Proteome analysis of the urine has shown that urine contains disease-specific information for a variety of urogenital system disorders, including prostate cancer (PCa). The aim of this study was to determine the protein components of urine from PCa patients. Urine from 8 patients with clinically and histologically confirmed PCa was analyzed by conventional 2D PAGE. The MS identification of the most prominent 125 spots from the urine map revealed 45 distinct proteins. According to Gene Ontology, the identified proteins are involved in a variety of biological processes, majority of them are secreted (71%), and half of them are enzymes or transporters. Comparison with the normal urine proteome revealed 11 proteins distinctive for PCa. Using Ingenuity Pathways Analysis, we have found 3 proteins (E3 ubiquitin-protein ligase rififylin, tumor protein D52, and thymidine phosphorylase) associated with cellular growth and proliferation (
Urine has become one of the most attractive biofluids in clinical proteomics because it can be obtained in large quantities, can be sampled noninvasively, and does not undergo significant proteolytic degradation compared with other biofluids [
Even though the urinary proteome is much less complex than the plasma proteome, it contains high number of proteins. The urinary proteome has been studied by almost any proteomics technology. The first proteomic profiling of the normal urine was performed in 1979 using two-dimensional electrophoresis (2D) [
Qualitative and quantitative changes in urinary proteome often point out to disease-related changes starting from urogenital diseases but also to some systemic diseases [
In this study, we describe proteomic map of urine from prostate cancer (PCa) patients using 2D PAGE/MS profiling. The determination of urinary proteome of PCa patients has created an initial database which can be used for comparison to normal urinary proteome database as well as to various cancer diseases urine proteome databases. The results from this study broaden the current knowledge in the field of urinary proteomics and provide some leads to understand the molecular bases of prostate cancer pathophysiology.
We analyzed 8 urine samples from patients with PCa prostate obtained from the University Clinic for Urology, University Clinical Centre “Mother Theresa,” Skopje, Republic of Macedonia. Informed consent for the use of these samples for research purposes was obtained from the patients in accordance with the Declaration of Helsinki. The study has been approved by the Ethics Committee of the Macedonian Academy of Sciences and Arts.
Patient’s clinical records including histology grading, tumor stage, and preoperative prostate-specific antigen (PSA) serum levels were reviewed to preselect the urine samples. Eight urine samples from patients with clinically confirmed and histologically graded tumors were chosen from the urine archive (Table
Clinical information of patients used to generate urine samples included in the study together with their PSA levels, histology grading and tumor stage.
Sample number | Patient number | Age | Diagnosis | Tumor stage | Gleason score | Preoperative PSA (ng/mL) |
---|---|---|---|---|---|---|
1 | PC-22 | 74 | PCA | pT2c pN0 pM0 | 7 (3 + 4) | 8.3 |
2 | PC-24 | 72 | PCA | pT2c pN0 pM0 | 7 (3 + 4) | 5.5 |
3 | PC-27 | 78 | PCA | pT2c pN0 pM0 | 5 (2 + 3) | 4.6 |
4 | PC-28 | 60 | PCA | pT2c pN0 pM0 | 7 (3 + 4) | 5.2 |
5 | PC-31 | 65 | PCA | pT3b pN1 pM0 | 7 (3 + 4) | 8.3 |
6 | PC-35 | 74 | PCA | pT2c pN0 pM0 | 7 (3 + 4) | 22.6 |
7 | PC-39 | 64 | PCA | pT2b pN1 pM0 | 9 (4 + 5) | 50.0 |
8 | PC-48 | 65 | PCA | pT2c pN0 pM0 | 7 (3 + 4) | 32.2 |
The first morning urine (3–10) mL was collected from the patients prior to clinical intervention and stored on ice for short period (<1 h). Samples were centrifuged at 1000
The stored urine samples were thawed and, for each sample, proteins were isolated in triplicate from 100
Pooled samples of total protein extract from urine were used. Equal amounts of proteins from each of the 8 samples were pooled to the total of 400
The gels were stained with Coomassie G-250. Gels were fixed in 30% (v/v) ethanol and 2% (v/v) phosphoric acid for 30 min with two exchanges of the fixing solution, washed three times with 2% (v/v) phosphoric acid for 10 min each, balanced in prestaining buffer (12% (w/v) (NH4)2SO4, 2% (v/v) phosphoric acid, and 18% (v/v) ethanol) for another 30 min, and stained in staining solution (0.01% (w/v) CBB G-250, 12% (w/v) (NH4)2SO4, 2% (v/v) phosphoric acid, and 18% (v/v) ethanol) for 72 h. The gels were stored in the staining solution until the spots of interests were manually picked.
The gels were scanned on an Ettan DIGE imager (GE Healthcare) and the resulting images were analyzed with ImageMaster 2D Platinum 7.0 (GE Healthcare, Little Chalfont, Buckinghamshire, UK) software. ImageMaster Platinum values of smooth, minimum area and saliency were 2, 5, and 50, respectively. Exclusion filter (vol >450) was applied to remove artificial spots and dust particles.
Ingel digestion was carried out manually with trypsin. Spots were first destained two times with a mixture of 50% (v/v) ACN for 15 min each and then once with 100 mM NH4HCO3 and 50% (v/v) ACN for 15 min. Spots were dried in vacuum centrifuge and then reduced with 100 mM NH4HCO3 containing 10 mM DTT for 45 min at 56°C and then alkylated with 54 mM iodoacetamide in 100 mM NH4HCO3 for 30 min in the dark, at room temperature. Gels pieces were washed with 100 mM NH4HCO3, shrunk with 50% ACN for 15 min, and dried in vacuum centrifuge. Gel particles were rehydrated with 20
For MS analysis, peptides were purified using Zip
For an overview of the cellular localization, molecular function, and biological processes in which identified proteins are included, we used the UniProt Knowledgebase (UniProtKB) and Gene Ontology (GO) database. The accession numbers of the identified proteins were imported into Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, USA) and functionally assigned to canonical pathways and the most significant networks generated from previous publications and public protein interaction databases. A
For this study, we selected urine samples from 8 patients with clinically and histologically confirmed PCa cancer (Table
Following 2D PAGE and staining,
List of the all protein spots from the PCa urinary proteome identified by MS.
Spot number | Protein name | SwissProt accession number | Mw (kDa) | pI | Mascot protein score |
|
Number of the matched peptides | % of sequence coverage |
---|---|---|---|---|---|---|---|---|
1 | Uromodulin OS = |
UROM_HUMAN | 72.64 | 5.05 | 158 |
|
18 | 22 |
2–14, 19–21 | Serotransferrin OS = |
TRFE_HUMAN | 79.29 | 6.8 | 243 |
|
22 | 40 |
15–18 | Alpha-1B-glycoprotein OS = |
A1BG_HUMAN | 54.79 | 5.56 | 93 |
|
12 | 35 |
22–24 | Serum albumin OS = |
ALBU_HUMAN | 71.31 | 5.92 | 246 |
|
26 | 48 |
25–28 | Alpha-1-antichymotrypsin OS = |
AACT_HUMAN | 47.79 | 5.33 | 82 |
|
12 | 31 |
Kininogen-1 OS = |
KNG1_HUMAN | 72.99 | 6.34 | 82 |
|
13 | 19 | |
29–33 | Ig alpha-1 chain C region OS = |
IGHA1_HUMAN | 38.49 | 6.08 | 62 |
|
5 | 18 |
34–37 | Alpha-2-HS-glycoprotein OS = |
FETUA_HUMAN | 40.00 | 5.43 | 95 |
|
15 | 43 |
38–43 | Alpha-1-antitrypsin OS = |
A1AT_HUMAN | 46.89 | 5.37 | 194 |
|
23 | 53 |
44–46 | Vitamin D-binding protein OS = |
VTDB_HUMAN | 54.52 | 5.40 | 135 |
|
12 | 50 |
47 | Antithrombin-III OS = |
ANT3_HUMAN | 53.02 | 6.32 | 85 |
|
8 | 21 |
48 | Alpha-amylase 1 OS = |
AMY1_HUMAN | 58.41 | 6.47 | 82 |
|
6 | 15 |
49, 67–69 | Fibrinogen beta chain OS = |
FIBB_HUMAN | 56.57 | 8.54 | 62 |
|
8 | 19 |
50-51 | U3 small nucleolar RNA-associated protein 18 homolog OS = |
UTP18_HUMAN | 62.42 | 8.93 | 75 |
|
6 | 13 |
52–54 | Leucine-rich alpha-2-glycoprotein OS = |
A2GL_HUMAN | 38.38 | 6.45 | 66 |
|
4 | 18 |
55 | Fibrinogen gamma chain OS = |
FIBG_HUMAN | 52.10 | 5.37 | 164 |
|
17 | 47 |
56 | Thymidine phosphorylase OS = |
TYPH_HUMAN | 50.32 | 5.36 | 64 |
|
5 | 25 |
57–62, 118–120 | Haptoglobin OS = |
HPT_HUMAN | 45.86 | 6.13 | 126 |
|
12 | 27 |
63 | Gelsolin OS = |
GELS_HUMAN | 86.04 (52.48) | 5.90 (5.34) | 80 |
|
7 | 16 |
64 | Apolipoprotein A-IV OS = |
APOA4_HUMAN | 45.37 | 5.28 | 82 |
|
8 | 22 |
65-66 | Actin, cytoplasmic 1 OS = |
ACTB_HUMAN | 42.05 | 5.29 | 64 |
|
7 | 24 |
Actin, cytoplasmic 2 OS = |
ACTG_HUMAN | 42.05 | 5.29 | 64 |
|
7 | 24 | |
70-71 | Fibrinogen alpha chain OS = |
FIBA_HUMAN | 95.65 (50) | 5.7 (4.65) | 91 |
|
10 | 15 |
72–75 | Zinc-alpha-2-glycoprotein OS = |
ZA2G_HUMAN | 34.46 | 5.71 | 104 |
|
8 | 29 |
76 | Endonuclease domain-containing 1 protein OS = |
ENDD1_HUMAN | 55.72 | 5.55 | 104 |
|
9 | 20 |
77 | E3 ubiquitin-protein ligase rififylin OS = |
RFFL_HUMAN | 41.74 | 5.33 | 59 |
|
5 | 26 |
78-79 | Inter-alpha-trypsin inhibitor heavy chain H4 OS = |
ITIH4_HUMAN | 103.52 (45) | 6.51 (5.15) | 88 |
|
11 | 13 |
80 | Quinone oxidoreductase-like protein 1 OS = |
QORL1_HUMAN | 39.07 | 5.49 | 66 |
|
4 | 12 |
81 | Interleukin enhancer-binding factor 2 OS = |
ILF2_HUMAN | 43.26 | 5.19 | 62 |
|
5 | 19 |
82 | Vesicular integral-membrane protein VIP36 OS = |
LMAN2_HUMAN | 40.54 | 6.46 | 80 |
|
7 | 21 |
83 | Transmembrane and immunoglobulin domain-containing protein 1 OS = |
TMIG1_HUMAN | 29.62 | 8.07 | 62 |
|
6 | 20 |
84–88 | Protein AMBP OS = |
AMBP_HUMAN | 39.87 | 5.95 | 130 |
|
15 | 37 |
89–92 | Prostaglandin-H2 D-isomerase OS = |
PTGDS_HUMAN | 21.24 | 7.66 | 64 |
|
8 | 33 |
93–97 | Ig kappa chain C region OS = |
IGKC_HUMAN | 28.82 | 6.73 | 77 |
|
7 | 29 |
98–102 | Apolipoprotein A-I OS = |
APOA1_HUMAN | 30.75 | 5.56 | 116 |
|
16 | 46 |
103 | Basement membrane-specific heparan sulphate proteoglycan core protein (perlecan) chain A, laminin-G-like domain 3 from human perlecan | PGBM_HUMAN | 468.83 (20.65) | 6.06 (5.47) | 204 |
|
15 | 88 |
104-105 | Ras-related protein Rab-36 OS = |
RAB36_HUMAN | 36.81 | 8.05 | 63 |
|
4 | 23 |
106 | Tumor protein D52 OS = |
TPD52_HUMAN | 24.35 | 4.79 | 63 |
|
4 | 22 |
107 | Alpha-1-acid glycoprotein 1 OS = |
A1AG1_HUMAN | 23.72 | 4.93 | 64 |
|
4 | 21 |
108–111 | Retinol-binding protein 4 OS = |
RET4_HUMAN | 23.34 | 5.76 | 109 |
|
9 | 49 |
112–115 | CD59 glycoprotein OS = |
CD59_HUMAN | 14.79 | 6.02 | 61 |
|
5 | 28 |
116 | Mannan-binding lectin serine protease 2 OS = |
MASP2_HUMAN | 77.19 (19.53) | 5.44 (5.75) | 127 |
|
8 | 37 |
117.121 | Secreted and transmembrane protein 1 OS = |
SCTM1_HUMAN | 27.30 | 7.00 | 75 |
|
6 | 22 |
122 | Transthyretin OS = |
TTHY_HUMAN | 15.99 | 5.52 | 160 |
|
10 | 73 |
123–125 | Uncharacterized protein KIAA2012 OS = |
K2012_HUMAN | 65.27 (50) | 6.09 (6.00) | 75 |
|
13 | 22 |
2D maps of the urine proteome from PCa patients obtained by 2D electrophoresis using IEF on pH 4–7 IPG strip and 12.5% SDS-PAGE. (a) Images of the three technical replicates of the pooled urine protein samples. The detected spots in the images are represented with green dots. Overall, 948 spots were reproducibly visualised in the three maps. (b) Representative 2D map of the urine proteome. All identified protein spots are marked with numbered arrows. Details of these proteins identified by MALDI MS are tabulated in Table
The identified proteins from PCa patient’s urine were classified by subcellular location, molecular function, biological function, and type of protein using the available data from the UniProt Knowledgebase (UniProtKB) and Gene Ontology (GO) database (Figure
Classification of the identified proteins in urine of PCa patients. The molecular function, biological processes in which they are involved, subcellular location, and type of protein were assessed by Gene Ontology search.
Comparison of the identified 45 proteins with proteins identified in normal urine by 2D PAGE/MS [
Functional characterization of the 11 identified proteins found only in PCa.
SwissProt accession number | Protein name | Gene name | Subcellular location | Type of protein | Biological function |
---|---|---|---|---|---|
ANT3_HUMAN | Antithrombin-III OS = |
SERPINC1 | Extracellular space | Enzyme | Serine protease inhibitor in plasma that regulates the blood coagulation cascade |
|
|||||
AMY1_HUMAN | Alpha-amylase 1 OS = |
AMY1 | Extracellular space | Enzyme | Carbohydrate metabolic process |
|
|||||
UTP18_HUMAN | U3 small nucleolar RNA-associated protein 18 homolog OS = |
UTP18 | Nucleus | Other | It is involved in nucleolar processing of pre-18S ribosomal RNA |
|
|||||
TYPH_HUMAN | Thymidine phosphorylase OS = |
TYMP | Extracellular space | Growth factor | It has a role in maintaining the integrity of the blood vessels, growth promoting activity on endothelial cells, angiogenic activity in vivo, and chemotactic activity on endothelial cells in vitro and catalyzes the reversible phosphorolysis of thymidine |
|
|||||
ENDD1_HUMAN | Endonuclease domain-containing 1 protein OS = |
ENDOD1 | Extracellular space | Enzyme | It may act as a DNase and a Rnase |
|
|||||
RFFL_HUMAN | E3 ubiquitin-protein ligase rififylin OS = |
RFFL | Cytoplasm | Enzyme | It regulates several biological processes through the ubiquitin-mediated proteasomal degradation of various target proteins and negatively regulates the tumor necrosis factor-mediated signaling pathway and p53/TP53 through its direct ubiquitination and targeting to proteasomal degradation |
|
|||||
QORL1_HUMAN | Quinone oxidoreductase-like protein 1 OS = |
CRYZL1 | Cytoplasm | Enzyme | Quinone metabolic process |
|
|||||
ILF2_HUMAN | Interleukin enhancer-binding factor 2 OS = |
ILF2 | Nucleus | Transcription regulator | It functions predominantly as a heterodimeric complex with ILF3. This complex may regulate transcription of the IL2 gene during T-cell activation |
|
|||||
TMIG1_HUMAN | Transmembrane and immunoglobulin domain-containing protein 1 OS = |
TMIGD1 | Other | Other | Integral component of membrane |
|
|||||
RAB36_HUMAN | Ras-related protein Rab-36 OS = |
RAB36 | Cytoplasm | Enzyme | Protein transport. It is probably involved in vesicular traffic |
|
|||||
TPD52_HUMAN | Tumor protein D52 OS = |
TPD52 | Cytoplasm | Other | B cell differentiation and anatomical structure morphogenesis and secretion |
Using Ingenuity Pathways Analysis (IPA) classification and networking, we found out that some of the 11 identified proteins in PCa are significantly associated with cancer and organism injury and abnormalities diseases and disorders. Four proteins (antithrombin-III, transmembrane and immunoglobulin domain-containing protein 1, tumor protein D52, and thymidine phosphorylase) are associated with different types of cancers (
Network associated with the 11 urinary proteins from patients with PCa according to IPA. Top protein network of functional associations between proteins was Cell Death and Survival, Cell-To-Cell Signaling and Interaction, and System Development and Function with score 30 (
Determination of protein map and composition of PCa patient’s urine may lead to an increased understanding of cancer pathophysiology. Using 2D PAGE/MALDI-TOF, we have identified a total of 125 protein spots belonging to 45 unique proteins in PCa patient’s urine. According to the molecular and functional data for these proteins, they can be classified into several groups:
Regarding the subcellular location of the identified proteins, our analysis revealed that extracellular proteins and plasma membrane proteins represent the majority in the PCa patient’s urine. This was expected for two reasons: first, the urine is in direct contact with several glands in the male urinary tract, and, second, substantial fraction of the urinary proteins is derived from plasma [
Thirty-four proteins in our study have been reported as constituents of the normal urine, while 11 proteins (antithrombin-III, alpha-amylase 1, U3 small nucleolar RNA-associated protein 18 homolog, thymidine phosphorylase, endonuclease domain-containing 1 protein, E3 ubiquitin-protein ligase rififylin, quinone oxidoreductase-like protein 1, interleukin enhancer-binding factor 2, transmembrane and immunoglobulin domain-containing protein 1, Ras-related protein Rab-36, and Tumor protein D52) were not reported in the normal urine proteome [
Thymidine phosphorylase (TYMP) is an enzyme involved in pyrimidine metabolism and also known to be a platelet-derived endothelial cell growth factor (PD-ECGF). TYMP is overexpressed in various tumors including prostate cancer and plays an important role in angiogenesis, tumor growth invasion, and metastasis [
Furthermore, IPA analysis pointed out functional associations between the 11 proteins. The network encompassed 10 out of 11 proteins connected through four nodes: ubiquitin C (UBC), tumor necrosis factor (TNF), transforming growth factor beta 1 (TGFB1), and interferon gamma (IFNG). Ubiquitination has been associated with protein degradation, DNA repair, cell cycle regulation, kinase modification, endocytosis, and regulation of other cell signaling pathways [
The rest of the regulatory nodes in the network are represented by cytokines (TFN, TGFB1, and IFNG). Cytokines are regulators of host responses to infection, immune responses, inflammation, and trauma [
The interaction of ubiquitin, cytokines, and urine proteins found in PCa patients in this study, as proposed by IPA network, having in mind dual nature of cytokines and ubiquitin in the cancer progression, may lead to deeper understanding of prostate cancer pathogenesis. The possible role of these proteins and their connection with the signal transduction cascade of prostate cancer remains to be solved in the future.
In summary, we have created an initial proteomic map of PCa patient’s human urine. The most prominent spots were successfully identified and analyzed in context of prostate cancer. Comparison with other published studies analyzing normal urine proteome pointed out several proteins that might have some role in the pathogenesis of prostate cancer. Moreover, IPA analysis showed significant association of our proteins with cancer and cellular growth and proliferation. The attempts to identify more low-abundant proteins in the urine from PCa patients by different strategies as well as comparison with urinary proteome from different cancer are underway. Although the presented urinary proteome map from patients with PCa showed limited number of proteins, the information regarding their position, molecular mass, possible posttranslational modifications, and presence of different protein fragments are useful addition to the present knowledge and provide some leads to understand the molecular bases of prostate cancer pathophysiology.
One-dimensional
Two-dimensional
Two-dimensional polyacrylamide gel electrophoresis
Acetonitrile
Bovine serum albumin
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate
Dalton
Dithiothreitol
Gene Ontology
2-Iodoacetamide
Ingenuity Pathways Analysis
Liquid chromatography
Matrix-assisted laser desorption/ionization-time of flight-time of flight
Mass spectrometry
Prostate-specific antigen
Sodium dodecyl sulfate
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Trifluoroacetic acid
Tris(hydroxymethyl)aminomethane
UniProt Knowledgebase.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by the funds for Science of the Macedonian Academy of Sciences and Arts (Grant no. 09-114/1, Biomarker Detection in Prostate Cancer with the Use of 2D-DIGE/MALDI MS Technology). The authors thank patients for the participation in the study, medical personnel at the University Clinic for Urology at the University Clinical Centre “Mother Theresa,” Skopje, Republic of Macedonia, for the collection of urine samples, and Katerina Markovska for the technical assistance.