Application Note 23

Metabolic Incorporation of Stable Isotope Labels into Glycans

Ron Orlando

Complex Carbohydrate Research Center, Departments of Biochemistry and Molecular Biology, and Chemistry
University of Georgia, Athens, GA 30602 USA

Glycosylation is one of the most common post-translational protein modifications in eukaryotic systems.^1-3 It has been estimated that 60-90% of all mammalian proteins are glycosylated at some point during their existence^1,3 and virtually all membrane and secreted proteins are glycosylated.² Glycoprotein glycans often play crucial roles in physiological events such as cell-cell recognition,^4-6 signal transduction,⁷ inflammation,⁸ and tumorigenesis.^9-13 Given the important physiological roles of protein glycosylation, numerous research groups have devoted significant effort to the character­ization of specific glycan structures, the identification of proteins that express each glycan, and the detailed study of how these structures change, e.g., as cells differentiate or as tumor cells progress. All of these efforts have given rise to the emerging field of glycomics.¹⁴

Mass spectrometry (MS) has developed into the analytical method of choice as it provides a rapid, sensitive, and reliable method to analyze complex mixtures.¹⁵ Glycomic studies typically involve the en toto release of the glycans followed by MS analysis of the glycans.^16,17 While this is a fruitful approach for qualitative characterization of the glycome, multiple issues arise when MS is used to obtain quantitative results. For instance, matrix effects, caused by phenomenon such as ion suppression from the presence of other compounds competing with or interfering with the ionization of the analyte, can alter the response from a particular analyte even when the analyte’s concentration does not change. Other issues that interfere with quantitative MS analyses result from variable instrument response, instrument-to-instrument variability, and differential losses during sample handling/processing. The success of the different approaches for relative quantitation depends on how well each of these sources of error is addressed.

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“My lab has utilized a range of CIL products, and we have been very pleased with their performance for quantitative / comparative proteomics and glycomics. The CIL sales staff is very helpful and extremely knowledgeable. They have aided us on experimental design numerous times, and without this help we would not have been able to do this work.”

– Ron Orlando