Plasma proteomics for biomarker discovery: A study in blue

Francesco Di Girolamo, Pier Giorgio Righetti

Research output: Contribution to journalArticle

Abstract

The performance of Cibacron Blue dye (HiTrapBlue or Affigel Blue) in depleting albumin from plasma, as a pre-treatment for biomarker searching in the low-abundance proteome, is here assessed. It is shown that (i) co-depletion of non-albumin species is an ever-present hazard; (ii) the only proper eluant able to release quantitatively the proteins bound to the dye is boiling 4% SDS-25mM DTT, an ion shock (2M NaCl) being quite ineffective in releasing the low-abundance species tightly bound to the dye moiety; (iii) the mechanism of dye-protein interaction, after an initial ion-ion docking, is a robust hydrophobic interaction, which progressively augments at lower and lower pH values; (iv) at pH 2.2 in the presence of 0.1% TFA, the blue resin behaves, for all practical purposes, just as a reverse-phase chromatography column, since all residual proteins present in plasma are completely harvested. However Cibacron Blue technology should not necessarily be discarded: As long as also the plasma fraction adsorbed is properly released and analyzed, together with the flow through, one should be able to perform a viable analysis of the low-abundance proteome.

Original languageEnglish
Pages (from-to)3638-3644
Number of pages7
JournalElectrophoresis
Volume32
Issue number24
DOIs
Publication statusPublished - Dec 2011

Keywords

  • Albumin depletion
  • Biomarkers
  • Cibacron blue
  • Elution protocols
  • Plasma

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry

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