An Automated RP–SCX Solid-Phase Extraction Procedure for Urinary Peptidomics Biomarker Discovery Studies

  • Crina I. A. Balog
  • Rico Derks
  • Oleg A. Mayboroda
  • André M. Deelder
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1023)

Abstract

Urine represents the most easily obtainable body fluid and consequently one of the most common samples in clinical chemistry. The majority of pathological changes in human organs may well be reflected in urine. In this way, urine analysis can aid in disease diagnosis, treatment monitoring, and prognosis. Currently, the most commonly used method for identification of new urine biomarkers involves centrifugation of the urine sample to collect either the soluble urine proteins or the urinary exosomes followed by 1 or 2 protein purification and separation steps before visualization and finally identification of potential biomarkers, usually by mass spectrometry.

Here we present a generally applicable, rapid, and robust method for screening large number of urine samples, resulting in a broad spectrum of native peptides, as a tool to be used for biomarker discovery. The method combines online sample pretreatment with a well-established mass spectrometric technique. Native peptides are extracted from urine samples on a miniaturized reverse-phase–strong cation exchange cartridge system. As the proper identification of native peptides often requires combination of data acquired on different mass analyzers, we have aimed at a procedure providing us with sufficient material to identify and characterize the differentially expressed markers.

Key words

Urine Peptidomics RP–SCX purification Native peptides 

Notes

Acknowledgments

This study was supported by the European Union, Sixth framework program, Specific Measures in support of International Cooperation (INCO), contract 517733.

References

  1. 1.
    Petricoin EF, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steinberg SM, Mills GB, Simone C, Fishman DA, Kohn EC, Liotta LA (2002) Use of proteomic patterns in serum to identify ovarian cancer. Lancet 359:572–577PubMedCrossRefGoogle Scholar
  2. 2.
    Adam BL, Qu Y, Davis JW, Ward MD, Clements MA, Cazares LH, Semmes OJ, Schellhammer PF, Yasui Y, Feng Z, Wright GL Jr (2002) Serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men. Cancer Res 62:3609–3614PubMedGoogle Scholar
  3. 3.
    De Noo ME, Deelder A, van der Werff M, Ozalp A, Mertens B, Tollenaar R (2006) MALDI-TOF serum protein profiling for the detection of breast cancer. Onkologie 29:501–506PubMedCrossRefGoogle Scholar
  4. 4.
    De Noo ME, Mertens BJ, Ozalp A, Bladergroen MR, van der Werff MP, van de Velde CJ, Deelder AM, Tollenaar RA (2006) Detection of colorectal cancer using MALDI-TOF serum protein profiling. Eur J Cancer 42:1068–1076PubMedCrossRefGoogle Scholar
  5. 5.
    Rai AJ, Zhang Z, Rosenzweig J, Shih I, Pham T, Fung ET, Sokoll LJ, Chan DW (2002) Proteomic approaches to tumor marker discovery. Arch Pathol Lab Med 126:1518–1526PubMedGoogle Scholar
  6. 6.
    Yanagisawa K, Shyr Y, Xu BJ, Massion PP, Larsen PH, White BC, Roberts JR, Edgerton M, Gonzalez A, Nadaf S, Moore JH, Caprioli RM, Carbone DP (2003) Proteomic patterns of tumour subsets in non-small-cell lung cancer. Lancet 362:433–439PubMedCrossRefGoogle Scholar
  7. 7.
    Davidsson P, Sjogren M (2005) The use of proteomics in biomarker discovery in neurodegenerative diseases. Dis Markers 21:81–92PubMedCrossRefGoogle Scholar
  8. 8.
    Gineste C, Ho L, Pompl P, Bianchi M, Pasinetti GM (2003) High-throughput proteomics and protein biomarker discovery in an experimental model of inflammatory hyperalgesia: effects of nimesulide. Drugs 63(Suppl 1):23–29PubMedCrossRefGoogle Scholar
  9. 9.
    Vlahou A, Schellhammer PF, Mendrinos S, Patel K, Kondylis FI, Gong L, Nasim S, Wright JG Jr (2001) Development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine. American Journal of Pathoogy 158:1491–1502CrossRefGoogle Scholar
  10. 10.
    Hortin GL, Sviridov D (2007) Diagnostic potential for urinary proteomics. Pharmacogenomics 8:237–255PubMedCrossRefGoogle Scholar
  11. 11.
    Kim MK, Warren TC, Kimball ES (1985) Purification and characterization of a low molecular weight transforming growth factor from the urine of melanoma patients. J Biol Chem 260:9237–9243PubMedGoogle Scholar
  12. 12.
    Meng QC, Balcells E, Dell’Italia L, Durand J, Oparil S (1995) Sensitive method for quantitation of angiotensin-converting enzyme (ACE) activity in tissue. Biochem Pharmacol 50:1445–1450PubMedCrossRefGoogle Scholar
  13. 13.
    Sato K, Hirata Y, Imai T, Iwashina M, Marumo F (1995) Characterization of immunoreactive adrenomedullin in human plasma and urine. Life Sci 57:189–194PubMedCrossRefGoogle Scholar
  14. 14.
    Walters AE, Myrdal PB, Pinsuwan S, Manka AM, Yalkowsky SH (1997) Determination of melanotan II in rabbit urine using solid-phase extraction sample preparation followed by reversed-phase high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 690:99–103PubMedCrossRefGoogle Scholar
  15. 15.
    Fierens C, Thienpont LM, Stockl D, Willekens E, De Leenheer AP (2000) Quantitative analysis of urinary C-peptide by liquid chromatography-tandem mass spectrometry with a stable isotopically labelled internal standard. J Chromatogr A 896:275–278PubMedCrossRefGoogle Scholar
  16. 16.
    Sanchez JC, Hochstrasser DF (1999) Preparation and solubilization of body fluids for 2-D. Methods Mol Biol 112:87–93PubMedGoogle Scholar
  17. 17.
    McKee JA, Kumar S, Ecelbarger CA, Fernandez-Llama P, Terris J, Knepper MA (2000) Detection of Na(+) transporter proteins in urine. J Am Soc Nephrol 11:2128–2132PubMedGoogle Scholar
  18. 18.
    Thongboonkerd V, McLeish KR, Arthur JM, Klein JB (2002) Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation. Kidney Int 62:1461–1469PubMedCrossRefGoogle Scholar
  19. 19.
    Marshall T, Williams KM (1998) Clinical analysis of human urinary proteins using high resolution electrophoretic methods. Electrophoresis 19:1752–1770PubMedCrossRefGoogle Scholar
  20. 20.
    Cutler P, Bell DJ, Birrell HC, Connelly JC, Connor SC, Holmes E, Mitchell BC, Monte SY, Neville BA, Pickford R, Polley S, Schneider K, Skehel JM (1999) An integrated proteomic approach to studying glomerular nephrotoxicity. Electrophoresis 20:3647–3658PubMedCrossRefGoogle Scholar
  21. 21.
    Ostergaard M, Wolf H, Orntoft TF, Celis JE (1999) Psoriasin (S100A7): a putative urinary marker for the follow-up of patients with bladder squamous cell carcinomas. Electrophoresis 20:349–354PubMedCrossRefGoogle Scholar
  22. 22.
    Heine G, Raida M, Forssmann WG (1997) Mapping of peptides and protein fragments in human urine using liquid chromatography-mass spectrometry. J Chromatogr A 776:117–124PubMedCrossRefGoogle Scholar
  23. 23.
    Cutillas PR, Norden AG, Cramer R, Burlingame AL, Unwin RJ (2003) Detection and analysis of urinary peptides by on-line liquid chromatography and mass spectrometry: application to patients with renal Fanconi syndrome. Clinical Science (London) 104:483–490CrossRefGoogle Scholar
  24. 24.
    Hughes NC, Wong EY, Fan J, Bajaj N (2007) Determination of carryover and contamination for mass spectrometry-based chromatographic assays. The American Association of Pharmaceutical Scientists journal 9:E353–E360Google Scholar
  25. 25.
    Balog CI, Hensbergen PJ, Derks R, Verweij JJ, van Dam GJ, Vennervald BJ, Deelder AM, Mayboroda OA (2009) Novel automated biomarker discovery work flow for urinary peptidomics. Clin Chem 55:117–125PubMedCrossRefGoogle Scholar
  26. 26.
    Beavis RC, Chaudhary T, Chait BT (1992) Alpha-cyano-4-hydroxycinnamic acid as a matrix for matrix-assisted laser desorption mass-spectrometry. Organic Mass Spectrometry 27:156–158CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Crina I. A. Balog
    • 1
  • Rico Derks
    • 1
  • Oleg A. Mayboroda
    • 1
  • André M. Deelder
    • 1
  1. 1.Department of ParasitologyLeiden University Medical CenterRC LeidenThe Netherlands

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