Anion-exchange chromatography using short monolithic columns as a complementary technique for human serum albumin depletion prior to human plasma proteome analysis.
In order to enable the detection of low abundance proteins from human plasma, it is necessary to remove high abundance proteins. Among them, human serum albumin and immunoglobulin G represent more than 75% of all such proteins. In this paper, the characterization of short monolithic columns was performed followed by the optimization of a multidimensional approach, known as conjoint liquid chromatography, to deplete human serum albumin and immunoglobulin G from a human plasma sample. Two different chromatographic modes were used: ion-exchange chromatography and affinity chromatography. A monolithic stationary phase (convective interaction media disk) bearing strong anion-exchange groups and another immobilized with protein G were placed in series into one housing. The optimal binding conditions were found that removed a majority of human serum albumin and immunoglobulin G from the human plasma sample. This method was compared to the depletion using a combination of pseudo-affinity and affinity columns. The results of the human serum albumin and immunoglobulin G depletion were confirmed by 2D electrophoresis. It has been shown that anion-exchange and affinity chromatography using convective interaction media monolithic columns can represent an efficient complementary technique for human serum albumin and immunoglobulin G removal from human plasma.
Petric
Tatjana Cerk
TC
Lek Pharmaceuticals d.d., Verovskova 57, SI-1526 Ljubljana, Slovenia.
Brne
Peter
P
Gabor
Bostjan
B
Govednik
Lidija
L
Barut
Milos
M
Strancar
Ales
A
Kralj
Lucija Zupancic
LZ
D015415:Biomarkers; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry; D007525:Isoelectric Focusing; D008024:Ligands; D010949:Plasma / Q000737:chemistry; D020543:Proteome / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry*
D015415:Biomarkers; D007074:Immunoglobulin G; D008024:Ligands; D020543:Proteome; D012709:Serum Albumin