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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.
© 2008 American Association for Clinical Chemistry
In the last 2 decades, mass spectrometry- based proteomics has emerged as an indispensable tool of modern biomedical science, but it was not before the publication of Petricoin et al. (1 ) that clinicians fully appreciated the benefits of this new technology. Petricoin and coauthors introduced the concept of protein profiling: the fusion of mass spectrometry with pattern recognition, where specific peak profiles, without knowledge of individual peak identity, were treated as biomarkers. A number of studies have demonstrated the applicability of serum profiling to a range of medical research questions, including diagnostics of a variety of cancers (1-6). Recently, protein profiling was adapted for biomarker discovery studies in cerebrospinal fluid (7, 8 ) and urine (9 ).
Apart from providing the possibility of noninvasive diagnostic testing, urine has...