Reduction and labeling of oligosaccharides/released N-glycans with Procainamide v1

Liquid Chromatography (LC)-Mass Spectrometry (MS) is a well-known tool for the characterization and quantification of N-glycans (1–5). The structure of glycans is highly fragile under the influence of MS ionization, and the unreduced N-acetylglucosamine (GlcNAc) terminus often creates complex chromatographic profiles. Derivatization of the terminus of N glycans is highly recommended in LC MS analysis. Derivatization not only significantly improves MS signal (6–9) but also avoids complex chromatographic behavior by converting unreduced GlcNAc into sugar alditols(10). Reductive amination is a common method for N-glycan derivatization (6). Procainamide (ProA) is a popular tertiary amine fluorescence tag with high ionization efficiency (8) and is a cost-effective option for research applications. This protocol describes in detail the labeling and reduction procedures for released N-glycans using ProA and sodium cyanoborohydride (NaBH₃CN), respectively. This protocol can also be used for other reductive amination tags, such as 2-aminobenzamide (2-AB) and 2-aminobenzoic acid (2-AA), and to derivatize other oligosaccharides with a reducing-sugar terminus._