Chemical Cross‐Linking Immobilized Concanavalin A for use in Proteomic Analyses

Abstract

Lectin affinity chromatography was used to reduce the amount of the abundant glycoprotein β‐conglycinin in total protein samples prepared from developing soybean (Glycine max L. Merrill cv. Jack) seeds. Electrophoretic analysis of both the concanavalin A‐Sepharose binding and non‐binding fraction revealed an abundant protein band at Mr 26,000. The amount of this protein was greatly increased when concanavalin A‐Sepharose was used with urea‐containing buffers. Peptide mass fingerprint analysis of this abundant protein band unequivocally identified it as concanavalin A (con A). A simple and gentle method was used to chemically cross‐link the con A subunits so that the lectin‐Sepharose retained the ability to bind high‐mannose type glycoproteins. The chemically cross‐linked con A‐Sepharose was stable in buffers that contained up to 8M urea, making this an affinity matrix suitable for use in electrophoresis‐based proteomic analyses.

Keywords:

• Chemical cross‐linking
• Concanavalin‐A
• Proteomic analysis

Acknowledgments

Monica Schmidt generously provided the developing soybean seeds. The authors acknowledge The Proteomics Center at the University of Missouri‐Columbia for gel electrophoresis and MS services.