Clinical Chemistry
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Clinical Chemistry 55: 117-125, 2009. First published November 6, 2008; 10.1373/clinchem.2008.108795
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(Clinical Chemistry. 2009;55:117-125.)
© 2009 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Novel Automated Biomarker Discovery Work Flow for Urinary Peptidomics

Crina I. Balog1,a, Paul J. Hensbergen1, Rico Derks1, Jaco J. Verweij1, Govert J. van Dam1, Birgitte J. Vennervald2, André M. Deelder1 and Oleg A. Mayboroda1

1 Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands; 2 DBL-Center for Health Research and Development, Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark.

aAddress correspondence to this author at: Department of Parasitology, Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, 2300 RC Leiden, The Netherlands. Fax +31-71-5266907; e-mail c.i.a.balog{at}lumc.nl.

Background: Urine is potentially a rich source of peptide biomarkers, but reproducible, high-throughput peptidomic analysis is often hampered by the inherent variability in factors such as pH and salt concentration. Our goal was to develop a generally applicable, rapid, and robust method for screening large numbers of urine samples, resulting in a broad spectrum of native peptides, as a tool to be used for biomarker discovery.

Methods: Peptide samples were trapped, desalted, pH-normalized, and fractionated on a miniaturized automatic reverse-phase strong cation exchange (RP-SCX) cartridge system. We analyzed eluted peptides using MALDI-TOF, Fourier transform ion cyclotron resonance, and liquid chromatography-iontrap mass spectrometry. We determined qualitative and quantitative reproducibility of the system and robustness of the method using BSA digests and urine samples, and we used a selected set of urine samples from Schistosoma haematobium-infected individuals to evaluate clinical applicability.

Results: The automated RP-SCX sample cleanup and fractionation system exhibits a high qualitative and quantitative reproducibility, with both BSA standards and urine samples. Because of the relatively high cartridge binding capacity (1–2 mL urine), eluted peptides can be measured with high sensitivity using multiple mass spectrometric techniques. As proof of principle, hemoglobin-derived peptides were identified in urine samples from S. haematobium-infected individuals, even when the microhematuria test was negative.

Conclusions: We present a practical, step-by-step method for screening and identification of urinary peptides. Alongside the analytical method evaluation on standard samples, we demonstrate its feasibility with actual clinical material.






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Copyright © 2009 by the American Association for Clinical Chemistry.