Efficacy of glycoprotein enrichment by microscale lectin affinity chromatography.
Reproducible and efficient affinity enrichment is increasingly viewed as an essential step in many investigations leading to the discovery of new biomarkers. In this work, we have evaluated the repeatability of lectin enrichment of glycoproteins from human blood serum through both qualitative and quantitative proteomic approaches. In a comprehensive evaluation of lectin binding, we have performed 30 separate microscale lectin affinity chromatography experiments, followed by a conventional sample purification, and LC-MS/MS analysis of the enriched glycoproteins. Two lectin affinity matrixes, both with Con A lectin, immobilized to the same solid support but differing in the amount of immobilized lectin, were investigated to characterize their binding properties. Both qualitative and quantitative data indicate acceptable repeatability and binding efficiency for the lectin materials received from two different commercial sources.
Madera
Milan
M
Department of Chemistry, National Center for Glycomics and Glycoproteomics, Indiana University, IN 47405, USA.
Mann
Benjamin
B
Mechref
Yehia
Y
Novotny
Milos V
MV
D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D003208:Concanavalin A / Q000737:chemistry*; D005260:Female; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D011485:Protein Binding; D020543:Proteome; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods
D006023:Glycoproteins; D037102:Lectins; D020543:Proteome; D003208:Concanavalin A