Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin affinity column.
Glycosylation is one the most common post-translational modifications (PTM) and glycoproteins play fundamental roles in a diversity of biological processes. The development of an analytical approach to the study of variation of glycosylation patterns in serum samples has been hindered by the structural heterogeneity of this post-translational modification and the complexity of serum proteome. We have used the ability of different lectins to recognize specific glycosylation motifs to develop a specific affinity system that can achieve a comprehensive capture of serum glycoproteins. In a preliminary investigation, we evaluated the ability of five commonly used immobilized lectins to capture glycoproteins from human serum. SDS-PAGE analysis showed each lectin was able to enrich a subset of the serum glycoproteome and overlaps in lectin specificity were indeed observed. Based on these results and with the goal of studying the extent of the human serum glycoproteome, we then developed a multi-lectin affinity column containing Concanavalin A (Con A), Wheat germ and Jacalin lectin. The selection of lectins was also based on the known N-linked and O-linked glycan structures that are considered representative of the serum proteome. We then demonstrated that the capture of glycoproteins was specific, efficient and reproducible with this multi-lectin column. The results obtained with this affinity step indicated that about 10% of human serum proteins are glycosylated (weight/weight) and, after removal of six high abundance proteins, including albumin, at least 50% of the remaining proteins were glycosylated. We then evaluated the use of this affinity column to monitor changes in the pattern of glycosylation in serum samples by a controlled, stepwise release of the captured proteins from the multi-lectin affinity column with specific displacers.
Yang
Ziping
Z
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
Hancock
William S
WS
D002846:Chromatography, Affinity / Q000379:methods*; D003208:Concanavalin A / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D037121:Plant Lectins / Q000737:chemistry*; D015203:Reproducibility of Results; D014909:Wheat Germ Agglutinins / Q000737:chemistry*
D006023:Glycoproteins; D037121:Plant Lectins; D014909:Wheat Germ Agglutinins; C043696:jacalin; D003208:Concanavalin A