Ultra-sensitive label-free deep profiling of N-glycans released from biomedically-relevant samples

Abstract Protein glycosylation profiles can serve as sensitive and specific disease biomarkers. Labeling procedures for improved separation and detectability of oligosaccharides have several drawbacks, including incomplete derivatization, side-products, noticeable desialylation/defucosylation, sample loss, and interference with downstream analysis. We developed a novel label-free workflow based on high sensitivity capillary zone electrophoresis-mass spectrometry (CZE-MS) for profiling of native underivatized released N-glycans that resulted in > 45-fold increase in signal intensity compared to conventional techniques. Qualitative and quantitative N-glycan profiling of purified human serum IgG, bovine serum fetuin, bovine pancreas ribonuclease B, blood-derived extracellular vesicle isolates, and total plasma resulted in detection of > 250, >400, > 150, >310, and > 520 N-glycans, respectively, using injected amounts equivalent to < 25 ng of model protein and nL-levels of plasma-derived samples. Compared to reported results for biological samples of similar amounts and complexity, the number of identified N-glycans was increased up to ~ 15-fold. Also, previously undetected highly sialylated N-glycans were identified and structurally characterized. For the first time, untreated sialic acid-linkage isomers were resolved in a single CZE-MS analysis.