Background: Low-abundance proteins are difficultly observed on the two-dimensional gel electrophoresis (2-DE)
maps of urine proteome, because they are usually obscured by high-abundance proteins such as albumin and
immunoglobulin. In this study, a novel fractionation method was developed for enriching low-abundance proteins
by removing high-abundance proteins and progressive elution with salts of various concentrations.
Results: Stepwise weak anion exchange (WAX) chromatography, which applied DEAE-Sephacel resin with nonfixed
volume elution, was used to fractionate urine proteome prior to performing 2-DE. Urine proteome was
separated into four fractions by progressively eluting the column with 0 M, 50 mM, 100 mM, and 1 M NaCl
solutions. Most of the heavy and light immunoglobulin chains appeared in the eluent. After the high-abundance
proteins were removed, various low-abundance proteins were enriched and could be easily identified. The
potential of this method for obtaining diversified fractionations was demonstrated by eluting the column
separately with Na2SO4 and MgCl2 solutions. The 2-DE maps of the fractions eluted with these different salt
solutions of identical ionic strength revealed markedly different stain patterns.
Conclusion: The present study demonstrated that this fractionation method could be applied for purposes of
enriching low-abundance proteins and obtaining diversified fractionations of urine, and potentially other
proteomes.
