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

Abstract

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.

Equalizer technology followed by DIGE‐based proteomics for detection of cellular proteins in artificial peritoneal dialysis effluents

“…In these studies the equalizer beads have been used in small sample volumes with high protein concentration. An increasing number of evidence, including publications in this journal, reports on the power and reproducibility of the equalizing technique . Unlike other biomaterials for which the equalizer bead technology has been applied, PDE can be obtained as waste material in bulk.…”

Postpolymerization modification of a hydroxy monolith precursor. Part I. Epoxy alkane and octadecyl isocyanate modified poly (hydroxyethyl methacrylate‐co‐pentaerythritol triacrylate) monolithic capillary columns for reversed‐phase capillary electrochromatography

“…In the selection of the final composition of the polymerization composition (in terms of % monomers, % porogens and initiator) used for preparing the optimized poly(HEMA‐co‐PETA) precursor monolith (i.e. OHM precursor), we relied on the experience gained in our laboratory from previous works on monoliths . The optimal monolith was free‐flowing thus suitable for post‐polymerization modification with alkyl ligands via the regio‐selective ring opening reaction of 1,2‐epoxyalkanes in the presence of BF 3 as the catalyst.…”

Liquid phase based separation systems for depletion, prefractionation, and enrichment of proteins in biological fluids and matrices for in‐depth proteomics analysis—An update covering the period 2011–2014

“…The CPLL technology was combined with other fractionations such as tandem iminodiacetic acid (IDA)-metal chelate columns (e.g., IDA-Zn 2+ , IDA-Ni 2+ and IDA-Cu 2+ ) in series with an RPC column [26]. After the equalization step, the treated proteins from human serum were fractionated on a series of IDA-metal columns arranged in the order: IDA-Zn 2+ →IDA-Ni 2+ →IDA-Cu 2+ followed by the RPC column.…”