Reduction of protein concentration range difference followed by multicolumn fractionation prior to 2-DE and LC-MS/MS profiling of serum proteins.

This article is concerned with the reduction of protein concentration range differences by the peptide beads library technology (ProteoMiner™ or "equalizer" technology), which in principle allows the enrichment of proteins to the same concentration level (i.e. protein equalizer) regardless of the original protein abundance in a given biological fluid such as human serum, which is the subject of our investigation. After the equalization step, the captured proteins from human serum were fractionated on a series of tandem monolithic columns with surface-bound iminodiacetic acid ligands to which three different metal ions, namely, Zn²+, Ni²+ and Cu²+ were immobilized to yield the so-called immobilized metal affinity chromatography columns. These three monolithic columns were connected to a reversed-phase column packed with polystyrene divinyl benzene beads. Aliquots taken from the four collected fractions from the four tandem columns were subsequently fractionated by 2-DE. Also, aliquots from the four collected fractions were tryptically digested and analyzed by LC-MS/MS. The strategy of subsequent fractionation on the four tandem columns after equalization allowed the identification of more proteins than simply using the equalization by ProteoMiner™ . The equalizer technology was compared to the immuno-subtraction approach. While the ProteoMiner™ technology is superior in terms of the overall number of captured proteins, it only complements the immuno-subtraction approach since the latter can capture the proteins that were not captured by the former.