Novel automated biomarker discovery work flow for urinary peptidomics.
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.
Balog
Crina I
CI
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands. c.i.a.balog@lumc.nl
Hensbergen
Paul J
PJ
Derks
Rico
R
Verweij
Jaco J
JJ
van Dam
Govert J
GJ
Vennervald
Birgitte J
BJ
Deelder
André M
AM
Mayboroda
Oleg A
OA
D000293:Adolescent; D001331:Automation; D015415:Biomarkers / Q000652:urine*; D002648:Child; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D002853:Chromatography, Liquid; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D010455:Peptides / Q000652:urine*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012553:Schistosomiasis haematobia / Q000652:urine*; D012680:Sensitivity and Specificity
D015415:Biomarkers; D010455:Peptides; D004247:DNA