Printer Friendly

Mapping and identification of the urine proteome of prostate cancer patients by 2D PAGE/MS.

1. Introduction

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 [1]. The urine contains water, glucose, salt, and proteins derived from plasma or the urogenital tract. It can be viewed as modified ultrafiltrate of plasma combined with proteins derived from kidney and urinary tract, with protein concentration approximately 1000-fold lower than in plasma itself [2].

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) [3]. Afterwards, 2D, liquid chromatography (LC) and capillary electrophoresis (CE), all of them coupled to mass spectrometry (MS), have been used extensively in the proteomics definition of the urine. With the advent of the high throughput proteomics platforms consisting of 1D SDS-PAGE or LC coupled with high resolution mass spectrometers such as LTQ-FT and LTQ-Orbitrap, the number of detected proteins in healthy urine reached from 1310 to 1823, depending on the technology used [4-6]. The number of detected proteins in normal urine using 2D PAGE/MS is relatively lower compared to the proteomics platforms mentioned above [7]. This is mainly due to two reasons: lack of ability to cope with the broad dynamic range of complex samples and with hydrophobic proteins. However, 2D PAGE/MS is still an indispensable platform in proteomics, particularly for the assessment of the molecular mass of any protein or protein fragments and posttranslational modifications [8].

Qualitative and quantitative changes in urinary proteome often point out to disease-related changes starting from urogenital diseases but also to some systemic diseases [9]. Proteomic analysis of urine has shown that it contains disease-specific information for various diseases. Up till now, urine has been used as a source of biomarkers for a number of kidney diseases and cancers related to the urogenital system such as bladder and prostate cancer, as well as various nonnephrological/urogenital diseases such as preeclampsia, stroke, coronary artery diseases, heart failure, acute appendicitis in children, and graft-versus-host disease reviewed extensively elsewhere [9,10].

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.

2. Materials and Methods

2.1. Urine Samples. 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 1), for the 2D PAGE/MS analysis. The preoperative serum PSA levels ranged from 4.6 to 50.0 ng/mL (mean PSA = 17.1 [+ or -] 16.6). The mean age of the selected patients was 69 [+ or -] 6.3 years and the mean Gleason score was 7.0 [+ or -] 1.1.

The first morning urine (3-10) mL was collected from the patients prior to clinical intervention and stored on ice for short period (<1h). Samples were centrifuged at 1000 xg, for 10 min, to remove cell debris and casts, aliquoted in 1.5 mL tubes, and stored at -80[degrees] C until use.

2.2. Preparation of Total Protein Extract from Urine. The stored urine samples were thawed and, for each sample, proteins were isolated in triplicate from 100 [micro]L urine using 2D Clean-UP Kit (GE Healthcare) according to the manufacturer's instructions. The pellets from each replicate were dissolved in 10 [micro]L of UTC buffer (8 M urea, 2 M thiourea, 4% CHAPS), pooled together for each sample, quantified by Bradford method [11] in duplicate against a standard curve of bovine serum albumin (BSA), and stored at -80[degrees]C until use.

2.3. 2D PAGE. 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 [micro]g of protein per gel. Pooled samples were analyzed in three technical replicates by conventional 2D PAGE analysis on immobilized pH gradient strips Immobiline DryStrip pH 4-7 (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and 12.5% SDS PAGE subsequently. For the immobilized pH gradient strips pH 4-7, we have used the rehydration buffer consisting of 8 M urea, 2M thiourea, 2% (w/v) CHAPS, 10 mM DTT, 1.2% (v/v) IPG-buffer pH 4-7, and trace of bromophenol blue. Isoelectric focusing of the rehydrated 24 cm immobilized pH gradient strips was performed on Ettan IPGphor 3 system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The strips were focused until total of 64.5 kVh was reached. The focused proteins in the immobilized pH gradient strips were immediately equilibrated in two incubation steps, each lasting 15 min, at room temperature. In the first step, the equilibration buffer (6M urea, 2% (w/v) SDS, 30% (v/v) glycerol, 50 mM tris, pH 8,6) was supplemented with 1% (w/v) DTT for reduction, followed by alkylation in the same buffer containing 4.7% (w/v) iodoacetamide instead of DTT. The second dimension was carried using Ettan DALTsix system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 2.5 W per gel for 30 min, followed by 16 W/gel for 4 h.

2.4. 2D PAGE Imaging and Analysis. 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) [(N[H.sub.4]).sub.2]S[O.sub.4], 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) [(N[H.sub.4]).sub.2]S[O.sub.4],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.

2.5. Mass Spectrometry: Ingel Tryptic Digestion. 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 NH HCO and 50% (v/v) ACN for 15 min. Spots were dried in vacuum centrifuge and then reduced with 100 mM N[H.sub.4]HC[O.sub.3] containing 10 mM DTT for 45 min at 56[degrees]C and then alkylated with 54 mM iodoacetamide in 100 mM N[H.sub.4]HC[O.sub.3] for 30 min in the dark, at room temperature. Gels pieces were washed with 100 mM N[H.sub.4] HC[O.sub.3], shrunk with 50% ACN for 15 min, and dried in vacuum centrifuge. Gel particles were rehydrated with 20 [micro]L of 0.01 [micro]g/[micro]L trypsin proteomics grade (Roche Diagnostics GmbH) in digestion buffer (95% 50 mM N[H.sub.4]HC[O.sub.3]/5% ACN) for 45 min at room temperature. The remaining enzyme supernatant was replaced with one gel volume of the digestion buffer and digestion was carried out at 37[degrees]C, overnight. After digestion, peptides were collected in separate tube, extracted once with 20 [micro]L of 50% ACN and twice with a mixture of 50% ACN/5% formic acid, dried in vacuum centrifuge, and reconstituted in 10 [micro]L of 0.1% TFA.

2.6. Mass Spectrometry: Identification. For MS analysis, peptides were purified using ZipTipC18 (Millipore Corporation) following the manufacturer's instructions and eluted in 2-3[micro]L of CHCA (4mg/mL in 50% ACN/0.1% TFA) directly onto a MALDI target plate (Shimadzu Biotech Kratos Analytical). Droplets were allowed to dry at room temperature. Samples analysis was performed using AXIMA Performance MALDI-TOF-TOF mass spectrometer (Shimadzu Biotech Kratos Analytical). Spectra acquisition and processing were performed using the MALDI-MS software (Shimadzu Biotech Kratos Analytical) version 2.9.3.20110624 in positive reflectron mode at mass range 1-5000 Da with a low mass gate at 500 Da and pulsed extraction optimized at 2300 Da. External calibration was performed based on monoisotopic values of five well-defined peptides: bradykinin fragment 1-5, angiotensin II human, [Glu1]-gibrinopeptide B human, adrenocorticotropic hormone fragment 1-17 human, and adrenocorticotropic hormone fragment 7-38 human (Sigma-Aldrich). External calibration mix (500 fmol/[micro]L) was diluted with the matrix in ratio 1 : 1 and applied onto the MALDI target plate at final concentration of 250 fmol per spot. Each mass spectrum was acquired by 500 laser profiles (five pulses per profile) collected across the whole sample. After filtering tryptic-, keratin-, and matrix-contaminant peaks, the resulting monoisotopic list of m/z values was submitted to the search engine Mascot (version 2.4.01, MatrixScience, UK) searching all human proteins and sequence information from Swiss-Prot (version 2014_05, 20265 sequences) and NCBInr (version 20140323, 276505 sequences). The following search parameters were applied: fixed modification-carbamidomethylation and variable modifications-methionine oxidation and N-terminal acetylation. Up to 1 missed tryptic cleavage was permitted and peptide mass tolerance of [+ or -]0.40 Da was used for all mass searches. Positive identification was based on a Mascot score greater than 56, above the significance level (P < 0.05). The reported proteins were always those with the highest number of peptide matches.

2.7. Functional Characterization of the Identified Proteins. 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 P value calculated with the right-tailed Fisher's exact test was used to yield a network's score and to rank networks according to their degree of association with our data set.

3. Results

For this study, we selected urine samples from 8 patients with clinically and histologically confirmed PCa cancer (Table 1). The mean age of the selected patients was 69 years ([+ or -]6.3), the mean Gleason score was 7.0 ([+ or -] 1.1), and the mean preoperative serum PSA level was 17.1 ([+ or -] 16.6). The average amount of purified proteins from 100 [micro]L urine ranged from 18.2 to 29.8 pg. The analysis of each patient urine proteome required substantial number of isolations and 2D PAGE gels for analysis. Therefore, we used pooled samples of total protein extract from 8 patients. The pooled samples also give more relevant picture of the PCa patient's urine proteome.

Following 2D PAGE and staining, 1085 [+ or -] 110 spots were detected and 948 of them were reproducibly visualized in the three technical replicates (Figure 1(a)). The most prominent 125 spots were picked up for the MS analysis. The position of these spots in the 2D urine map is shown in Figure 1(b). The MS identification of these spots revealed that they belong to 45 distinct proteins (Table 2). A number of proteins were presented on 2D map as a horizontal row of multiple spots (with small changes in pI and molecular weight), likely caused by variable posttranslational modifications (PTMs). All identified proteins were in the expected molecular size (14-80 kDa) and pI value (4.93 to 8.07) and the majority of it were high-molecular weight proteins (>30 kDa). Representative 2D map showed no evidence of protein degradation that usually presents as vertical streaking of the protein stain.

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 2). Regarding molecular function, 53% of all identified proteins in PCa patient's urine have binding function, followed by transport and catalytic activity. Approximately half of the identified proteins are involved in regulation of biological processes, developmental processes, and cellular processes. Half of the proteins were enzymes and transporters and the rest belonged to transcription regulators, growth factors, cytokines, and other protein types. The majority of the identified proteins (71%) were secreted and had extracellular space location.

Comparison of the identified 45 proteins with proteins identified in normal urine by 2D PAGE/MS [7] showed that 34 proteins are found in the normal urine as well, while 11 have not been reported. The functional characteristics of the 11 proteins are given in Table 3.

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 (p = 4.17 x [10.sup.-4] - 3.21 x [10.sup.-2]), while 2 proteins (antithrombin-III and thymidine phosphorylase) are associated with organism injury and abnormalities (p = 8.35 x [10.sup.-4] - 4.54 x [10.sup.-2]). In the molecular and cellular function classification, we have found 3 proteins (E3 ubiquitin-protein ligase rififylin, tumor protein D52, and thymidine phosphorylase) associated with cellular growth and proliferation (p = 8.35 x [10.sup.-4] - 3.41 x [10.sup.-2]). The 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 (p = [10.sup.-30]). The network encompassed 10 from 11 proteins closely connected through four major nodes: ubiquitin C (UBC), tumor necrosis factor (TNF), transforming growth factor beta 1 (TGFB1), and interferon gamma (IFNG) (Figure 3).

4. Discussion

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: kidney secretory and structural proteins: uromodulin (formerly Tamm-Horsfall protein), vesicular integral-membrane protein (VIP), gelsolin, and actin cytoplasmic 1 and 2, basement membrane-specific heparin sulphate proteoglycan core protein; serum constitutional and transport proteins: albumin, vitamin D binding protein, leucine-rich [alpha]2 glycoprotein, transthyretin, plasma retinol-binding protein, haptoglobin B chain, ubiquitin, [alpha]-1 acid glycoprotein 1, and tumor protein D52; coagulation factors: fibrinogen a chain, kininogen-1, fibrinogen [beta] chain, fibrinogen [gamma] chain, CD59 glycoprotein, and antithrombin-III; complement fractions: mannan-binding lectin serine protease, immunoglobulins with heavy and light chains, secreted and transmembrane protein 1, protein AMBP, and transmembrane and immunoglobulin domain-containing protein 1; proteases and inhibitors: [alpha]1 antitrypsin and [alpha]1 antitrypsin fragment, [alpha]1 antichymotrypsin, and inter-[alpha] trypsin inhibitor heavy chain H4; enzymes: prostaglandin-H2-isomerase, alpha-amylase 1, thymidine phosphorylase, endonuclease domain-containing 1 protein, quinone oxidoreductase-like protein 1, E3 ubiquitin-protein ligase rififylin, and Ras-related protein Rab-36; metal binding proteins: serum transferrin, aHS-glycoprotein, and zinc-[alpha]-2-glycoprotein; transcriptional regulators: U3 small nucleolar RNA-associated protein 18 and interleukin enhancer-binding factor 2; and lipoprotein metabolism: apolipoprotein.

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 [4, 7]. Among diverse biological functions in which the identified set of urinary proteins is involved, signal transducer activity was unexpected as this function was not present in the observed proportion in proteins from the plasma proteome [4]. On the other hand, it was expected that the PCa patient's urine contains substantial amount of immune response proteins, proteins involved in response to stimuli, signalling and adhesion molecules, as cancers may be considered a state of constant inflammation of the organism [12].

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 [7]. Of them, thymidine phosphorylase, E3 ubiquitin-protein ligase, and tumor protein D52 are involved in processes of angiogenesis, tumor growth, or metastasis and the rest are proteins with different physiological functions and no reported involvement in cancer development or progression. IPA analysis of the 11 proteins also pointed out these 3 proteins as significantly associated with cellular growth and proliferation.

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 [13]. E3 ubiquitin-protein ligase (RFFL) has been implicated in regulation of p53 tumor suppressor stability [14,15]. Major physiological function of RFFL is promotion of p53 destruction and, as a result of it, it is frequently found overexpressed in a variety of human cancers [16]. The tumor protein D52 (TPD52) is frequently and strongly upregulated in many human cancer types and this trend is observed in various urogenital cancers among which is prostate cancer as well 17].

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 [18, 19]. Human neurodegenerative, infectious diseases and tumorigenesis have been associated with alterations in ubiquitin pathways [20]. Ubiquitin has been detected in a variety of normal and cancerous tissues. Variations in the distribution and intensity of ubiquitin in benign and malignant conditions of the human prostate have been observed, leading to conclusion that it has implication in tumor pathogenesis of prostate cancer [21]. Our results are in concordance with this finding since seven out of ten proteins in the network, found in PCa, are directly connected to UBC.

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 [22]. The biological activity of these cytokines is conventionally associated with antitumor mechanisms during cell-mediated adaptive immune response. Despite this, a number of reports suggest that their role in carcinogenesis is complex, having both tumor suppressor and oncogenic activities [23-25].

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.

5. Conclusions

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.

Abbreviations

1D:              One-dimensional
2D:              Two-dimensional
2D PAGE:         Two-dimensional polyacrylamide gel
                 electrophoresis
ACN:             Acetonitrile
BSA:             Bovine serum albumin
CHAPS:           3-[(3-Cholamidopropyl)
                 dimethylammonio]-1-propanesulfonate
Da:              Dalton
DTT:             Dithiothreitol
GO:              Gene Ontology
IAA:             2-Iodoacetamide
IPA:             Ingenuity Pathways Analysis
LC:              Liquid chromatography
MALDI-TOF-TOF:   Matrix-assisted laser
                 desorption/ionization-time of
                 flight-time of flight
MS:              Mass spectrometry
PSA:             Prostate-specific antigen
SDS:             Sodium dodecyl sulfate
SDS-PAGE:        Sodium dodecyl sulfate
                 polyacrylamide gel electrophoresis
TFA:             Trifluoroacetic acid
Tris:            Tris(hydroxymethyl)aminomethane
UniProtKB:       UniProt Knowledgebase.


http://dx.doi.org/10.1155/2014/594761

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was supported by the funds for Science of the Macedonian Academy of Sciences and Arts (Grant no. 09114/1, 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.

References

[1] S. Decramer, A. G. de Peredo, B. Breuil et al., "Urine in clinical proteomics," Molecular and Cellular Proteomics, vol. 7, no. 10, pp. 1850-1862, 2008.

[2] G. L. Hortin and D. Sviridov, "Diagnostic potential for urinary proteomics," Pharmacogenomics, vol. 8, no. 3, pp. 237-255,2007

[3] N. G. Anderson, N. L. Anderson, and S. L. Tollaksen, "Proteins in human urine. I. Concentration and analysis by two-dimensional electrophoresis," Clinical Chemistry, vol. 25, no. 7, pp. 1199-1210, 1979.

[4] J. Adachi, C. Kumar, Y. Zhang, J. V. Olsen, and M. Mann, "The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins," Genome Biology, vol. 7, no. 9, article R80, 2006.

[5] Q. R. Li, K. X. Fan, R. X. Li et al., "A comprehensive and nonprefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine," Rapid Communications in Mass Spectrometry, vol. 24, no. 6, pp. 823832, 2010.

[6] A. Marimuthu, R. N. O'Meally, R. Chaerkady et al., "A comprehensive map of the human urinary proteome," Journal of Proteome Research, vol. 10, no. 6, pp. 2734-2743, 2011.

[7] G. Candiano, L. Santucci, A. Petretto et al., "2D-electrophoresis and the urine proteome map: where do we stand?" Journal of Proteomics, vol. 73, no. 5, pp. 829-844, 2010.

[8] S. Magdeldin, S. Enany, Y. Yoshida et al., "Basics and recent advances of two dimensional-polyacrylamide gel electrophoresis," Clinical Proteomics, vol. 11, no. 1, article 16, 2014.

[9] E. R. Suarez, J. Siwy, P. Zurbig, and H. Mischak, "Urine as a source for clinical proteome analysis: from discovery to clinical application," Biochimica et Biophysica Acta, vol. 1844, no. 5, pp. 884-898, 2014.

[10] A. Albalat, H. Mischak, and W. Mullen, "Clinical application of urinary proteomics/peptidomics," Expert Review of Proteomics, vol. 8, no. 5, pp. 615-629, 2011.

[11] M. M. Bradford, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding," Analytical Biochemistry, vol. 72, no. 1-2, pp. 248-254,1976.

[12] C. Gabay and I. Kushner, "Acute-phase proteins and other systemic responses to inflammation," The New England Journal of Medicine, vol. 340, no. 6, pp. 448-454, 1999.

[13] S. Akiyama, T. Furukawa, T. Sumizawa et al., "The role of thymidine phosphorylase, an angiogenic enzyme, in tumor progression," Cancer Science, vol. 95, no. 11, pp. 851-857, 2004.

[14] R. Honda, H. Tanaka, and H. Yasuda, "Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53," The FEBS Letters, vol. 420, no. 1, pp. 25-27,1997

[15] J. D. Oliner, J. A. Pietenpol, S. Thiagalingam, J. Gyuris, K. W. Kinzler, and B. Vogelstein, "Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53," Nature, vol. 362, no. 6423, pp. 857-860, 1993.

[16] E. Meulmeester, Y. Pereg, Y. Shiloh, and A. G. Jochemsen, "ATM-mediated phosphorylations inhibit Mdmx/Mdm2 stabilization by HAUSP in favor of p53 activation," Cell Cycle, vol. 4, no. 9, pp. 1166-1170, 2005.

[17] P. Tennstedt, C. Bolch, G. Strobel et al., "Patterns of TPD52 overexpression in multiple human solid tumor types analyzed by quantitative PCR," International Journal of Oncology, vol. 44, no. 2, pp. 609-615, 2014.

[18] M. J. Clague and S. Urbe, "Ubiquitin: same molecule, different degradation pathways," Cell, vol. 143, no. 5, pp. 682-685, 2010.

[19] D. Mukhopadhyay and H. Riezman, "Proteasome-independent functions of ubiquitin in endocytosis and signaling," Science, vol. 315, no. 5809, pp. 201-205, 2007

[20] B. M. Kessler, "Ubiquitin-omics reveals novel networks and associations with human disease," Current Opinion in Chemical Biology, vol. 17, no. 1, pp. 59-65, 2013.

[21] Z. M. Bataineh and O. Habbal, "Immunoreactivity of ubiqitin in human prostate gland," Neuroendocrinology Letters, vol. 27, no. 4, pp. 517-522, 2006.

[22] C. A. Dinarello, "Proinflammatory cytokines," Chest, vol. 118, no. 2, pp. 503-508, 2000.

[23] L. Bertazza and S. Mocellin, "The dual role of tumor necrosis factor (TNF) in cancer biology," Current Medicinal Chemistry, vol. 17, no. 29, pp. 3337-3352, 2010.

[24] M. P. de Caestecker, E. Piek, and A. B. Roberts, "Role of transforming growth factor-signaling in cancer," Journal of the National Cancer Institute, vol. 92, no. 17, pp. 1388-1402, 2000.

[25] M. R. Zaidi and G. Merlino, "The two faces of interferon-y in cancer," Clinical Cancer Research, vol. 17, no. 19, pp. 6118-6124, 2011.

Sanja Kiprijanovska, (1) Sotir Stavridis, (2) Oliver Stankov, (2) Selim Komina, (3) Gordana Petrusevska, (3) Momir Polenakovic, (1) and Katarina Davalieva (1)

(1) Research Centre for Genetic Engineering and Biotechnology "Georgi D Efremov", Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000 Skopje, Macedonia

(2) University Clinic for Urology, University Clinical Centre "Mother Theresa", 1000 Skopje, Macedonia

(3) Institute of Pathology, Medical Faculty, University of "St. Cyril and Methodius", 1000 Skopje, Macedonia

Correspondence should be addressed to Katarina Davalieva; katarina@manu.edu.mk

Received 26 June 2014; Revised 1 August 2014; Accepted 2 August 2014; Published 20 August 2014

Academic Editor: Petra Zurbig

TABLE 1: Clinical information of patients used to generate urine
samples included in the study together with their PSA levels,
histology grading and tumor stage.

Sample   Patient   Age   Diagnosis   Tumor stage     Gleason
number   number                                       score

1         PC-22    74       PCA      pT2c pN0 pM0   7 (3 + 4)
2         PC-24    72       PCA      pT2c pN0 pM0   7 (3 + 4)
3         PC-27    78       PCA      pT2c pN0 pM0   5 (2 + 3)
4         PC-28    60       PCA      pT2c pN0 pM0   7 (3 + 4)
5         PC-31    65       PCA      pT3b pN1 pM0   7 (3 + 4)
6         PC-35    74       PCA      pT2c pN0 pM0   7 (3 + 4)
7         PC-39    64       PCA      pT2b pN1 pM0   9 (4 + 5)
8         PC-48    65       PCA      pT2c pN0 pM0   7 (3 + 4)

Sample   Preoperative
number   PSA (ng/mL)

1            8.3
2            5.5
3            4.6
4            5.2
5            8.3
6            22.6
7            50.0
8            32.2

Table 2: List of the all protein spots from the PCa urinary
proteome identified by MS.

Spot number     Protein name            SwissProt       Mw (kDa)
                                        accession
                                         number

1               Uromodulin OS = Homo   UROM_HUMAN        72.64
                sapiens GN = UMOD PE
                = 1 SV = 1

2-14,19-21      Serotransferrin OS =   TRFE_HUMAN        79.29
                Homo sapiens GN = TF
                PE = 1 SV = 3

15-18           Alpha-1B-              A1BG_TUMAN        54.79
                glycoprotein OS =
                Homo sapiens GN =
                A1BG PE = 1 SV = 4

22-24           Serum albumin OS =     ALBU_HUMAN        71.31
                Homo sapiens GN =
                ALB PE = 1 SV = 2

25-28           Alpha-1-               AACT_HUMAN        47.79
                antichymotrypsin OS
                = Homo sapiens GN =
                SERPINA3 PE = 1 SV =
                2

                Kininogen-1 OS =       KNG1_HUMAN        72.99
                Homo sapiens GN =
                KNG1 PE = 1 SV = 2

29-33           Ig alpha-1 chain C     IGHA1_HUMAN       38.49
                region OS = Homo
                sapiens GN = IGHA1
                PE = 1 SV = 2

34-37           Alpha-2-HS-            FETUA_HUMAN       40.00
                glycoprotein OS =
                Homo sapiens GN =
                AHSG PE = 1 SV = 1

38-43           Alpha-1-antitrypsin    A1AT_HUMAN        46.89
                OS = Homo sapiens GN
                = SERPINA1 PE = 1 SV
                = 3

44-46           Vitamin D-binding      VTDB_HUMAN        54.52
                protein OS = Homo
                sapiens GN = GC PE =
                1 SV = 1

47              Antithrombin-III OS    ANT3_HUMAN        53.02
                = Homo sapiens GN =
                SERPINC1 PE = 1 SV =
                1

48              Alpha-amylase 1 OS =   AMY1_HUMAN        58.41
                Homo sapiens GN =
                AMY1A PE = 1 SV = 2

49, 67-69       Fibrinogen beta        FIBB_HUMAN        56.57
                chain OS = Homo
                sapiens GN = FGB PE
                = 1 SV = 2 U3 small
                nucleolar
                RNA-associated
                protein

50-51           18 homolog OS = Homo   UTP18_HUMAN       62.42
                sapiens GN = UTP18
                PE = 1 SV = 3

52-54           Leucine-rich alpha-    A2GL_HUMAN        38.38
                2-glycoprotein OS =
                Homo sapiens GN =
                LRG1 PE = 1 SV = 2

55              Fibrinogen gamma       FIBG_HUMAN        52.10
                chain OS = Homo
                sapiens GN = FGG PE
                = 1 SV = 3

56              Thymidine              TYPH_HUMAN        50.32
                phosphorylase OS =
                Homo sapiens GN =
                TYMP PE = 1 SV = 2

57-62,118-120   Haptoglobin OS =        HPT_HUMAN        45.86
                Homo sapiens GN = HP
                PE = 1 SV = 1

63              Gelsolin OS = Homo     GELS_HUMAN    86.04 (52.48)
                sapiens GN = GSN PE
                = 1 SV = 1
                (fragment)

64              Apolipoprotein A-IV    APOA4_HUMAN       45.37
                OS = Homo sapiens GN
                = APOA4 PE = 1 SV =
                3

65-66           Actin, cytoplasmic 1   ACTB_HUMAN        42.05
                OS = Homo sapiens GN
                = ACTB PE = 1 SV = 1

                Actin, cytoplasmic 2   ACTG_HUMAN        42.05
                OS = Homo sapiens GN
                = ACTG1 PE = 1 SV =
                1

70-71           Fibrinogen alpha       FIBA_HUMAN      95.65 (50)
                chain OS = Homo
                sapiens GN = FGA PE
                = 1 SV = 2
                (fragment)

72-75           Zinc-alpha-2-          ZA2G_HUMAN        34.46
                glycoprotein OS =
                Homo sapiens GN =
                AZGP1 PE = 1 SV = 2
                Endonuclease domain-
                containing 1 protein

76              OS = Homo sapiens GN   ENDD1_HUMAN       55.72
                = ENDOD1 PE = 1 SV =
                2

77              E3 ubiquitin-          RFFL_HUMAN        41.74
                protein ligase
                rififylin OS = Homo
                sapiens GN = RFFL PE
                = 1 SV = 1 Inter-
                alpha-trypsin
                inhibitor heavy
                chain

78-79           H4 OS = Homo sapiens   ITIH4_HUMAN    103.52 (45)
                GN = ITIH4 PE = 1 SV
                = 4 (fragment)

80              Quinone                QORL1_HUMAN       39.07
                oxidoreductase-like
                protein 1 OS = Homo
                sapiens GN = CRYZL1
                PE = 1 SV = 2

81              Interleukin            ILF2_HUMAN        43.26
                enhancer-binding
                factor 2 OS = Homo
                sapiens GN = ILF2 PE
                = 1 SV = 2 Vesicular
                integral-membrane
                protein

82              VIP36 OS = Homo        LMAN2_HUMAN       40.54
                sapiens GN = LMAN2
                PE = 1 SV = 1

83              Transmembrane and      TMIG1_HUMAN       29.62
                immunoglobulin
                domain-containing
                protein 1 OS = Homo
                sapiens GN = TMIGD1
                PE = 2 SV = 1

84-88           Protein AMBP OS =      AMBP_HUMAN        39.87
                Homo sapiens GN =
                AMBP PE = 1 SV = 1

89-92           Prostaglandin-H2 D-    PTGDS_HUMAN       21.24
                isomerase OS = Homo
                sapiens GN = PTGDS
                PE = 1 SV = 1

93-97           Ig kappa chain C       IGKC_HUMAN        28.82
                region OS = Homo
                sapiens GN = IGKC PE
                = 1 SV = 1
                (immunoglobulin
                kappa light chain
                VLJ region)

98-102          Apolipoprotein A-I     APOA1_HUMAN       30.75
                OS = Homo sapiens GN
                = APOA1 PE = 1 SV =
                1

103             Basement               PGBM_HUMAN    468.83 (20.65)
                membrane-specific
                heparan sulphate
                proteoglycan core
                protein (perlecan)
                chain A,
                laminin-G-like
                domain 3 from human
                perlecan

104-105         Ras-related protein    RAB36_HUMAN       36.81
                Rab-36 OS = Homo
                sapiens GN = RAB36
                PE = 2 SV = 2

106             Tumor protein D52 OS   TPD52_HUMAN       24.35
                = Homo sapiens GN =
                TPD52 PE = 1 SV = 2

107             Alpha-1-acid           AlAG1_HUMAN       23.72
                glycoprotein 1 OS =
                Homo sapiens GN =
                ORM1 PE = 1 SV = 1

108-111         Retinol-binding        RET4_HUMAN        23.34
                protein 4 OS = Homo
                sapiens GN = RBP4 PE
                = 1 SV = 3

112-115         CD59 glycoprotein OS   CD59_HUMAN        14.79
                = Homo sapiens GN =
                CD59 PE = 1 SV = 1
                Mannan-binding
                lectin serine
                protease 2

116             OS = Homo sapiens GN   MASP2_HUMAN   77.19 (19.53)
                = MASP2 PE = 1 SV =
                4 (chainA, human
                MBL-associated
                protein 19)

117.121         Secreted and           SCTM1_HUMAN       27.30
                transmembrane
                protein 1 OS = Homo
                sapiens GN = SECTM1
                PE = 1 SV

122             Transthyretin OS =     TTHY_HUMAN        15.99
                Homo sapiens GN =
                TTR PE = 1 SV = 1

123-125         Uncharacterized        K2012_HUMAN     65.27 (50)
                protein KIAA2012 OS
                = Homo sapiens GN =
                KIAA2012 PE = 2 SV =
                1 (fragment)

Spot number     Protein name               pI        Mascot    P value
                                                     protein
                                                      score

1               Uromodulin OS = Homo      5.05         158     3.2E-12
                sapiens GN = UMOD PE
                = 1 SV = 1

2-14,19-21      Serotransferrin OS =       6.8         243     1.0E-20
                Homo sapiens GN = TF
                PE = 1 SV = 3

15-18           Alpha-1B-                 5.56         93      9.3E-06
                glycoprotein OS =
                Homo sapiens GN =
                A1BG PE = 1 SV = 4

22-24           Serum albumin OS =        5.92         246     5.1E-21
                Homo sapiens GN =
                ALB PE = 1 SV = 2

25-28           Alpha-1-                  5.33         82      3.4E-05
                antichymotrypsin OS
                = Homo sapiens GN =
                SERPINA3 PE = 1 SV =
                2

                Kininogen-1 OS =          6.34         82      3.6E-05
                Homo sapiens GN =
                KNG1 PE = 1 SV = 2

29-33           Ig alpha-1 chain C        6.08         62      1.3E-02
                region OS = Homo
                sapiens GN = IGHA1
                PE = 1 SV = 2

34-37           Alpha-2-HS-               5.43         95      6.0E-06
                glycoprotein OS =
                Homo sapiens GN =
                AHSG PE = 1 SV = 1

38-43           Alpha-1-antitrypsin       5.37         194     8.1E-16
                OS = Homo sapiens GN
                = SERPINA1 PE = 1 SV
                = 3

44-46           Vitamin D-binding         5.40         135     6.4E-10
                protein OS = Homo
                sapiens GN = GC PE =
                1 SV = 1

47              Antithrombin-III OS       6.32         85      6.1E-05
                = Homo sapiens GN =
                SERPINC1 PE = 1 SV =
                1

48              Alpha-amylase 1 OS =      6.47         82      1.2E-03
                Homo sapiens GN =
                AMY1A PE = 1 SV = 2

49, 67-69       Fibrinogen beta           8.54         62      1.3E-03
                chain OS = Homo
                sapiens GN = FGB PE
                = 1 SV = 2 U3 small
                nucleolar
                RNA-associated
                protein

50-51           18 homolog OS = Homo      8.93         75      6.6E-04
                sapiens GN = UTP18
                PE = 1 SV = 3

52-54           Leucine-rich alpha-       6.45         66      5.3E-03
                2-glycoprotein OS =
                Homo sapiens GN =
                LRG1 PE = 1 SV = 2

55              Fibrinogen gamma          5.37         164     8.1E-13
                chain OS = Homo
                sapiens GN = FGG PE
                = 1 SV = 3

56              Thymidine                 5.36         64      7.4E-03
                phosphorylase OS =
                Homo sapiens GN =
                TYMP PE = 1 SV = 2

57-62,118-120   Haptoglobin OS =          6.13         126     5.1E-09
                Homo sapiens GN = HP
                PE = 1 SV = 1

63              Gelsolin OS = Homo     5.90 (5.34)     80      2.0E-03
                sapiens GN = GSN PE
                = 1 SV = 1
                (fragment)

64              Apolipoprotein A-IV       5.28         82      1.2E-04
                OS = Homo sapiens GN
                = APOA4 PE = 1 SV =
                3

65-66           Actin, cytoplasmic 1      5.29         64      7.0E-03
                OS = Homo sapiens GN
                = ACTB PE = 1 SV = 1

                Actin, cytoplasmic 2      5.29         64      7.0E-03
                OS = Homo sapiens GN
                = ACTG1 PE = 1 SV =
                1

70-71           Fibrinogen alpha       5.7 (4.65)      91      1.6E-05
                chain OS = Homo
                sapiens GN = FGA PE
                = 1 SV = 2
                (fragment)

72-75           Zinc-alpha-2-             5.71         104     8.1E-07
                glycoprotein OS =
                Homo sapiens GN =
                AZGP1 PE = 1 SV = 2
                Endonuclease domain-
                containing 1 protein

76              OS = Homo sapiens GN      5.55         104     8.1E-07
                = ENDOD1 PE = 1 SV =
                2

77              E3 ubiquitin-             5.33         59      2.7E-02
                protein ligase
                rififylin OS = Homo
                sapiens GN = RFFL PE
                = 1 SV = 1 Inter-
                alpha-trypsin
                inhibitor heavy
                chain

78-79           H4 OS = Homo sapiens   6.51 (5.15)     88      2.9E-05
                GN = ITIH4 PE = 1 SV
                = 4 (fragment)

80              Quinone                   5.49         66      4.9E-02
                oxidoreductase-like
                protein 1 OS = Homo
                sapiens GN = CRYZL1
                PE = 1 SV = 2

81              Interleukin               5.19         62      3.0E-02
                enhancer-binding
                factor 2 OS = Homo
                sapiens GN = ILF2 PE
                = 1 SV = 2 Vesicular
                integral-membrane
                protein

82              VIP36 OS = Homo           6.46         80      1.9E-04
                sapiens GN = LMAN2
                PE = 1 SV = 1

83              Transmembrane and         8.07         62      2.0E-02
                immunoglobulin
                domain-containing
                protein 1 OS = Homo
                sapiens GN = TMIGD1
                PE = 2 SV = 1

84-88           Protein AMBP OS =         5.95         130     2.0E-09
                Homo sapiens GN =
                AMBP PE = 1 SV = 1

89-92           Prostaglandin-H2 D-       7.66         64      7.7E-03
                isomerase OS = Homo
                sapiens GN = PTGDS
                PE = 1 SV = 1

93-97           Ig kappa chain C          6.73         77      5.8E-03
                region OS = Homo
                sapiens GN = IGKC PE
                = 1 SV = 1
                (immunoglobulin
                kappa light chain
                VLJ region)

98-102          Apolipoprotein A-I        5.56         116     5.1E-08
                OS = Homo sapiens GN
                = APOA1 PE = 1 SV =
                1

103             Basement               6.06 (5.47)     204     1.1E-15
                membrane-specific
                heparan sulphate
                proteoglycan core
                protein (perlecan)
                chain A,
                laminin-G-like
                domain 3 from human
                perlecan

104-105         Ras-related protein       8.05         63      9.7E-03
                Rab-36 OS = Homo
                sapiens GN = RAB36
                PE = 2 SV = 2

106             Tumor protein D52 OS      4.79         63      1.1E-02
                = Homo sapiens GN =
                TPD52 PE = 1 SV = 2

107             Alpha-1-acid              4.93         64      8.8E-03
                glycoprotein 1 OS =
                Homo sapiens GN =
                ORM1 PE = 1 SV = 1

108-111         Retinol-binding           5.76         109     2.6E-07
                protein 4 OS = Homo
                sapiens GN = RBP4 PE
                = 1 SV = 3

112-115         CD59 glycoprotein OS      6.02         61      1.6E-02
                = Homo sapiens GN =
                CD59 PE = 1 SV = 1
                Mannan-binding
                lectin serine
                protease 2

116             OS = Homo sapiens GN   5.44 (5.75)     127     5.5E-08
                = MASP2 PE = 1 SV =
                4 (chainA, human
                MBL-associated
                protein 19)

117.121         Secreted and              7.00         75      6.1E-04
                transmembrane
                protein 1 OS = Homo
                sapiens GN = SECTM1
                PE = 1 SV

122             Transthyretin OS =        5.52         160     2.0E-12
                Homo sapiens GN =
                TTR PE = 1 SV = 1

123-125         Uncharacterized        6.09 (6.00)     75      5.8E-04
                protein KIAA2012 OS
                = Homo sapiens GN =
                KIAA2012 PE = 2 SV =
                1 (fragment)

Spot number     Protein name            Number of      % of
                                       the matched   sequence
                                        peptides     coverage

1               Uromodulin OS = Homo       18           22
                sapiens GN = UMOD PE
                = 1 SV = 1

2-14,19-21      Serotransferrin OS =       22           40
                Homo sapiens GN = TF
                PE = 1 SV = 3

15-18           Alpha-1B-                  12           35
                glycoprotein OS =
                Homo sapiens GN =
                A1BG PE = 1 SV = 4

22-24           Serum albumin OS =         26           48
                Homo sapiens GN =
                ALB PE = 1 SV = 2

25-28           Alpha-1-                   12           31
                antichymotrypsin OS
                = Homo sapiens GN =
                SERPINA3 PE = 1 SV =
                2

                Kininogen-1 OS =           13           19
                Homo sapiens GN =
                KNG1 PE = 1 SV = 2

29-33           Ig alpha-1 chain C          5           18
                region OS = Homo
                sapiens GN = IGHA1
                PE = 1 SV = 2

34-37           Alpha-2-HS-                15           43
                glycoprotein OS =
                Homo sapiens GN =
                AHSG PE = 1 SV = 1

38-43           Alpha-1-antitrypsin        23           53
                OS = Homo sapiens GN
                = SERPINA1 PE = 1 SV
                = 3

44-46           Vitamin D-binding          12           50
                protein OS = Homo
                sapiens GN = GC PE =
                1 SV = 1

47              Antithrombin-III OS         8           21
                = Homo sapiens GN =
                SERPINC1 PE = 1 SV =
                1

48              Alpha-amylase 1 OS =        6           15
                Homo sapiens GN =
                AMY1A PE = 1 SV = 2

49, 67-69       Fibrinogen beta             8           19
                chain OS = Homo
                sapiens GN = FGB PE
                = 1 SV = 2 U3 small
                nucleolar
                RNA-associated
                protein

50-51           18 homolog OS = Homo        6           13
                sapiens GN = UTP18
                PE = 1 SV = 3

52-54           Leucine-rich alpha-         4           18
                2-glycoprotein OS =
                Homo sapiens GN =
                LRG1 PE = 1 SV = 2

55              Fibrinogen gamma           17           47
                chain OS = Homo
                sapiens GN = FGG PE
                = 1 SV = 3

56              Thymidine                   5           25
                phosphorylase OS =
                Homo sapiens GN =
                TYMP PE = 1 SV = 2

57-62,118-120   Haptoglobin OS =           12           27
                Homo sapiens GN = HP
                PE = 1 SV = 1

63              Gelsolin OS = Homo          7           16
                sapiens GN = GSN PE
                = 1 SV = 1
                (fragment)

64              Apolipoprotein A-IV         8           22
                OS = Homo sapiens GN
                = APOA4 PE = 1 SV =
                3

65-66           Actin, cytoplasmic 1        7           24
                OS = Homo sapiens GN
                = ACTB PE = 1 SV = 1

                Actin, cytoplasmic 2        7           24
                OS = Homo sapiens GN
                = ACTG1 PE = 1 SV =
                1

70-71           Fibrinogen alpha           10           15
                chain OS = Homo
                sapiens GN = FGA PE
                = 1 SV = 2
                (fragment)

72-75           Zinc-alpha-2-               8           29
                glycoprotein OS =
                Homo sapiens GN =
                AZGP1 PE = 1 SV = 2
                Endonuclease domain-
                containing 1 protein

76              OS = Homo sapiens GN        9           20
                = ENDOD1 PE = 1 SV =
                2

77              E3 ubiquitin-               5           26
                protein ligase
                rififylin OS = Homo
                sapiens GN = RFFL PE
                = 1 SV = 1 Inter-
                alpha-trypsin
                inhibitor heavy
                chain

78-79           H4 OS = Homo sapiens       11           13
                GN = ITIH4 PE = 1 SV
                = 4 (fragment)

80              Quinone                     4           12
                oxidoreductase-like
                protein 1 OS = Homo
                sapiens GN = CRYZL1
                PE = 1 SV = 2

81              Interleukin                 5           19
                enhancer-binding
                factor 2 OS = Homo
                sapiens GN = ILF2 PE
                = 1 SV = 2 Vesicular
                integral-membrane
                protein

82              VIP36 OS = Homo             7           21
                sapiens GN = LMAN2
                PE = 1 SV = 1

83              Transmembrane and           6           20
                immunoglobulin
                domain-containing
                protein 1 OS = Homo
                sapiens GN = TMIGD1
                PE = 2 SV = 1

84-88           Protein AMBP OS =          15           37
                Homo sapiens GN =
                AMBP PE = 1 SV = 1

89-92           Prostaglandin-H2 D-         8           33
                isomerase OS = Homo
                sapiens GN = PTGDS
                PE = 1 SV = 1

93-97           Ig kappa chain C            7           29
                region OS = Homo
                sapiens GN = IGKC PE
                = 1 SV = 1
                (immunoglobulin
                kappa light chain
                VLJ region)

98-102          Apolipoprotein A-I         16           46
                OS = Homo sapiens GN
                = APOA1 PE = 1 SV =
                1

103             Basement                   15           88
                membrane-specific
                heparan sulphate
                proteoglycan core
                protein (perlecan)
                chain A,
                laminin-G-like
                domain 3 from human
                perlecan

104-105         Ras-related protein         4           23
                Rab-36 OS = Homo
                sapiens GN = RAB36
                PE = 2 SV = 2

106             Tumor protein D52 OS        4           22
                = Homo sapiens GN =
                TPD52 PE = 1 SV = 2

107             Alpha-1-acid                4           21
                glycoprotein 1 OS =
                Homo sapiens GN =
                ORM1 PE = 1 SV = 1

108-111         Retinol-binding             9           49
                protein 4 OS = Homo
                sapiens GN = RBP4 PE
                = 1 SV = 3

112-115         CD59 glycoprotein OS        5           28
                = Homo sapiens GN =
                CD59 PE = 1 SV = 1
                Mannan-binding
                lectin serine
                protease 2

116             OS = Homo sapiens GN        8           37
                = MASP2 PE = 1 SV =
                4 (chainA, human
                MBL-associated
                protein 19)

117.121         Secreted and                6           22
                transmembrane
                protein 1 OS = Homo
                sapiens GN = SECTM1
                PE = 1 SV

122             Transthyretin OS =         10           73
                Homo sapiens GN =
                TTR PE = 1 SV = 1

123-125         Uncharacterized            13           22
                protein KIAA2012 OS
                = Homo sapiens GN =
                KIAA2012 PE = 2 SV =
                1 (fragment)

TABLE 3: Functional characterization of the 11 identified proteins
found only in PCa.

SwissProt     Protein name             Gene      Subcellular
accession                              name       location
number

ANT3.HUMAN    Antithrombin-III OS    SERPINC1   Extracellular
              = Homo sapiens GN =                   space
              SERPINC1 PE = 1 SV =
              1

AMY1.HUMAN    Alpha-amylase 1 OS =     AMY1     Extracellular
              Homo sapiens GN =                     space
              AMY1A PE = 1 SV = 2

UTP18.HUMAN   U3 small nucleolar      UTP18        Nucleus
              RNA-associated
              protein 18 homolog
              OS = Homo sapiens GN
              = UTP18 PE = 1 SV =
              3

TYPH.HUMAN    Thymidine                TYMP     Extracellular
              phosphorylase OS =                    space
              Homo sapiens GN =
              TYMP PE = 1 SV = 2

ENDD1.HUMAN   Endonuclease            ENDOD1    Extracellular
              domain-containing 1                   space
              protein OS = Homo
              sapiens GN = ENDOD1
              PE = 1 SV = 2

RFFL_HUMAN    E3 ubiquitin-protein     RFFE       Cytoplasm
              ligase rififylin OS
              = Homo sapiens GN =
              RFFL PE = 1 SV = 1

QORFl_HUMAN   Quinone                 CRYZE1      Cytoplasm
              oxidoreductase-like
              protein 1 OS = Homo
              sapiens GN = CRYZE1
              PE = 1 SV = 2

IFF2.HUMAN    Interleukin              IEF2        Nucleus
              enhancer-binding
              factor 2 OS = Homo
              sapiens GN = ILF2 PE
              = 1 SV = 2

TMIG1_HUMAN   Transmembrane and       TMIGD1        Other
              immunoglobulin
              domain-containing
              protein 1 OS = Homo
              sapiens GN = TMIGD1
              PE = 2 SV = 1

RAB36JTUMAN   Ras-related protein     RAB36       Cytoplasm
              Rab-36 OS = Homo
              sapiens GN = RAB36
              PE = 2 SV = 2

TPD52.HUMAN   Tumor protein D52 OS    TPD52       Cytoplasm
              = Homo sapiens GN =
              TPD52 PE = 1 SV = 2

SwissProt     Type of protein   Biological function
accession
number

ANT3.HUMAN        Enzyme        Serine protease
                                inhibitor in plasma
                                that regulates the
                                blood coagulation
                                cascade

AMY1.HUMAN        Enzyme        Carbohydrate
                                metabolic process

UTP18.HUMAN        Other        It is involved in
                                nucleolar processing
                                of pre-18S ribosomal
                                RNA

TYPH.HUMAN     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       Enzyme        It may act as a
                                DNase and a Rnase

RFFL_HUMAN        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

QORFl_HUMAN       Enzyme        Quinone metabolic
                                process

IFF2.HUMAN     Transcription    It functions
                 regulator      predominantly as a
                                heterodimeric
                                complex with IEF3.
                                This complex may
                                regulate
                                transcription of the
                                IE2 gene during T-
                                cell activation

TMIG1_HUMAN        Other        Integral component
                                of membrane

RAB36JTUMAN       Enzyme        Protein transport.
                                It is probably
                                involved in
                                vesicular traffic

TPD52.HUMAN        Other        B cell
                                differentiation and
                                anatomical structure
                                morphogenesis and
                                secretion

Figure 2: 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.

Molecular function

Transport                  17%
Unknown                     4%
Binding                    53%
Catalytic activity         15%
Organization of
 cellular compartment       4%

Biological function

Immune response            15%
Unknown                     3%
Cell adhesion               3%
Developmental processes     4%
Regulation of biological
   processes               22%
Metabolic processes        11%
Cellular processes         15%
Response to stimulus       10%
Signalling                  6%
Cellular component
   Organization or
   Localization            11%

Type of protein

Enzyme                     20%
Other                      44%
Transporter                27%
Cytokine                    5%
Growth factor               2%
Transcription
   Regulator                2%

Subcellular location

Cytoplasm                  18%
Plasma membrane             7%
Nucleus                     4%
Extracellular space        71%

Note: Table made from pie chart.
COPYRIGHT 2014 Hindawi Limited
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Research Article
Author:Kiprijanovska, Sanja; Stavridis, Sotir; Stankov, Oliver; Komina, Selim; Petrusevska, Gordana; Polena
Publication:International Journal of Proteomics
Date:Jan 1, 2014
Words:7579
Previous Article:Characterisation of the proteome of Leptospira interrogans serovar Canicola as a resource for the identification of common serovar immunogenic...
Next Article:A method to determine lysine acetylation stoichiometries.
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters