Ultrapure-grade urea, ammonium bicarbonate, ammonium acetate, calcium chloride and Tris were from BioShop Canada, Inc. (Burlington, ON, Canada). Ultrapure-grade iodoacetamide, DTT, 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). Solid-phase extraction C18 MacroSpin™ Columns were from The Nest Group, Inc. (Southboro, MA, USA).
EPS-urine collection and concentration
All samples were collected from patients and utilized after informed consent following Institutional Review Board-approved protocols at Urology of Virginia 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 by performing a gentle massage of the prostate gland during DRE prior to biopsy, as previously described 7
. 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.
To generate sample pools from non-cancer and cancer patients, 10–20 ml of urine and EPS-urine were collected from the same individual an hour before the DRE massage, herein denoted as urine, and after DRE, herein denoted as EPS-urine. Urine and EPS-urine from a group of 5 patients with PCa and 5 biopsy negative, non-cancer individuals (), were pooled together to generate a sample panel comprising 4 different conditions: U_NC: urine non-cancer; EPS-U_NC: EPS-urine non-cancer; U_Ca: urine cancer; EPS-U_Ca: EPS-urine cancer. After collection, samples were stored on ice for no longer than 1 hour. Each sample was aliquoted and stored at −80°C until use.
Clinical information for the urine and EPS-urine pooled samples (NC and PCa) analyzed by MudPIT. Urine and EPS-urine sample pairs were collected from the same patient and pooled together as described in Materials and Methods. (Serum PSA in ng/ml)
Individual EPS-urines were obtained from an independent cohort of 11 different patients: 5 with low-grade PCa and 6 with biopsy negative benign conditions (BPH) (). Following collection, 9 ml of EPS-urine was centrifuged at 14,000 g to remove the cell pellet/sediment. The supernatant was recovered and concentrated using an Amicon Ultra-15 Centrifugal Filter (3 kDa cutoff; Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Approximately 500 μl of each concentrated EPS-urine sample was recovered from the filter device and stored at −80°C until use.
Table 2 Clinical information for the PCa (5 samples) and BPH (6 samples) individual EPS-urines analyzed by MudPIT. Serum PSA values are from the time of initial diagnosis. The treatment column indicates the clinical course followed for each cancer patient (DVP (more ...)
Protein digestion and peptide preparation
For MS analysis, all samples were first quantified using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, San Jose, CA) and volumes corresponding to 100 μg of total protein for the pools and 150 μg of total protein for the individual EPS-urines were resuspended in 50 μl of 8 M urea, 2mM DTT, 50 mM Tris-HCl, pH 8.5, and incubated at 37 °C with constant shaking for 30 min. Carbamidomethylation was performed by incubating samples with 8 mM of iodoacetamide for 30 min at 37 °C in the dark. Samples were then diluted to approximately 1.5 M urea using 100 mM ammonium bicarbonate, pH 8.5. Calcium chloride was added to a final concentration of 2 mM and the protein mixture was digested with trypsin (1:40 trypsin to protein ratio) at 37 °C overnight. The digested peptide mixture was purified with C18 MacroSpin™ columns and concentrated by vacuum centrifugation and reconstituted to a volume of 40 μl with 0.1 % formic acid. Samples were stored at − 80 °C until used for MudPIT analysis.
Individual EPS-urines (5 PCa and 6 BPH) were analyzed in triplicate using a fully automated 9-cycle MudPIT procedure as previously described 12, 13
. A quaternary HPLC pump was interfaced with a linear ion-trap mass spectrometer (LTQ, Thermo Fisher Scientific, San Jose, CA) equipped with a nanoelectrospray source (Proxeon Biosystems, Odense, Denmark). The pooled urine and EPS-urine samples were analyzed in triplicate on a LTQ Orbitrap XL, using a modified 5-cycle MudPIT, as previously described 14
Protein identification and data analysis
Raw data obtained from all MudPIT runs were converted to m/z XML using ReAdW and searched by X!Tandem against a locally installed version of the human UniProt complete human proteome (www.uniprot.org
) protein sequence database (version 2010_06; number of entries 20,295). A target/decoy search was performed to experimentally estimate the number of false-positive identifications (<1% estimated FDR) and an in-house protein grouping algorithm was applied to satisfy the principles of parsimony 15–17
The following parameters were applied according to the instrument used:
LTQ analyses of individual EPS-urines
The search was performed with a fragment ion mass tolerance of 0.4 Da and a parent ion mass tolerance of 4 Da. Complete tryptic digest was assumed. Carbamidomethylation of cysteine was specified as fixed and oxidation of methionine as a variable modification. Only proteins identified with two unique high-quality peptide identifications per triplicate were considered, as previously reported 15–17
(11 decoy proteins identified; FDR ~1%). Each sample (n = 11) was examined by 3 technical replicates (33 total MudPIT analyses).
LTQ-Orbitrap XL analyses of pooled urines and EPS-urines
The search was performed with a fragment ion mass tolerance of 0.4 Da and a parent ion mass tolerance of ± 10 ppm. Complete tryptic digest was assumed. Carbamidomethylation of cysteine was specified as fixed and oxidation of methionine as variable modification. Only proteins identified with two unique high quality peptide identifications per analyzed sample were considered, as previously reported 14, 18, 19
(2 decoy proteins identified; FDR ~0.5%). Each sample pool was analyzed by ≥3 technical replicates (13 total MudPIT analyses).
Protein relative abundance was calculated using the QSpec algorithm 20
. Proteins were considered to be up-regulated in the pooled EPS-urine samples versus the urine samples if they complied with the following parameters: false discovery rate (FDR) <0.05 and fold change (FC) 2, based on the QSpec algorithm 20
Gene Ontology annotation and data comparison
Functional annotations (Gene Ontology terms, KEGG pathways, and Swiss Prot entries) were assigned using the Database for Annotation, Visualization and Integrated Discovery (DAVID, bioinformatics resources v6.7; http://david.abcc.ncifcrf.gov/
. Unique proteins detected in the EPS-urine dataset 7, 8
were compared to the UniProt database and the top five significantly over-represented categories were reported (p-value <0.001). Comparisons of the present EPS-urine dataset to urine 8
and direct-EPS 7
datasets was accomplished using ProteinCenter (Proxeon Biosystems, Odense, Denmark). Proteins were sequence-aligned against each other and only proteins with at least 95% sequence identity were considered to match (i.e. protein clusters).
Prostate-enriched proteins characterization
The BioGPS portal (http://biogps.org/
was used to map identified proteins against available mRNA microarray datasets. We selected 25 major organ systems among those available in BioGPS and linked our proteins via gene accessions. The expression level for each gene was based on averaged probe intensities, and the significant enrichment in prostate tissue (>2 fold change) was calculated as a log2
ratio compared to the other selected tissues. The random sampling analysis was carried out using the unpaired one-tailed Student’s t-test. A p-value ≤0.05 was considered statistically significant. The alphabetical roster of the selected organs is as follows: bone marrow, colon, heart, hypothalamus, kidney, liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivary gland, skeletal muscle, skin, small intestine, smooth muscle, spinal cord, testis, thalamus, thymus, thyroid, uterus, whole blood, and whole brain.
The UniProt database (http://www.uniprot.org/
was used to assign sub-cellular localization to the 49 proteins enriched in EPS-urine pooled samples. We manually reported annotations and grouped the 49 proteins into three main categories: secreted, membrane, and intracellular (which includes cytoplasmic, nuclear, and lysosomal). Identified proteins were also screened against the Human Protein Atlas database (HPA; http://www.proteinatlas.org/
) 10, 11
for availability of antibodies and to examine their prostate tissue expression patterns.
SDS-PAGE and Western blot analysis on urine and EPS-urine pools
For Western blotting, 40 μg of total proteins were separated on 8 or 10% SDS-PAGE gels and blotted on PVDF membranes (0.2 μm; Bio-Rad Laboratories, Hercules, CA). Membranes were blocked with 5% milk in TBS-Tween (0.2%) for 1 hour at room temperature and subsequently incubated overnight at 4°C with the following primary antibodies: anti-Lactoferrin (1:1000 #ab10110; Abcam, Cambridge, UK), anti–CD10 (MME 1:1000 #ab951; Abcam, Cambridge, UK), anti–TIMP1 (1:2000 #RP1-TIMP1; Triple Point Biologics, Forest Grove, OR), anti–CD13 (ANPEP 1:500 #ab7417; Abcam, Cambridge, UK), anti–TGM4 (1:500 #sc55791; Santa Cruz Biotechnology, Santa Cruz, CA), anti–14-3-3σ (1:250 #ab14123; Abcam, Cambridge, UK), and anti–PARK7 (1:1000 #ab11251; Abcam, Cambridge, UK). After three 10-minute washes with TBS-Tween (0.2%), membranes were incubated with anti-mouse/anti-rabbit/anti-goat IgG-HRP secondary antibody (Invitrogen, Carlsbad, CA) at a dilution of 1:25,000 for 1 hour at room temperature, washed and visualized with the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, San Jose, CA).