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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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Ed. by Gillery, Philippe / Greaves, Ronda / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter

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Volume 55, Issue 2


An integrated proteomic and peptidomic assessment of the normal human urinome

Ashley Di Meo
  • Department of Laboratory Medicine, and the Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON, Canada
  • Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada
  • Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Ihor Batruch / Arsani G. Yousef / Maria D. Pasic
  • Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
  • Department of Laboratory Medicine, St. Joseph’s Health Centre, Toronto, ON, Canada
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Eleftherios P. Diamandis
  • Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada
  • Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
  • Department of Clinical Biochemistry, University Health Network, Toronto, ON, Canada
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ George M. Yousef
  • Corresponding author
  • Department of Laboratory Medicine, and the Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON, Canada
  • Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2016-07-09 | DOI: https://doi.org/10.1515/cclm-2016-0390



Urine represents an ideal source of clinically relevant biomarkers as it contains a large number of proteins and low molecular weight peptides. The comprehensive characterization of the normal urinary proteome and peptidome can serve as a reference for future biomarker discovery. Proteomic and peptidomic analysis of urine can also provide insight into normal physiology and disease pathology, especially for urogenital diseases.


We developed an integrated proteomic and peptidomic analytical protocol in normal urine. We employed ultrafiltration to separate protein and peptide fractions, which were analyzed separately using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on the Q-Exactive mass spectrometer.


By analyzing six urines from healthy individuals with advanced age, we identified 1754 proteins by proteomic analysis and 4543 endogenous peptides, arising from 566 proteins by peptidomic analysis. Overall, we identified 2091 non-redundant proteins by this integrated approach. In silico protease activity analysis indicated that metalloproteases are predominantly involved in the generation of the endogenous peptide signature. In addition, a number of proteins that were detected in normal urine have previously been implicated in various urological malignancies, including bladder cancer and renal cell carcinoma (RCC).


We utilized a highly sensitive proteomics approach that enabled us to identify one of the largest sets of protein identifications documented in normal human urine. The raw proteomics and peptidomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD003595.

This article offers supplementary material which is provided at the end of the article.

Keywords: mass spectrometry; peptide sequence alignment; protease activity analysis; urinary peptidome; urinary proteome


  • 1.

    Blaine J, Chonchol M, Levi M. Renal control of calcium, phosphate, and magnesium homeostasis. Clin J Am Soc Nephrol 2015;10:1257–72.Google Scholar

  • 2.

    Tojo A. The role of the kidney in protein metabolism: the capacity of tubular lysosomal proteolysis in nephrotic syndrome. Kidney Int 2013;84:861–3.Google Scholar

  • 3.

    Bauca JM, Martinez-Morillo E, Diamandis EP. Peptidomics of urine and other biofluids for cancer diagnostics. Clin Chem 2014;60:1052–61.Google Scholar

  • 4.

    Dallas DC, Guerrero A, Parker EA, Robinson RC, Gan J, German JB, et al. Current peptidomics: applications, purification, identification, quantification, and functional analysis. Proteomics 2015;15:1026–38.Google Scholar

  • 5.

    Jurgens M, Appel A, Heine G, Neitz S, Menzel C, Tammen H, et al. Towards characterization of the human urinary peptidome. Comb Chem High Throughput Screen 2005;8:757–65.Google Scholar

  • 6.

    Di Meo A, Pasic MD, Yousef GM. Proteomics and peptidomics: moving toward precision medicine in urological malignancies. Oncotarget 2016. .CrossrefGoogle Scholar

  • 7.

    Sigdel TK, Salomonis N, Nicora CD, Ryu S, He J, Dinh V, et al. The identification of novel potential injury mechanisms and candidate biomarkers in renal allograft rejection by quantitative proteomics. Mol Cell Proteomics 2014;13:621–31.Google Scholar

  • 8.

    Frantzi M, Metzger J, Banks RE, Husi H, Klein J, Dakna M, et al. Discovery and validation of urinary biomarkers for detection of renal cell carcinoma. J Proteomics 2014;98:44–58.Google Scholar

  • 9.

    Rocchetti MT, Centra M, Papale M, Bortone G, Palermo C, Centonze D, et al. Urine protein profile of IgA nephropathy patients may predict the response to ACE-inhibitor therapy. Proteomics 2008;8:206–16.Google Scholar

  • 10.

    Davalieva K, Kiprijanovska S, Komina S, Petrusevska G, Zografska NC, Polenakovic M. Proteomics analysis of urine reveals acute phase response proteins as candidate diagnostic biomarkers for prostate cancer. Proteome Sci 2015;13:2.Google Scholar

  • 11.

    Smith CR, Batruch I, Bauca JM, Kosanam H, Ridley J, Bernardini MQ, et al. Deciphering the peptidome of urine from ovarian cancer patients and healthy controls. Clin Proteomics 2014;11:23.Google Scholar

  • 12.

    Frantzi M, Latosinska A, Fluhe L, Hupe MC, Critselis E, Kramer MW, et al. Developing proteomic biomarkers for bladder cancer: towards clinical application. Nat Rev Urol 2015;12:317–30.Google Scholar

  • 13.

    Davis MT, Auger PL, Patterson SD. Cancer biomarker discovery via low molecular weight serum profiling--are we following circular paths? Clin Chem 2010;56:244–7.Google Scholar

  • 14.

    Good DM, Thongboonkerd V, Novak J, Bascands JL, Schanstra JP, Coon JJ, et al. Body fluid proteomics for biomarker discovery: lessons from the past hold the key to success in the future. J Proteome Res 2007;6:4549–55.Google Scholar

  • 15.

    Mischak H, Julian BA, Novak J. High-resolution proteome/peptidome analysis of peptides and low-molecular-weight proteins in urine. Proteomics Clin Appl 2007;1:792.Google Scholar

  • 16.

    Thongboonkerd V, McLeish KR, Arthur JM, Klein JB. Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation. Kidney Int 2002;62: 1461–9.Google Scholar

  • 17.

    Pieper R, Gatlin CL, McGrath AM, Makusky AJ, Mondal M, Seonarain M, et al. Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots. Proteomics 2004;4:1159–74.Google Scholar

  • 18.

    Sun W, Li F, Wu S, Wang X, Zheng D, Wang J, et al. Human urine proteome analysis by three separation approaches. Proteomics 2005;5:4994–5001.Google Scholar

  • 19.

    Li QR, Fan KX, Li RX, Dai J, Wu CC, Zhao SL, et al. A comprehensive and non-prefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine. Rapid Commun Mass Spectrom 2010;24:823–32.Google Scholar

  • 20.

    Adachi J, Kumar C, Zhang Y, Olsen JV, Mann M. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biol 2006;7:R80.Google Scholar

  • 21.

    Marimuthu A, O'Meally RN, Chaerkady R, Subbannayya Y, Nanjappa V, Kumar P, et al. A comprehensive map of the human urinary proteome. J Proteome Res 2011;10:2734–43.Google Scholar

  • 22.

    Santucci L, Candiano G, Petretto A, Bruschi M, Lavarello C, Inglese E, et al. From hundreds to thousands: Widening the normal human Urinome (1). J Proteomics 2015;112:53–62.Google Scholar

  • 23.

    Santucci L, Candiano G, Petretto A, Bruschi M, Lavarello C, Inglese E, et al. From hundreds to thousands: Widening the normal human Urinome. Data Brief 2014;1:25–8.Google Scholar

  • 24.

    Yang X, Hu L, Ye M, Zou H. Analysis of the human urine endogenous peptides by nanoparticle extraction and mass spectrometry identification. Anal Chim Acta 2014;829:40–7.Google Scholar

  • 25.

    Fiedler GM, Baumann S, Leichtle A, Oltmann A, Kase J, Thiery J, et al. Standardized peptidome profiling of human urine by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Clin Chem 2007;53:421–8.Google Scholar

  • 26.

    Mischak H, Kolch W, Aivaliotis M, Bouyssie D, Court M, Dihazi H, et al. Comprehensive human urine standards for comparability and standardization in clinical proteome analysis. Proteomics Clin Appl 2010;4:464–78.Google Scholar

  • 27.

    Liu X, Chinello C, Musante L, Cazzaniga M, Tataruch D, Calzaferri G, et al. Intraluminal proteome and peptidome of human urinary extracellular vesicles. Proteomics Clin Appl 2015;9:568–73.Google Scholar

  • 28.

    Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 2011;10:1794–805.Google Scholar

  • 29.

    Luber CA, Cox J, Lauterbach H, Fancke B, Selbach M, Tschopp J, et al. Quantitative proteomics reveals subset-specific viral recognition in dendritic cells. Immunity 2010;32:279–89.Google Scholar

  • 30.

    Cox J, Hubner NC, Mann M. How much peptide sequence information is contained in ion trap tandem mass spectra? J Am Soc Mass Spectrom 2008;19:1813–20.Google Scholar

  • 31.

    Klein J, Eales J, Zurbig P, Vlahou A, Mischak H, Stevens R. Proteasix: a tool for automated and large-scale prediction of proteases involved in naturally occurring peptide generation. Proteomics 2013;13:1077–82.Google Scholar

  • 32.

    Guerrero A, Dallas DC, Contreras S, Chee S, Parker EA, Sun X, et al. Mechanistic peptidomics: factors that dictate specificity in the formation of endogenous peptides in human milk. Mol Cell Proteomics 2014;13:3343–51.Google Scholar

  • 33.

    Nagaraj N, Mann M. Quantitative analysis of the intra- and inter-individual variability of the normal urinary proteome. J Proteome Res 2011;10:637–45.Google Scholar

  • 34.

    Mutowo-Meullenet P, Huntley RP, Dimmer EC, Alam-Faruque Y, Sawford T, Jesus Martin M, et al. Use of Gene Ontology Annotation to understand the peroxisome proteome in humans. Database (Oxford) 2013;2013:bas062.Google Scholar

  • 35.

    Lachmann PJ. Lupus and desoxyribonuclease. Lupus 2003;12:202–6.Google Scholar

  • 36.

    Bakun M, Senatorski G, Rubel T, Lukasik A, Zielenkiewicz P, Dadlez M, et al. Urine proteomes of healthy aging humans reveal extracellular matrix (ECM) alterations and immune system dysfunction. Age 2014;36:299–311.Google Scholar

  • 37.

    Liu KQ, Liu ZP, Hao JK, Chen L, Zhao XM. Identifying dysregulated pathways in cancers from pathway interaction networks. BMC Bioinformatics 2012;13:126.Google Scholar

  • 38.

    Overall CM, Blobel CP. In search of partners: linking extracellular proteases to substrates. Nat Rev Mol Cell Biol 2007;8:245–57.Google Scholar

  • 39.

    Lyons PJ, Fricker LD. Peptidomic approaches to study proteolytic activity. Curr Protoc Protein Sci 2011;Chapter 18:Unit 18.13.

  • 40.

    Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 2007;8:221–33.Google Scholar

  • 41.

    Vu TH, Werb Z. Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev 2000;14:2123–33.Google Scholar

  • 42.

    Klein T, Bischoff R. Physiology and pathophysiology of matrix metalloproteases. Amino Acids 2011;41:271–90.Google Scholar

  • 43.

    Turk B. Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov 2006;5:785–99.Google Scholar

  • 44.

    Szarvas T, vom Dorp F, Ergun S, Rubben H. Matrix metalloproteinases and their clinical relevance in urinary bladder cancer. Nat Rev Urol 2011;8:241–54.Google Scholar

  • 45.

    Barkan DT, Hostetter DR, Mahrus S, Pieper U, Wells JA, Craik CS, et al. Prediction of protease substrates using sequence and structure features. Bioinformatics 2010;26:1714–22.Google Scholar

  • 46.

    Chen YT, Chen CL, Chen HW, Chung T, Wu CC, Chen CD, et al. Discovery of novel bladder cancer biomarkers by comparative urine proteomics using iTRAQ technology. J Proteome Res 2010;9:5803–15.Google Scholar

  • 47.

    Chen CL, Lin TS, Tsai CH, Wu CC, Chung T, Chien KY, et al. Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics. J Proteomics 2013;85:28–43.Google Scholar

  • 48.

    Kawata N, Nagane Y, Hirakata H, Ichinose T, Okada Y, Yamaguchi K, et al. Significant relationship of matrix metalloproteinase 9 with nuclear grade and prognostic impact of tissue inhibitor of metalloproteinase 2 for incidental clear cell renal cell carcinoma. Urology 2007;69:1049–53.Google Scholar

  • 49.

    Sato A, Nagase H, Obinata D, Fujiwara K, Fukuda N, Soma M, et al. Inhibition of MMP-9 using a pyrrole-imidazole polyamide reduces cell invasion in renal cell carcinoma. Int J Oncol 2013;43:1441–6.Google Scholar

  • 50.

    Gaut JP, Crimmins DL, Lockwood CM, McQuillan JJ, Ladenson JH. Expression of the Na+/K+-transporting ATPase gamma subunit FXYD2 in renal tumors. Mod Pathol 2013;26:716–24.Google Scholar

  • 51.

    Langner C, Ratschek M, Rehak P, Schips L, Zigeuner R. Expression of MUC1 (EMA) and E-cadherin in renal cell carcinoma: a systematic immunohistochemical analysis of 188 cases. Mod Pathol 2004;17:180–8.Google Scholar

  • 52.

    Aubert S, Fauquette V, Hemon B, Lepoivre R, Briez N, Bernard D, et al. MUC1, a new hypoxia inducible factor target gene, is an actor in clear renal cell carcinoma tumor progression. Cancer Res 2009;69:5707–15.Google Scholar

  • 53.

    Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 2014;13:2513–26.Google Scholar

  • 54.

    Romanova EV, Dowd SE, Sweedler JV. Quantitation of endogenous peptides using mass spectrometry based methods. Curr Opin Chem Biol 2013;17:801–8.Google Scholar

  • 55.

    Olsen JV, Mann M. Status of large-scale analysis of post-translational modifications by mass spectrometry. Mol Cell Proteomics 2013;12:3444–52.Google Scholar

  • 56.

    Thompson RH, Ordonez MA, Iasonos A, Secin FP, Guillonneau B, Russo P, et al. Renal cell carcinoma in young and old patients–is there a difference? J Urol 2008;180:1262–6; discussion 6.Google Scholar

  • 57.

    Taylor JA, 3rd, Kuchel GA. Bladder cancer in the elderly: clinical outcomes, basic mechanisms, and future research direction. Nat Clin Pract Urol 2009;6:135–44.Google Scholar

  • 58.

    Nkuipou-Kenfack E, Bhat A, Klein J, Jankowski V, Mullen W, Vlahou A, et al. Identification of ageing-associated naturally occurring peptides in human urine. Oncotarget 2015;6: 34106–17.Google Scholar

About the article

Corresponding author: George M. Yousef, MD, PhD, FRCPC (Path), Department of Laboratory Medicine, and the Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael's Hospital, 30 Bond Street, Toronto, ON, M5B 1W8, Canada, Phone: +1-416-864-6060, Ext: 77605, Fax: +416-864-5648

Received: 2016-05-03

Accepted: 2016-06-09

Published Online: 2016-07-09

Published in Print: 2017-02-01

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: This work was supported by grants from the Canadian Institute of Health Research (MOP 119606), Kidney Foundation of Canada (KFOC130030), Kidney Cancer Research Network of Canada and Prostate Cancer Canada Movember Discovery Grants (D2013-39).

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 55, Issue 2, Pages 237–247, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2016-0390.

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