Project PXD000117

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PRIME-XS Project
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Human exosome proteome


Proteomics study of human urine exosomes. This data describes the proteomic complement of the human exosomal urine fraction from 10 healthy volunteers (5 male, 5 female) aged 23 to 36 years. 50 µg protein from each sample were fragmented on a 4%/12% SDS-polyacrylamide gel. Following staining, each gel track was separated into 28 equal sections, which were processed individually. Peptides were separated by reverse-phase chromatography (Dionex, Sunnyvale, CA) and LC-MS/MS was performed using an Eksigent NanoLC-1D Plus (Eksigent Technologies, Dublin, CA) HPLC system and an LTQ Orbitrap mass spectrometer (ThermoFisher, Waltham, MA). Peptides from each gel segment were analysed twice: (1) with a dynamic exclusion list and (2) a fixed exclusion list for the abundant protein uromodulin which was superimposed on a dynamic exclusion. MS data were processed using SEQUEST Bioworks Browser (version 3.3.1 SP1, ThermoFisher) to generate MS/MS peak lists. Combined peak list files were submitted to the MASCOT search algorithm (version 2.2.1, Matrix Science, London UK) and searched against the IPI-Human database.

Sample Processing Protocol

Exosomes were isolated from 10 healthy volunteer urine samples as previously described. The five men and five women ages 23–36 years were on no regular medication and had not consumed antibiotics or other medication within the previous 1 month. Current UTI was excluded. Briefly, subjects urinated directly into a container with protease inhibitors, including PMSF (500 μl 0.5 M solution), leupeptin (450 μg), and sodium azide (15 ml 100 mM solution). Urine was centrifuged within 30 minutes of collection (Beckman AVANTI J26-XP centrifuge; JA-17 fixed angle rotor; polyallomer 50-ml centrifuge bottles) for 20 minutes at 17,000×g. The supernatant was passed through a sterile 0.22-µm filter and ultracentrifuged (Beckman Optim L-100 XP VAC Ultracentrifuge; Ti45 fixed-angle titanium rotor; Beckman 70-ml polycarbonate ultracentrifuge bottles) for 135 minutes at 235,000×g and 4°C. Each ultracentrifugation pellet was suspended in 50 µl suspension buffer (250 mM sucrose and 10 mM triethanolamine [pH 7.6]) and pooled with the other pellets from the same urine sample. To avoid confounding effects on bacterial growth, urine samples for these experiments were collected without protease inhibitors or sodium azide, triethanolamine was removed from the suspension buffer, and exosomal pellets were washed one time by resuspension and ultracentrifugation. Protein from resuspended exosomal pellets was concentrated by precipitation and quantified using a Bradford protein binding colorimetric assay (Bio-Rad). Protein pellets were stored at −80°C until use. For MS, protein pellets were suspended in Laemmli sample buffer and incubated at 95°C; 50 µg protein from each sample was fragmented on a 4%/12% SDS–polyacrylamide gel. After staining, each gel track was separated into 28 equal sections, which were processed individually for the remainder of the workflow. After destaining, proteins were reduced and alkylated in gel, washed with NH4HCO3, and dehydrated in acetonitrile, and proteins were digested with modified trypsin. Liquid chromatography–MS/MS was performed using an Eksigent NanoLC-1D Plus (Eksigent Technologies) HPLC system and an LTQ Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Peptides were separated by reverse-phase chromatography (Dionex). Peptides were loaded onto a 5-cm C18 precolumn (300 μm inner diameter; LC Packings) from the autosampler. Peptides were eluted onto the analytical column using gradients for solvent A (water+0.1% formic acid) and B (acetonitrile+0.1% formic acid) of 5%–50% B over 40 minutes. A New-Objective nanospray source was used for electrospray ionization. m/z Values of eluting ions were measured in the Orbitrap mass analyzer with a mass range of 350–1600, and the resolution was set at 7500.

Data Processing Protocol

Peptides from each gel segment were run two times. All segments were run with dynamic exclusion. From these runs, a fixed exclusion list was generated for the abundant protein uromodulin and superimposed on a dynamic exclusion list as described elsewhere. Data from these two sets of runs were combined. MS data were processed using the SEQUEST Bioworks Browser (version 3.3.1 SP1; Thermo Fisher Scientific) to generate MS/MS peak lists. Combined peak list files were submitted to the MASCOT search algorithm (version 2.2.1; Matrix Science) and searched against the IPI-Human Database, version 4.3. Spectra were rescored using MASCOT-Percolator, a machine learning tool that minimizes false discoveries and incorporates target decoy searching. Protein identification required two or more unique peptides, with a false discovery rate of 0.1. Single peptide identifications were included if the MASCOT-percolator posterior error probability was <0.01. In addition, we included proteins based on an in-house protein type I error estimator termed espresso (Supplemental Material ). The cellular location of proteins was evaluated by searching the Ensembl gene identifier for each protein against the Human Protein Atlas Database ( ). Renal expression was evaluated using the bioGPS Gene Portal System ( ). Comparison of exosomal proteins with previous reports was made using the Exocarta exosomal protein database. Enrichment scoring was performed using the DAVID bioinformatics tool. This method provides a measure (by tests of proportions) of whether functional categories are overrepresented within a gene list compared with what is expected from stochastic sampling of the entire human gene set.


Laurent Gatto, Department of Biochemistry, University of Cambridge
Kathryn Lilley, University of Cambridge ( lab head )

Submission Date


Publication Date





Not available

Experiment Type

Bottom-up proteomics

Assay count



    Hiemstra TF, Charles PD, Gracia T, Hester SS, Gatto L, Al-Lamki R, Floto RA, Su Y, Skepper JN, Lilley KS, Karet Frankl FE. Human urinary exosomes as innate immune effectors. J Am Soc Nephrol. 2014 Sep;25(9):2017-27 PubMed: 24700864


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# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 28027 Exosomes in Huma Urine 1098 6851 663 23133 4738
2 28028 Exosomes in Huma Urine 986 5605 547 15158 3485
3 28029 Exosomes in Huma Urine 1941 8630 540 29909 4709
4 28030 Exosomes in Huma Urine 1272 4671 352 17786 2328
5 28031 Exosomes in Huma Urine 1465 13551 814 30013 8944
6 28032 Exosomes in Huma Urine 1789 8609 1018 21755 5036
7 28033 Exosomes in Huma Urine 1634 15679 977 30995 9638
8 28034 Exosomes in Huma Urine 1626 6372 816 22124 3856
9 28035 Exosomes in Huma Urine 3188 17382 983 33733 8803
10 28036 Exosomes in Huma Urine 1663 7072 521 21288 3835