Coupled affinity-hydrophobic monolithic column for on-line removal of immunoglobulin G, preconcentration of low abundance proteins and separation by capillary zone electrophoresis.
A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 microm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1cm monolith was reached after loading 48 ng of protein. Finally, the BuMA-co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins.
Armenta
Jenny M
JM
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602-5700, USA.
Gu
Binghe
B
Thulin
Craig D
CD
Lee
Milton L
ML
D045304:Cytochromes c / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification; D008689:Methacrylates / Q000138:chemical synthesis / Q000737:chemistry; D008855:Microscopy, Electron, Scanning; D009113:Muramidase / Q000737:chemistry; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry; D052616:Solid Phase Extraction; D014362:Trypsinogen / Q000737:chemistry
D007074:Immunoglobulin G; D008689:Methacrylates; D011108:Polymers; D011506:Proteins; D012709:Serum Albumin; C004919:ethylene dimethacrylate; D014362:Trypsinogen; D045304:Cytochromes c; D009113:Muramidase; C016284:butyl methacrylate