Supplemental Materials and Methods
Materials
Ammonium bicarbonate, ammonium acetate, calcium chloride and Tris were from
BioShop Canada, Inc. (Burlington, ON, Canada). Ultrapure-grade iodoacetamide, DTT,
2,2,2-Trifluoroethanol and formic acid were from Sigma-Aldrich. HPLC-grade solvents
(methanol, acetonitrile, and water) were obtained from Thermo Fisher Scientific (San
Jose, CA). Trifluoroacetic acid was from J.T. Baker (Phillipsburg, NJ, USA). Mass
spectrometry-grade trypsin gold was from Promega (Madison, WI, USA).
Expressed prostatic secretion collection
All samples were collected from patients and utilized after informed consent following
Institutional Review Board-approved protocols at Urology of Virginia, Sentara Medical
School, and the Eastern Virginia Medical School along with the Research Ethics Board of
the University Health Network. All personal information or identifiers beyond diagnosis
and lab results were not available to the laboratory investigators. EPS-urine samples
were collected performing a gentle massage of the prostate gland during DRE prior to
biopsy, as previously described [1, 2]. The massage consisted of three strokes on each
side of the median sulcus of the prostate and the expressed fluid from the glandular
network of the prostate was subsequently voided in urine. Pools of 2 ml from each
sample of EPS-urine were derived from 12 patients classified as having low grade,
Gleason 6, organ-confined prostate cancer and 12 non-cancer samples, confirmed as
biopsy negative for prostate cancer. The average PSA levels were 4.6 ng/ml in serum
1
and 30.9 g/ml in EPS for non-cancer and 6.2 ng/ml in serum and 31.6 g/ml in EPS for
cancer subjects. All the additional patient information is summarized in Supplemental
Table 1.
Exosome preparation
To generate EPS-urine pools from non-cancer and cancer patients, 2 ml of each sample
were pooled together up to 24 ml of total volume. Exosomes were isolated by
differential centrifugation at 25,000 g for 30 minutes [3]. The supernatant was saved
and the pellet was re-suspended in 1ml sucrose/dithiothreitol solution (250 mM
sucrose/200 mg/ml DTT) and centrifuged at 25,000 g for 15 min [3]. This supernatant
was added to the first, and the sample was further centrifuged at 100,000 g for 5 hrs.
The pelleted exosomes were washed twice with PBS and re-suspended in 0.5 ml PBS.
Lipid composition of the total EPS-urine exosomes were predominantly species of
sphingomyelin
(~50%)
>
phosphatidylethanolamine
>
phosphatidylinositol
>
phosphatidylcholine > phosphatidylserine (data not shown), consistent with previous
reports of prostate exosome lipid composition [4, 5].
Protein solubilization and peptide preparation
The exosomes isolated from cancer and non-cancer EPS-urines were processed using
2,2,2-trifluoroethanol (TFE), an organic solvent used for extracting membrane proteins
[6]. Briefly, exosomes in 200 μL of 50 % TFE in PBS were incubated with constant shaking
at 60 °C for 1 hour. Proteins were reduced by adding 5 mM DTT (Ultrapure-grade,
2
Sigma-Aldrich, Oakville, ON, Canada) and alkylated with 25 mM iodoacetamide
(Ultrapure-grade, Sigma-Aldrich, Oakville, ON, Canada) for 30 minutes at room
temperature in the dark. The samples were diluted 5-fold with 100 mM ammonium
bicarbonate, pH 8.0, and digested with mass spectrometry-grade trypsin (Promega,
Madison, WI, USA) in 2mM calcium chloride at 37°C overnight. To stop the digestion, 20
μl of 90% formic acid (Ultrapure-grade, Sigma-Aldrich, Oakville, ON, Canada) were
added to the samples. TFE solution-digested samples were then centrifuged at 14,000 g
for 10 min at 4 °C and the resulting supernatant, containing the peptide mixture, was
solid phase extracted using Agilent Cleanup C18 Pipette Tips (Agilent, Palo Alto, CA,
USA), according to the manufacturer’s instructions. Eluted peptide mixtures were
vacuum dried and reconstituted in 5% acetonitrile 0.1% formic acid for mass
spectrometry analyses.
Nano Flow Liquid Chromatography and Tandem Mass Spectrometry analysis
For all samples, peptide concentrations were quantified using a NanoDrop
spectrophotometer (Thermo Fisher Scientific, San Jose, CA) and the volume
corresponding to 1 µg of total peptides was injected onto the chromatography column.
Chromatography was performed using a nano-flow ultra-performance liquid
chromatography (UPLC) system (Proxeon Biosystems, Odense, Denmark) with a 50 cm
heated C18 reverse phase column (Proxeon Biosystems, Odense, Denmark), interfaced
to a Q-Exactive tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA)
equipped with an EasySpray nanoelectrospray source (Proxeon Biosystems, Odense,
3
Denmark). Acidified peptide mixtures (2 μg) were automatically loaded from a 96-well
microplate autosampler and separated with a linear gradient at a flow rate of 250nl/min
using the blow gradient (buffer A = 0.1% formic acid in HPLC grade water; buffer B =
0.1% formic acid in HPLC grade acetonitrile).
Chromatographic gradient: shown is the time in minutes to ramp up to a certain %
buffer B.
Time (min)
%B
1
5
231
30
8
40
3
80
17
80
Peptides identification and data analysis
Raw data were analyzed using the MaxQuant computational proteomics platform
(version 1.3.0.3) with a fragment ion mass tolerance of 0.4 Da and a parent ion mass
tolerance of ±10ppm. Complete tryptic digest was assumed. Carbamidomethylation of
cysteine was specified as fixed modification and oxidation of methionine as a variable
modification. Only proteins identified with two unique and high quality peptide
identifications per analyzed sample were considered, as previously reported [7]. A
target/decoy search was performed to experimentally estimate the number of false-
4
positive identifications (<1% estimated FDR) [8]. Functional annotations (Gene Ontology
terms) were assigned using the Database for Annotation, Visualization and Integrated
Discovery (DAVID, bioinformatics resources v6.7; http://david.abcc. ncifcrf.gov/) [9]
using the UniProt database as background. Comparison of the present EPS-urine
exosomal data set to other publicly available data sets [2, 10-12] was accomplished
using ProteinCenter (Proxeon Biosystems, Odense, Denmark). Only proteins with ≥95%
sequence homology were considered matches (i.e. protein clusters).
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