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Glycosylationis a common co- and post-translational protein modification,having a large influence on protein properties like conformation andsolubility. Furthermore, glycosylation is an important determinantof efficacy and clearance of biopharmaceuticals such as immunoglobulinG （IgG）. Matrix-assisted laser desorption/ionization （MALDI）-time-of-flight（TOF）-mass spectrometry （MS） shows potential for the site-specificglycosylation analysis of IgG at the glycopeptide level. With thisapproach, however, important information about glycopeptide sialylationis not duly covered because of in-source and metastable decay of thesialylated species. Here, we present a highly repeatable sialic acidderivatization method to allow subclass-specific MALDI-TOF-MS analysisof tryptic IgG glycopeptides. The method, employing dimethylamidationwith the carboxylic acid activator 1-ethyl-3-（3-dimethylamino）-propyl）-carbodiimide（EDC） and the catalyst 1-hydroxybenzotriazole （HOBt）, results in differentmasses for the functionally divergent α2,3- and α2,6-linkedsialic acids. Respective lactonization and dimethylamidation leadsto their direct discrimination in MS and importantly, both glycanand peptide moieties reacted in a controlled manner. In addition,stabilization allowed the acquisition of fragmentation spectra informativewith respect to glycosylation and peptide sequence. This was in contrastto fragmentation spectra of underivatized samples, which were dominatedby sialic acid loss. The method allowed the facile discriminationand relative quantitation of IgG Fc sialylation in therapeutic IgGsamples. The method has considerable potential for future site- andsialic acid linkage-specific glycosylation profiling of therapeuticantibodies, as well as for subclass-specific biomarker discovery inclinical IgG samples derived from plasma.
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