Metabolic Incorporation of Stable Isotope Labels into Glycans

with amino acids in cell culture (SILAC), which provides an elegant method for the incorporation of an isotopic label into proteins prior to MS-based proteomics.18 In a SILAC experiment, two cell populations are grown in culture media that are identical except that one of them contains a “light”(natural abundance) and the other a “heavy”(isotopically enriched) form of a particular amino acid (unlabeled and L-Lysine•2HCl (C6, 99%) (CIL #CLM-2247-H) and L-Arginine•HCl (C6, 99%) (CIL #CLM-2265-H), for example). The isotopically heavy amino acids are supplied to cell cultures in place of the natural amino acids, and they become incorporated into all newly synthesized proteins. After multiple cell divisions, each instance of the particular amino acids is replaced by its “heavy” isotope-containing analog. An advantage of this approach is that the cells are mixed together immediately after cell lysis, thereby proteins from both cell types are subjected to the exact same experimental conditions during the sample handling, digestion, purification and separation steps. For this reason, SILAC is often considered the “gold standard” for comparitive quantitative proteomic analyses.19 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 existence1,3 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 inflammation8 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