Development of Isotopically Labeled Glycoproteins for use as Internal Standards

Project Summary/Abstract The ability to accurately quantitate the glycan chains attached to glycoproteins has wide-ranging implications. Numerous studies over the past 40 years have demonstrated that abnormal glycosylation occurs in virtually all types of human cancers, and demonstrates the potential of using glycan markers in either a diagnostic or a prognostic manner. The glycosylation on recombinant protein therapeutics is also known to have profound effects, with one of the better-known examples being the increased serum half-life of erythropoietin (EPO) resulting from glycoengineering. Hence, the quantification of glycoprotein glycans plays important roles from the discovery of new diagnostic/prognostic markers to the development of various therapeutic agents. A current impediment for performing quantitative glycomics is the shortage of widely available standard glycoproteins and isotopically labeled reagents to enable accurate quantitation. The issue with glycan quantitation was highlighted by inter-laboratory studies conducted by the Human Proteome Organization (HUPO) and the Association of Biomolecular Resource Facilities (ABRF). Both of these studies demonstrated errors greater than several hundred percent in the analysis of mid-to-low level glycans were compared across participating laboratories. The inability to accurately quantitate low-level glycans is particularly worrisome since it is often glycans of low abundance that have the greatest impact, as is seen with the therapeutic human intravenous immunoglobulin G (IVIg). The focus of this proposal is to develop a collection of well-characterized standard isotopically labeled glycoproteins to enable the accurate, robust, and reproducible analysis of N- and O-linked glycans at the relative and potentially absolute level. A collection of glycoproteins was selected to cover the needs of both biopharmaceutical and academic researchers. Here, a known quantity of the isotopically labeled glycoprotein can be added directly to the sample, analyzed by any standard procedure that includes mass spectrometry, and glycan quantitation will be provided by comparing the ratios of the native to isotopically labeled ions.Project Narrative The accurate quantification of glycoprotein glycans is important for numerous fields ranging from the discovery of new diagnostic/prognostic markers to the development of various therapeutic agents. A current impediment for performing these analyses is the shortage of widely available reference standards to enable accurate quantitation. The focus of this proposal is to develop a well- characterized standard glycoprotein with isotopically labeled glycans to enable the accurate, robust, and reproducible analysis of N- and O-linked glycans at the relative and potentially absolute level.