Comparison of different depletion strategies for improving resolution of the human urine proteome

Cinzia Magagnotti, Isabella Fermo, Rose Mary Carletti, Maurizio Ferrari, Angela Bachi

Research output: Contribution to journalArticle

Abstract

Background: Urine, being an ultrafiltrate of plasma, is a rich source for biomarker discovery. Since potential new disease markers are often present in low concentrations, a prefractionation/enrichment step could be useful in the discovery process. To enhance the detection of low-abundance proteins, three immuno-affinity depletion approaches were evaluated. Methods: To remove the most abundant proteins from a human urine sample, GenWay Spin IgY-12 kit, HPLC Agilent Hu-PL7 and a home-made column vs. human serum albumin [immuno-affinity column (IAC)] were compared. Quantification of total proteins, 2-D gel electrophoresis (2-DE), Progenesis gel images analysis and mass spectrometric proteins identification were applied to evaluate these strategies. Results: Reproducibility of depletion columns, by estimating protein content of unbound fractions, were: 343±20.0 μg, 5.8%; 186.3±13.3 μg, 7.2%; 292±20.6 μg, 8.8% [mean± standard deviation (SD), CV%], for GenWay , Agilent and IAC methods, respectively. To isolate urinary protein after depletion, ethanol precipitation provided the highest recovery (80%). Applying 2-DE and Progenesis analysis, the number of spots visualized on the gels was 468±21, 331±7, 368±22 and 304±7 (mean±SD) for GenWay , Agilent, IAC, and the undepleted urine pool sample, respectively, with a significant difference p™ kit followed by ethanol precipitation was found to be the most efficient method for exploring the urine proteome.

Original languageEnglish
Pages (from-to)531-535
Number of pages5
JournalClinical Chemistry and Laboratory Medicine
Volume48
Issue number4
DOIs
Publication statusPublished - Apr 2010

Keywords

  • Biomarkers
  • Proteins depletion
  • Proteomics
  • Urine

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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