A workflow for identification of isobaric isoforms of glycans using off-line MALDI-MS system

Nowadays, analysis of glycosylation is one of the indispensable approaches for a development of antibody-drug and biomarker discovery, because a large number of proteins in eukaryotes are glycosylated and they play various roles in physiological function like a molecular recognizing. Therefore, a well-established workflow for characterization of glycosylation has been a one of the growing demands. The author and co-workers has reported a system for identification of glycan structures using an observational MSⁿ spectral library obtained by MALDI-QIT-TOF MS. We demonstrates a practical workflow combined with the spectral library and off-line separation system for the glycan structure characterization.
In this study, glycans released from proteins with N-glycosidase were pyridylaminated and purified by cellulose and graphite carbon tip. A small aliquot of a purified glycan mixture was subjected to MALDI MS analysis directly and a list of candidate precursor ions was generated by the library search system, which is revised to correspond to various chemical labeling methods (Accurate Glycan Analyzer 2; AGA2 / Shimadzu Corp.). The remaining sample was provided to a nano HPLC for the separation of isomers and eluted glycans were automatically fractionated onto a MALDI plate by a spotter (Accuspot; Shimadzu Corp.). Then, a Mass spectrum corresponding to each fraction time was obtained automatically, and the intensity maps of the mass values as a set of candidates for next MSn analysis were created. Finally, MSⁿ analysis was performed semi-automatically on all the found precursors and isomer structures were identified by AGA2.
We applied the workflow to human immunoglobulin and erythropoietin, and obtained successful results.
The result of these glycoproteins indicates that our practical workflow could be applicable to multiple purposes including an biomarker discovery, quality assurance study of antibody-drug, and other screening researches of glycosylation.