Analytical and Bioanalytical Chemistry

, Volume 407, Issue 12, pp 3393–3404 | Cite as

Quantitative urinary proteomics using stable isotope labelling by peptide dimethylation in patients with prostate cancer

  • Chunhui Li
  • Tuo Zang
  • Karolina Wrobel
  • Jeffrey T.-J. Huang
  • Ghulam NabiEmail author
Research Paper


Prostate cancer (PCa) is the most commonly diagnosed malignancy in men. The current prevalent diagnosis method, prostate-specific antigen (PSA) screening test, has low sensitivity, specificity and is poor at predicting the grade of disease. Thus, new biomarkers are urgently needed to improve the PCa diagnosis and staging for the management of patients. The aim of this study is to investigate the first voided urinary sample after massage for biomarker discovery for PCa. In this work, untargeted metabolomic profiling of the first voided urinary sample after massage from 28 confirmed prostate cancer patients, 20 benign enlarged prostate patients and 6 healthy volunteers was performed using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Single and multiple peptide protein and cross-linking molecules were identified using PEAKS software. Analytical and diagnostic performance was tested using the Student’s t test, Benjamini Hochberg correction and the receiver operating characteristic (ROC) curves. Using differential display analysis to compare peptides and cross-linking molecules of urinary samples between patients with benign, enlarged prostate and malignant cancer, we identified multiple peptides derived from osteopontin (SPP1) and prothrombin (F2) that are lower in PCa patients than in benign and enlarged prostate. The diagnosis accuracies of SPP1 and F2 peptides are 0.65–0.77 and 0.68–0.72, respectively. In addition to this, there are significant differences between PCa and benign/enlarged prostate patients in pyridinoline (PYD) and deoxypyridinoline (DPD) (p value = 0.001). Differences also, as shown in the excretion of these molecules for different stages of PCa (p value = 0.04) as the level of DPD and DPD/PYD ratio, were high in patients with locally advanced tumours. The study underscores the importance of proteomics analysis, and our results demonstrate that a urinary-based in depth proteomic approach allows the potential identification of dysregulated pathways and diagnostic biomarkers.


Prostate cancer Cancer diagnostic markers Urine Proteomics analysis Expressed prostatic secretions 



We thank Dr. Sheila Sharp at Biomarker and Drug Analysis Core Facility for performing LC-MS/MS analysis. We acknowledge help of Christine Birtles in collection of urinary samples from patients attending prostate access clinic. Also Special thanks to TENOVUS, Scotland (Tayside) for funding this study.


  1. 1.
    Anonymous. Prostate cancer mortality statistics. URL:, 2013
  2. 2.
    Center MM, Jemal A, Lortet-Tieulent J, Ward E, Ferlay J, Brawley O, Bray F (2012) International variation in prostate cancer incidence and mortality rates. Eur Urol 61:1079–1092CrossRefGoogle Scholar
  3. 3.
    Schroder FH (2011) Stratifying risk—the U.S. Preventive Services Task Force and prostate-cancer screening. N Engl J Med 365:1953–1955CrossRefGoogle Scholar
  4. 4.
    Thompson IM, Pauler DK, Goodman PJ, Tangen CM, Lucia MS, Parnes HL, Minasian LM, Ford LG, Lippman SM, Crawford ED, Crowley JJ, Coltman CA Jr (2004) Prevalence of prostate cancer among men with a prostate-specific antigen level < or =4.0 ng per milliliter. N Engl J Med 350:2239–2246CrossRefGoogle Scholar
  5. 5.
    Schroder FH, Hugosson J, Roobol MJ, Tammela TL, Ciatto S, Nelen V, Kwiatkowski M, Lujan M, Lilja H, Zappa M, Denis LJ, Recker F, Paez A, Maattanen L, Bangma CH, Aus G, Carlsson S, Villers A, Rebillard X, van der Kwast T, Kujala PM, Blijenberg BG, Stenman UH, Huber A, Taari K, Hakama M, Moss SM, de Koning HJ, Auvinen A (2012) Prostate-cancer mortality at 11 years of follow-up. N Engl J Med 366:981–990CrossRefGoogle Scholar
  6. 6.
    Calabria F, Chiaravalloti A, Tavolozza M, Ragano-Caracciolo C, Schillaci O (2013) Evaluation of extraprostatic disease in the staging of prostate cancer by F-18 choline PET/CT: can PSA and PSA density help in patient selection? Nucl Med Commun 34(8):733–40CrossRefGoogle Scholar
  7. 7.
    Quanico J, Franck J, Dauly C, Strupat K, Dupuy J, Day R, Salzet M, Fournier I, Wisztorski M (2013) Development of liquid microjunction extraction strategy for improving protein identification from tissue sections. J Proteome 79:200–218, ISSN 1874-3919CrossRefGoogle Scholar
  8. 8.
    Wiese H, Gelis L, Wiese S, Reichenbach C, Jovancevic N, Osterloh M, Meyer HE, Neuhaus EM, Hatt HH, Radziwill G, Warscheid B, Quantitative phosphoproteomics reveals the protein tyrosine kinase Pyk2 as a central effector of olfactory receptor signaling in prostate cancer cells, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Available online 9 September 2014, ISSN 1570-9639, doi: 10.1016/j.bbapap.2014.09.002
  9. 9.
    Grossman HB, Soloway M, Messing E, Katz G, Stein B, Kassabian V, Shen Y (2006) Surveillance for recurrent bladder cancer using a point-of-care proteomic assay. JAMA 295:299–305CrossRefGoogle Scholar
  10. 10.
    Kiprijanovska S, Stavridis S, Stankov O et al (2014) Mapping and identification of the urine proteome of prostate cancer patients by 2D PAGE/MS. Int J Proteomics 2014:594761. doi: 10.1155/2014/594761 CrossRefGoogle Scholar
  11. 11.
    Woodson K, O’Reilly KJ, Hanson JC, Nelson D, Walk EL, Tangrea JA (2008) The usefulness of the detection of GSTP1 methylation in urine as a biomarker in the diagnosis of prostate cancer. J Urol 179(2):508–11CrossRefGoogle Scholar
  12. 12.
    Sreekumar A, Poisson LM, Rajendiran TM, Khan AP, Cao Q, Yu JD, Laxman B, Mehra R, Lonigro RJ, Li Y et al (2013) Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature 2009, 457, 910–914. Int J Mol Sci 14(7):13893–908. doi: 10.3390/ijms140713893 CrossRefGoogle Scholar
  13. 13.
    Cernei N, Heger Z, Gumulec J, Zitka O, Masarik M, Babula P, Eckschlager T, Stiborova M, Kizek R, Adam V (2013) Sarcosine as a potential prostate cancer biomarker—a review. Int J Mol Sci 14(7):13893–908. doi: 10.3390/ijms140713893 CrossRefGoogle Scholar
  14. 14.
    Fujita K, Ewing CM, Chan DY, Mangold LA, Partin AW, Isaacs WB, Pavlovich CP (2009) Endoglin (CD105) as a urinary and serum marker of prostate cancer. Int J Cancer 124:664–669CrossRefGoogle Scholar
  15. 15.
    Kollermann J, Schlomm T, Bang H, Schwall GP, von Eichel-Streiber C, Simon R, Schostak M, Huland H, Berg W, Sauter G, Klocker H, Schrattenholz A (2008) Expression and prognostic relevance of annexin A3 in prostate cancer. Eur Urol 54:1314–1323CrossRefGoogle Scholar
  16. 16.
    Bussemakers MJ, van Bokhoven A, Verhaegh GW, Smit FP, Karthaus HF, Schalken JA, Debruyne FM, Ru N, Isaacs WB (1993) DD3: a new prostate-specific gene, highly overexpressed in prostate cancer. Cancer Res 59:5975–5979Google Scholar
  17. 17.
    Deras IL, Aubin SM, Blase A, Day JR, Koo S, Partin AW, Ellis WJ, Marks LS, Fradet Y, Rittenhouse H, Groskopf J (2008) PCA3: a molecular urine assay for predicting prostate biopsy outcome. J Urol 179:1587–1592CrossRefGoogle Scholar
  18. 18.
    Andriole GL, Crawford ED, Grubb RL 3rd, Buys SS, Chia D, Church TR, Fouad MN, Gelmann EP, Kvale PA, Reding DJ, Weissfeld JL, Yokochi LA, O'Brien B, Clapp JD, Rathmell JM, Riley TL, Hayes RB, Kramer BS, Izmirlian G, Miller AB, Pinsky PF, Prorok PC, Gohagan JK, Berg CD, PLCO Project Team (2009) Mortality results from a randomized prostate-cancer screening trial. N Engl J Med 360:1310–1319CrossRefGoogle Scholar
  19. 19.
    Schroder FH, Hugosson J, Roobol MJ, Tammela TL, Ciatto S, Nelen V, Kwiatkowski M, Lujan M, Lilja H, Zappa M, Denis LJ, Recker F, Berenguer A, Maattanen L, Bangma CH, Aus G, Villers A, Rebillard X, van der Kwast T, Blijenberg BG, Moss SM, de Koning HJ, Auvinen A, ERSPC Investigators (2009) Screening and prostate-cancer mortality in a randomized European study. N Engl J Med 360:1320–1328CrossRefGoogle Scholar
  20. 20.
    Goessl C, Muller M, Heicappell R, Krause H, Miller K (2001) DNA-based detection of prostate cancer in blood, urine, and ejaculates. Ann N Y Acad Sci 945:51–58CrossRefGoogle Scholar
  21. 21.
    Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6:359–362CrossRefGoogle Scholar
  22. 22.
    Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJ (2009) Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4:484–494CrossRefGoogle Scholar
  23. 23.
    Albarbarawi O, Barton A, Lin Z, Takahashi E, Buddharaju A, Brady J, Miller D, Palmer CN, Huang JT (2010) Measurement of urinary total desmosine and isodesmosine using isotope-dilution liquid chromatography-tandem mass spectrometry. Anal Chem 82:3745–3750CrossRefGoogle Scholar
  24. 24.
    Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 57(1):289–300Google Scholar
  25. 25.
    Atrih A, Mudaliar MVA, Zakikhani P, Lamon D, Huang TJ, Bray SE, Barton G, Fleming S, Nabi G (2014) Quantitative proteomics in resected renal cancer tissue for the discovery of biomarkers and pathway profiling. Br J Cancer 110(6):1622–33CrossRefGoogle Scholar
  26. 26.
    Shi T, Gao Y, Quek SI, Fillmore TL, Nicora CD, Su D, Zhao R, Kagan J, Srivastava S, Rodland KD, Liu T, Smith RD, Chan DW, Camp DG 2nd, Liu AY, Qian WJ (2013) A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum. J Proteome Res 13(2):875–82CrossRefGoogle Scholar
  27. 27.
    Lee SW, Lee KI, Kim JY (2005) Revealing urologic diseases by proteomic techniques. J Chromatogr B Analyt Technol Biomed Life Sci 815(1–2):203–13CrossRefGoogle Scholar
  28. 28.
    Craig AM, Bowden GT, Chambers AF, Spearman MA, Greenberg AH, Wright JA, McLeod M, Denhardt DT (1990) Secreted phosphoprotein mRNA is induced during multi-stage carcinogenesis in mouse skin and correlates with the metastatic potential of murine fibroblasts. Int J Cancer 46:133–137CrossRefGoogle Scholar
  29. 29.
    Rudland PS, Platt-Higgins A, El-Tanani M, De Silva RS, Barraclough R, Winstanley JH, Howitt R, West CR (2002) Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res 62:3417–3427Google Scholar
  30. 30.
    El-Tanani MK, Yuen HF, Shi Z, Platt-Higgins A, Buckley NE, Mullan PB, Harkin DP, Johnston PG, Rudland PS (2010) Osteopontin can act as an effector for a germline mutation of BRCA1 in malignant transformation of breast cancer-related cells. Cancer Sci 101:1354–1360CrossRefGoogle Scholar
  31. 31.
    Tuck AB, Chambers AF, Allan AL (2007) Osteopontin overexpression in breast cancer: knowledge gained and possible implications for clinical management. J Cell Biochem 102:859–868CrossRefGoogle Scholar
  32. 32.
    Ke HL, Chang LL, Yang SF, Lin HH, Li CC, Wu DC, Wu WJ (2011) Osteopontin overexpression predicts poor prognosis of upper urinary tract urothelial carcinoma. Urol Oncol 29:703–709CrossRefGoogle Scholar
  33. 33.
    Petrik D, Lavori PW, Cao H, Zhu Y, Wong P, Christofferson E, Kaplan MJ, Pinto HA, Sutphin P, Koong AC, Giaccia AJ, Le QT (2006) Plasma osteopontin is an independent prognostic marker for head and neck cancers. J Clin Oncol 24:5291–5297CrossRefGoogle Scholar
  34. 34.
    Schneider S, Yochim J, Brabender J, Uchida K, Danenberg KD, Metzger R, Schneider PM, Salonga D, Holscher AH, Danenberg PV (2004) Osteopontin but not osteonectin messenger RNA expression is a prognostic marker in curatively resected non-small cell lung cancer. Clin Cancer Res 10:1588–1596CrossRefGoogle Scholar
  35. 35.
    Weber GF, Zawaideh S, Hikita S, Kumar VA, Cantor H, Ashkar S (2002) Phosphorylation-dependent interaction of osteopontin with its receptors regulates macrophage migration and activation. J Leukoc Biol 72:752–761Google Scholar
  36. 36.
    Ye B, Skates S, Mok SC, Horick NK, Rosenberg HF, Vitonis A, Edwards D, Sluss P, Han WK, Berkowitz RS, Cramer DW (2006) Proteomic-based discovery and characterization of glycosylated eosinophil-derived neurotoxin and COOH-terminal osteopontin fragments for ovarian cancer in urine. Clin Cancer Res 12:432–441CrossRefGoogle Scholar
  37. 37.
    Overgaard J, Eriksen JG, Nordsmark M, Alsner J, Horsman MR (2005) Danish Head and Neck Cancer Study Group. Plasma osteopontin, hypoxia, and response to the hypoxia sensitiser nimorazole in radiotherapy of head and neck cancer: results from the DAHANCA 5 randomised double-blind placebo-controlled trial. Lancet Oncol 6:757–764CrossRefGoogle Scholar
  38. 38.
    Prager AJ (2011) Urinary osteopontin as a marker of localised and metastatic prostate cancer. J Clin Oncol 29: suppl; abstr e15147Google Scholar
  39. 39.
    Vergis R, Corbishley CM, Norman AR, Bartlett J, Jhavar S, Borre M, Heeboll S, Horwich A, Huddart R, Khoo V, Eeles R, Cooper C, Sydes M, Dearnaley D, Parker C (2008) Intrinsic markers of tumour hypoxia and angiogenesis in localised prostate cancer and outcome of radical treatment: a retrospective analysis of two randomised radiotherapy trials and one surgical cohort study. Lancet Oncol 9(4):342–51CrossRefGoogle Scholar
  40. 40.
    Hotte SJ, Winquist EW, Stitt L, Wilson SM, Chambers AF (2002) Plasma osteopontin: associations with survival and metastasis to bone in men with hormone-refractory prostate carcinoma. Cancer 95:506–512CrossRefGoogle Scholar
  41. 41.
    Jain A, McKnight DA, Fisher LW, Humphreys EB, Mangold LA, Partin AW, Fedarko NS (2009) Small integrin-binding proteins as serum markers for prostate cancer detection. Clin Cancer Res 15:5199–5207CrossRefGoogle Scholar
  42. 42.
    Thoms JW, Dal Pra A, Anborgh PH, Christensen E, Fleshner N, Menard C, Chadwick K, Milosevic M, Catton C, Pintilie M, Chambers AF, Bristow RG (2012) Plasma osteopontin as a biomarker of prostate cancer aggression: relationship to risk category and treatment response. Br J Cancer 107:840–846CrossRefGoogle Scholar
  43. 43.
    Rittling SRCA (2004) Role of osteopontin in tumour progression. Br J Cancer 90:1877–1881CrossRefGoogle Scholar
  44. 44.
    Weber GF, Lett GS, Haubein NC (2010) Osteopontin is a marker for cancer aggressiveness and patient survival. Br J Cancer 103:861–869CrossRefGoogle Scholar
  45. 45.
    Rud AK, Boye K, Oijordsbakken M, Lund-Iversen M, Halvorsen AR, Solberg SK, Berge G, Helland A, Brustugun OT, Mælandsmo GM (2013) Osteopontin as a prognostic marker in patients with small cell lung carcinoma. BMC Cancer 11Google Scholar
  46. 46.
    Radjabi AR, Sawada K, Jagadeeswaran S, Eichbichler A, Kenny HA, Montag A, Bruno K, Lengyel E (2008) Thrombin induces tumor invasion through the induction and association of matrix metalloproteinase-9 and beta1-integrin on the cell surface. J Biol Chem 283:2822–2834CrossRefGoogle Scholar
  47. 47.
    Nierodzik ML, Karpatkin S (2006) Thrombin induces tumor growth, metastasis, and angiogenesis: evidence for a thrombin-regulated dormant tumor phenotype. Cancer Cell 10:355–362CrossRefGoogle Scholar
  48. 48.
    Hu L, Lee M, Campbell W, Perez-Soler R, Karpatkin S (2004) Role of endogenous thrombin in tumor implantation, seeding, and spontaneous metastasis. Blood 104:2746–2751CrossRefGoogle Scholar
  49. 49.
    Patel A, Bhavan R, Somani B, Nabi G (2010) Correlation of percentage changes in platelet counts with recurrence rate following radical nephrectomy. Indian J Urol 26:183–187CrossRefGoogle Scholar
  50. 50.
    Miller GJ, Bauer KA, Howarth DJ, Cooper JA, Humphries SE, Rosenberg RD (2004) Increased incidence of neoplasia of the digestive tract in men with persistent activation of the coagulant pathway. J Thromb Haemost 2:2107–2114CrossRefGoogle Scholar
  51. 51.
    Sorensen HT, Pedersen L, Mellemkjaer L, Johnsen SP, Skriver MV, Olsen JH, Baron JA (2005) The risk of a second cancer after hospitalisation for venous thromboembolism. Br J Cancer 93:838–841CrossRefGoogle Scholar
  52. 52.
    Kakkar AK, Levine MN, Kadziola Z, Lemoine NR, Low V, Patel HK, Rustin G, Thomas M, Quigley M, Williamson RC (2004) Low molecular weight heparin, therapy with dalteparin, and survival in advanced cancer: the fragmin advanced malignancy outcome study (FAMOUS). J Clin Oncol 22:1944–1948CrossRefGoogle Scholar
  53. 53.
    Lee AY, Rickles FR, Julian JA, Gent M, Baker RI, Bowden C, Kakkar AK, Prins M, Levine MN (2005) Randomized comparison of low molecular weight heparin and coumarin derivatives on the survival of patients with cancer and venous thromboembolism. J Clin Oncol 23:2123–2129CrossRefGoogle Scholar
  54. 54.
    Kohli M, Williams K, Yao JL, Dennis RA, Huang J, Reeder J, Ricke WA (2011) Thrombin expression in prostate: a novel finding. Cancer Ivest 29:62–67CrossRefGoogle Scholar
  55. 55.
    Chay CH, Cooper CR, Gendernalik JD, Dhanasekaran SM, Chinnaiyan AM, Rubin MA, Schmaier AH, Pienta KJ (2002) A functional thrombin receptor (PAR1) is expressed on bone-derived prostate cancer cell lines. Urology 60:760–765CrossRefGoogle Scholar
  56. 56.
    Shi X, Gangadharan B, Brass LF, Ruf W, Mueller BM (2004) Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis. Mol Cancer Res 2:395–402Google Scholar
  57. 57.
    Lipton A, Demers L, Daniloff Y, Curley E, Hamilton C, Harvey H, Witters L, Seaman J, Van der Giessen R, Seyedin S (1993) Increased urinary excretion of pyridinium cross-links in cancer patients. Clin Chem 39:614–618Google Scholar
  58. 58.
    Pecherstorfer M, Zimmer-Roth I, Schilling T, Woitge HW, Schmidt H, Baumgartner G, Thiebaud D, Ludwig H, Seibel MJ (1995) The diagnostic value of urinary pyridinium cross-links of collagen, serum total alkaline phosphatase, and urinary calcium excretion in neoplastic bone disease. J Clin Endocrinol Metab 80:97–103Google Scholar
  59. 59.
    Vinholes J, Guo CY, Purohit OP, Eastell R, Coleman RE (1996) Metabolic effects of pamidronate in patients with metastatic bone disease. Br J Cancer 73:1089–1095CrossRefGoogle Scholar
  60. 60.
    Lipton A, Cook R, Saad F, Major P, Garnero P, Terpos E, Brown JE, Coleman RE (2008) Normalization of bone markers is associated with improved survival in patients with bone metastases from solid tumors and elevated bone resorption receiving zoledronic acid. Cancer 113:193–201CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Chunhui Li
    • 1
  • Tuo Zang
    • 2
  • Karolina Wrobel
    • 2
  • Jeffrey T.-J. Huang
    • 2
  • Ghulam Nabi
    • 1
    Email author
  1. 1.Academic Section of Urology, Division of Imaging TechnologyUniversity of Dundee, Ninewells HospitalDundeeUK
  2. 2.Medical Research Institute, Jacqui Wood Cancer CentreUniversity of DundeeDundeeUK

Personalised recommendations