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.