Polysialylated N‑Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations, and

ABSTRACT: The N-glycan diversity of human serum glycopro-teins, i.e., the human blood serum N-glycome, is both complex and constrained by the range of glycan structures potentially synthesiz-able by human glycosylation enzymes. The known glycome, however, has been further limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to include polysialylated N-glycans. Sample preparation improvements included acidified, microwave-accelerated, PNGase F N-glycan release to promote lactonization, and sodium borohydride reduction, that were both optimized to improve quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. Online separations were improved b