Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins.
We report on the preparation of an improved multi-lectin affinity support for HPLC separations. We combined the selectivity of three different lectins: concanavalin A (ConA), wheat germ agglutinin (WGA), and jacalin (JAC). Each lectin was first covalently immobilized onto a polymeric matrix and then the three lectin media were combined in equal ratios. The beads were packed into a column to produce a mixed-bed multi-lectin HPLC column (high-performance multi-lectin affinity chromatography (HP-M-LAC)) for fast chromatographic affinity separations. The support was characterized with respect to kinetics of immobilization, ligand density, and binding capacity for human plasma glycoproteins. A high lectin density (15 mg/mL of beads) was found to be optimal for the binding of glycoproteins from human plasma. A single clinical sample can be fractionated in less than 10 min per run, making this a useful sample preparation tool for proteomics/glycoproteomics studies associated with disease abnormalities.
Kullolli
Majlinda
M
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
Hancock
William S
WS
Hincapie
Marina
M
D000327:Adsorption; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D003208:Concanavalin A / Q000737:chemistry*; D005260:Female; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000737:chemistry*; D008024:Ligands; D037121:Plant Lectins / Q000737:chemistry*; D011485:Protein Binding; D012680:Sensitivity and Specificity; D013499:Surface Properties; D013997:Time Factors; D014909:Wheat Germ Agglutinins / Q000737:chemistry*
D006023:Glycoproteins; D037102:Lectins; D008024:Ligands; D037121:Plant Lectins; D014909:Wheat Germ Agglutinins; C043696:jacalin; D003208:Concanavalin A