine-256–phosphorylated AQP2 in urine may provide an improved means of assessing the state of vasopressin activation using phospho-specific antibodies.

	CONCISE METHODS

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Urinary Exosome Isolation
Urine was collected from eight healthy humans: Four men (aged 22 to 33) and four women (aged 24 to 35) (National Institute of Diabetes and Digestive and Kidney Diseases Clinical Research Protocol 00-DK-0107). Fifty milliliters per subject was collected and mixed together. The urinary exosome isolation procedure is shown in Figure 4. Protease inhibitors were added (1.67 ml of 100 mM NaN₃, 2.5 ml of 11.5 mM 4-[2-aminoethyl] benzenesulfonyl fluoride, and 50 µl of 1 mM leupeptin). The mixed sample was centrifuged at 17,000 x g for 10 min at 4°C. The 17,000 x g supernatant was ultracentrifuged at 200,000 x g for 1 h at 25°C. The ultracentrifugation step was repeated 3 additional times, adding new 17,000 x g supernatant volume each time to each of the 12 tubes. Each of the 12 pellets was suspended with 50 µl of "isolation solution" (10 mM triethanolamine and 250 mM sucrose). The suspensions were pooled together.

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Differential centrifugation procedure for the isolation of urinary exosomes from urine.

In-Gel Trypsin Digestion
The protein concentration was determined using the Bradford Assay. This sample was solubilized in Laemmli sample buffer (1.5% SDS, 6% glycerol/10 mM Tris HCl, and 60 mg/ml DTT). Proteins in the exosome sample were separated by 1D SDS-PAGE using a Bio-Rad Ready Gel 4 to 15% polyacrylamide gradient gel with 125 µg distributed among two lanes. The gel was stained with Colloidal Coomassie Blue (GelCode Blue Stain Reagent; Pierce, Rockford IL) for 10 min and destained using ddH₂O (2 x 30 min). The gel was divided from top to bottom into 40 1-mm strips over the entire molecular weight range of the gel. Each strip was diced into small pieces (1 mm³) and placed into labeled centrifuge tubes.

The gels pieces were destained by adding 100 µl of 25 mM ammonium bicarbonate (NH₄HCO₃)/50% acetonitrile (ACN) for 10 min and were dried using a SpeedVac. The samples were reduced in a solution of 10 mM DTT and 25 mM NH₄HCO₃ at 56°C for 1 h. The samples were alkylated in a solution containing 55 mM iodoacetamide and 25 mM NH₄HCO₃ in the dark at room temperature for 45 min. The gel pieces were washed with 25 mM NH₄HCO₃ and dehydrated in a solution containing 25 mM NH₄HCO₃ and 50% ACN. The samples were dried using the SpeedVac. The samples were rehydrated in a solution containing 12.5 ng/µl trypsin (V5113; Promega, Madison, WI) in 25 mM NH₄HCO₃ and digested overnight at 37°C. Peptides were extracted using 50% ACN/0.1% formic acid (FA). The extracted samples were dried using the SpeedVac to remove ACN and then reconstituted with 0.1% FA. All 40 peptide samples were desalted using C₁₈ Zip Tips (Millipore, Billerica, MA) before analysis by mass spectrometry.

Nanospray LC-MS/MS
A high-sensitivity linear ion trap mass spectrometer, LTQ (Thermo Electron Corp.) equipped with a nanoelectrospray ion source was used to acquire m/z ratios in both precursor ions (MS1) and fragmented ions (MS2) scans. To reduce further the sample complexity before mass analysis, we injected the tryptic peptides extracted from each gel slice using an Agilent 1100 nanoflow system (Agilent Technologies, Palo Alto, CA) into a reversed-phase liquid chromatographic column (PicoFrit, Biobasic C₁₈; New Objective, Woodburn, MA). This LC-MS/MS method allows the acquisition of raw data files that are the MS/MS scans of the five highest intensity peaks after fragmentation with collision-induced dissociation in the LTQ mass analyzer.

Analysis of Data
The raw data files were searched against the NCBI Reference Sequences (RefSeq) human protein database by using BIOWORKS software (Thermo Finnigan). BIOWORKS utilizes SEQUEST, which is a program that "finds database candidate sequences whose theoretical spectra are compared with the experimental spectrum."²⁷ To identify thoroughly peptide sequences, we searched the raw data files using the target-decoy approach and InsPecT.

In addition, we analyzed the data in a two-step process. The first step was to assess and minimize false-discovery peptide identifications using the target-decoy approach, manual inspection of spectra, and InsPecT. The second step was to assess and eliminate ambiguous protein identifications.

Target-Decoy
To apply the target-decoy database searching strategy,¹³ we used the NHLBI Proteomics Core Facility in-house software to create a composite database containing the forward and reverse sequences of the nonredundant NCBI Reference Sequences (RefSeq) human protein database released on January 26, 2007. We used the forward sequences as the target database and the reversed sequences as the decoy database. We searched the raw data files against this composite database. After the search, we assessed the FDR by the number of peptides matched from the reversed sequences. The parameters that determine the stringency of the filtering criteria include XCorr, Sp rank, and delta Cn. These parameters were incrementally adjusted, thereby reducing the false-discovery identifications until a target FDR was achieved. In our case, the data were filtered to a target of 2% FDR, and the actual FDR was 1.91%. The filter settings used were min Xcorr rank 1, min Sp rank 10, min delta Cn 0.08, charge + 1 min Xcorr 2.37, charge + 2 min Xcorr 2.87, and charge + 3 min Xcorr 3.37.

InsPecT
We performed an additional analysis of the tandem mass spectrometry data using the InsPecT tool.²⁸ InsPecT uses de novo sequencing to generate sequence information (tag filters) from the experimental data. The tag filters are used to search the human protein database, nonredundant NCBI Reference Sequences (RefSeq) human protein database released on January 26, 2007, and identify peptide sequences that match with the experimental data. The size of the tag filters are three peptides in length on average. As shown in Figure 5, the tag filter generated for the protein CHMP1A matches the experimental data accurately. The peptide sequences identified using the tag filters are then scored to estimate that the top match is correct.²⁸ The score procedure computes the P value for each peptide sequence by "comparing the match quality score to the distribution of quality scores for incorrect matches." For these data, we accept only peptide matches with P 0.05.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Spectrum generated by InsPecT for CHMP1A protein (NP_002759). The peptide sequence is RVYAENAIRK. The tag region for the b ions and the y ions are shown by the black solid lines.

Elimination of Ambiguous Protein Identifications
Once proteins were identified using the approaches described, we needed to determine whether all identifications corresponded to unique gene products. An "ambiguous identification" is defined as an identification for which the peptide sequence that is used to determine the protein identity is found in multiple proteins that are not splice variants of the same gene (Figure 6).

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Criteria to disambiguate data set. (A) An unambiguous identification when a peptide sequence was a 100% match without gaps to one and only one protein. (B) An unambiguous identification when a peptide sequence was a 100% match without gaps to more than one protein, but these proteins are splice-variant products of one unique gene. (C) An ambiguous identification when a peptide sequence was a 100% match without gaps to more than one protein deriving from more than one gene, and the identification was based only on that single peptide.

Patients with Bartter Syndrome Type I
We collected spot urine samples from two patients with clinically diagnosed Bartter syndrome type I. The patients were enrolled in the institutional review board–approved protocol 76-HG-0238. We obtained written informed consent from the parents and/or patient. We collected urine samples from healthy humans and used them as controls. We processed all samples using the differential centrifugation method to isolate human urinary exosomes described already. Each sample was prepared for immunoblotting by solubilizing in Laemmli buffer (1.5% SDS, 6% glycerol, 10 mM Tris HCl, and 60 mg/ml DTT). The samples, patient 1 and patient 2, and the control samples, control 1 and control 2, were loaded onto a 1-D SDS-PAGE gel on the basis of time as measured by creatinine excretion. The proteins were transferred to Immobilon-P (Millipore) membranes, blocked, and probed with antigen-specific NKCC2 and NCC primary antibodies. We incubated the blots with species-specific fluorescence secondary antibodies (Alexa 688) and visualized them using the Odyssey Infrared Imaging System (LiCor, Lincoln, NE).

Phosphopeptide Enrichment and LC-MS/MS Analysis
We collected urine specimens (200 ml) from six healthy humans, three men and three women. We processed the specimens 400 ml/d for 3 d and pooled them. The exosome isolation was as described previously except that phosphatase inhibitors 10 mM NaF (Sigma, St. Louis, MO), 20 mM β-glycerol phosphate (Fluka, St. Louis, MO), and 1 mM sodium orthovanadate (Sigma) were added. The pellet was resuspended in 6 M guanidine HCl/50 mM NH₄HCO₃.

The sample was concentrated using a Centricon tube at 13,500 x g, with a starting volume of 420 µl and a final volume of 55 µl. The sample was reduced with 50 mM DTT for 1 h at 56°C. The sample was alkylated by addition of 100 mM iodoacetamide for 1 h (dark) at room temperature and was digested with trypsin overnight at 37°C. The sample was centrifuged at 16,000 x g for 20 min. The supernatant was kept, and 100% FA was added to inactivate the trypsin. The sample was desalted on a 1-ml HLB column (Waters Oasis, Milford, MA) by positive displacement via a syringe with a luer adapter. The sample was eluted with two elution buffers. Elution buffer 1 contained 50% ACN and 0.1% FA, and elution buffer 2 contained 90% ACN and 0.1% FA. The eluents, 50 and 90%, were dried using the SpeedVac.

Phosphopeptides were enriched from the samples using the Pierce Phosphopeptide Isolation Kit (cat. no. 89853) according to the manufacturer's protocol. Phosphopeptide samples were desalted using C₁₈ ZipTips (Millipore) before analysis by mass spectrometry.

Phosphopeptide samples were analyzed on an Agilent 1100 nanoflow system (Agilent Technologies) LC connection to a Finnigan LTQ FT mass spectrometer (Thermo Electron) equipped with a nanoelectrospray ion source as described previously.¹¹ The five most intense ions were sequentially isolated and fragmented (MS2) in the linear ion trap using collision-induced dissociation. The data-dependent neutral loss algorithm in XCALIBUR software was used to trigger an MS3 scan when a neutral loss of 98.0, 49.0, or 32.7 Da was detected among the two most intense fragment ions in a given MS2 spectrum.

Analysis of Phosphopeptide Data Sets
We searched MS raw data files against a composite database containing the forward and reversed peptide sequence of the Human RefSeq Database from January 26, 2007. Putative phosphopeptides were selected and filtered to produce MS2 and MS3 data sets with target FPR of 2% (high stringency) and 20% (low stringency) via the PhosphoPIC program.⁴ This software was also used to merge MS2 and MS3 data sets into a single file to facilitate subsequent data analysis. Phosphopeptides identified in MS2 spectra were submitted for automated phosphorylation site assignment using the Ascore algorithm.¹³ A site with an Ascore 19 (>99% confidence) was considered to be unambiguously assigned. Phosphopeptides present only in MS3 spectra were checked manually. We used Scansite (http://scansite.mit.edu/motifscan_seq.phtml) to determine the phosphorylation motif for the identified sites. We searched the PhosphoSite database (http://www.phosphosite.org) to determine whether the sites were novel or previously identified.

	Disclosures

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None.

	Acknowledgments

 
We thank Brian Ruttenberg for valuable contribution in developing the code/program used to disambiguate the proteomic data.

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

Supplemental information for this article is available online at http://www.jasn.org/.

	REFERENCES

Top Abstract Introduction RESULTS DISCUSSION CONCISE METHODS Disclosures REFERENCES

 

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