title: Novel automated biomarker discovery work flow for urinary peptidomics.
abstract: 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.
authors.affiliation: Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands. c.i.a.balog@lumc.nl
authors.forename: Crina I
authors.initials: CI
authors.lastname: Balog
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hensbergen
authors.forename: Rico
authors.initials: R
authors.lastname: Derks
authors.forename: Jaco J
authors.initials: JJ
authors.lastname: Verweij
authors.forename: Govert J
authors.initials: GJ
authors.lastname: van Dam
authors.forename: Birgitte J
authors.initials: BJ
authors.lastname: Vennervald
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Oleg A
authors.initials: OA
authors.lastname: Mayboroda
mesh_terms: 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
chemical_list: D015415:Biomarkers; D010455:Peptides; D004247:DNA