Monitoring glycosylation pattern changes of glycoproteins using multi-lectin affinity chromatography.
Previously, we reported that the distribution of glycoproteins into the lectin displacement fractions of a multi-lectin affinity column was determined by the glycosylation patterns of the proteins. This distribution was observed by the sequential use of displacers specific to the lectins in the column. In this study we have evaluated the multi-lectin column (containing Concanavalin A, Wheat germ agglutinin and Jacalin lectin) to screen glycoproteins with known glycosylation pattern changes. The presence of a glycoprotein in a given displacer fraction was determined by LC-MS/MS analysis of a tryptic digest. We have used the enzyme neuraminidase to modify the oligosaccharide chains present in human transferrin, and used the enzymes, neuraminidase and fucosidase, to modify glycoproteins present in human serum. Then, by comparison with the untreated samples, we demonstrated a distribution shift of the enzyme-treated serum glycoproteins in the displacement fractions isolated from the multi-lectin column. The fractions were analyzed by a protein assay, Sequest rank comparison and peak area measurement from the extracted ion chromatogram. The results indicated that the multi-lectin affinity column (M-LAC) is sensitive to changes in the content of sialic acid and fucosyl residues present in serum glycoproteins, and has the potential to be used to screen serum proteins for glycosylation changes due to disease. In addition, the use of a glycosidase to induce specific structural changes in glycoproteins can support the development of multi-lectin column formats specific for detecting changes in the glycoproteome of certain diagnostic fluids and types of disease.
Yang
Ziping
Z
Department of Chemistry and Chemical Biology, Barnett Institute, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
Hancock
William S
WS
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D037102:Lectins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity
D006023:Glycoproteins; D037102:Lectins