title: 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.
authors.affiliation: Department of Chemistry, Oklahoma State University, Stillwater, OK 74078, USA.
authors.forename: Subhashini
authors.initials: S
authors.lastname: Selvaraju
authors.forename: Ziad
authors.initials: Z
authors.lastname: El Rassi
mesh_terms: D001798:Blood Proteins / Q000032:analysis* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D003300:Copper; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D009532:Nickel; D010446:Peptide Fragments / Q000032:analysis / Q000145:classification / Q000378:metabolism; D019151:Peptide Library; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism; D015032:Zinc
chemical_list: D001798:Blood Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D003300:Copper; D009532:Nickel; D014357:Trypsin; D015032:Zinc