Protein glycosylation alters with numerous physiological events, like disease, ageing and pregnancy, and is therefore considered to be a potential source of clinical biomarkers. To exploit protein glycosylation for biomarker discovery, both in-depth and high-throughput (HTP) analysis methods are needed. MALDI-TOF-MS provides a suitable HTP platform to study protein glycosylation on both the oligosaccharide and the glycopeptide level, however it shows biases in ionization and metastable decay for the analysis of sialylated glycoconjugates. To stabilize sialic acids and prevent ionization bias, we developed an array of reaction conditions that do not only stabilize sialic acids, but in addition do this in a sialic acid linkage-specific manner. In a two-step reaction, we used the carboxylic acid activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and catalyst 1-hydroxybenzotriazole (HOBt) in combination with the nucleophiles ethanol (step 1) and ammonia (step 2) to induce ethyl ester formation on alpha2,6-linked sialic acids of oligosaccharides and amide formation on alpha2,3-linked sialic acids, allowing linkage-specific MALDI-TOF-MS analysis. This reaction could additionally be combined with N-glycan reducing-end labeling for ultra-high sensitivity profiling by capillary-electrophoresis coupled to MS. For MALDI-TOF-MS analysis of IgG glycopeptides, we found derivatization using dimethylamine more suitable than ethanol, as the latter resulted in a diverse range of reaction products of the peptide portion. The reaction with dimethylamine and ammonia additionally found application in the in situ sialic acid derivatization on FFPE tissues, allowing MALDI-TOF-MS imaging of N-glycans after enzymatic release. We present a comprehensive toolbox for the sialic acid linkage-specific analysis of oligosaccharides and glycoconjugates. The rapid ethyl esterification reaction is used for released glycan analysis giving in-depth information about the glycome of complex samples such as plasma or cells. The dimethylamidation reaction is suitable for the analysis of IgG glycopeptides and in situ glycan imaging on tissue. We currently work on applying these methods in a clinical setting.

Linkage-specific sialic acid derivatization strategies for mass spectrometric profiling of glycans and glycopeptides in complex samples and tissues

256th National Meeting (2018)

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