Toward Automatic Inference of Glycan Linkages Using MS ⁿ and Machine Learning-Proof of Concept Using Sialic Acid Linkages

Glycosidic linkages in oligosaccharides play essential roles in determining their chemical properties and biological activities. MSn has been widely used to infer glycosidic linkages but requires a substantial amount of starting material, which limits its application. In addition, there is a lack of rigorous research on what MSn protocols are proper for characterizing glycosidic linkages. In this work, to deliver high-quality experimental data and analysis results, we propose a machine learning-based framework to establish appropriate MSn protocols and build effective data analysis methods. We demonstrate the proof-of-principle by applying our approach to elucidate sialic acid linkages (alpha 2'-3' and alpha 2'-6') in a set of sialyllactose standards and NIST sialic acid-containing N-glycans as well as identify several protocol configurations for producing high-quality experimental data. Our companion data analysis method achieves nearly 100% accuracy in classifying alpha 2'-3' vs alpha 2'-6' using MS5, MS4, MS3, or even MS2 spectra alone. The ability to determine glycosidic linkages using MS2 or MS3 is significant as it requires substantially less sample, enabling linkage analysis for quantity-limited natural glycans and synthesized materials, as well as shortens the overall experimental time. MS2 is also more amenable than MS3/4/5 to automation when coupled to direct infusion or LC-MS. Additionally, our method can predict the ratio of alpha 2'-3' and alpha 2'-6' in a mixture with 8.6% RMSE (root-mean-square error) across data sets using MS5 spectra. We anticipate that our framework will be generally applicable to analysis of other glycosidic linkages.