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Oct 1, 2009 (Vol. 29, No. 17)

Advances in Quantitative Proteomics

Alternatives Designed to Overcome the Limitations of 2-D Electrophoresis Are Under Development

  • Protein Glycosylation Characterization

    Tomas Rejtar, Ph.D., research assistant professor at Northeastern University’s Barnett Institute, along with Professor Barry Karger and colleagues, described the development of an analytical method for intact glycoproteins based on high-resolution capillary electrophoresis separation coupled to MS, which was developed in collaboration with Momenta Pharmaceuticals.

    Using this method, the investigators analyzed and compared glycoproteins obtained from different sources and discovered significant differences in glycoform abundance and composition among the proteins.

    To perform the analyses, the scientists used a capillary electrophoresis (CE)-MS instrument with a pressurized liquid junction-based interface coupled to an LTQ-FT instrument. Proteins used to evaluate the analytic performance of this system included the alpha-subunit of human chorionic gonadotropin with two glycosylation sites.

    High-resolution capillary electrophoresis enabled separation of intact protein glycoforms differing in the number of sialic acids as well as those with varying neutral glycan content. Analysis of the same sample showed that relative abundance of individual glycoforms could be quantitated by CE-MS with a relative standard deviation of less than 10%. The method can also be combined with enzymatic deglycosylation of the intact proteins to assign glycans to specific sites for further glycoprotein characterization, Dr. Rejtar said.

    This method has the potential for application in the rapid profiling of glycosylation in therapeutic proteins, according to Dr. Rejtar. Unlike traditional methods that rely on analysis of released glycans, direct analysis of intact glycoproteins by CE-MS allows fast monitoring of changes in glycosylation or other modification with limited sample preparation, he added.



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