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

Alterations of protein glycosylation 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 analyses. Here, we develop a label-free workflow based on high sensitivity capillary zone electrophoresis-mass spectrometry (CZE-MS) for profiling of native underivatized released N-glycans. Our workflow provides a >45-fold increase in signal intensity compared to the conventional CZE-MS approaches used for N-glycan analysis. 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 results in the 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 is increased up to ~15-fold, enabling highly sensitive analysis of sample amounts as low as sub-0.2 nL of plasma volume equivalents. Furthermore, highly sialylated N-glycans are identified and structurally characterized, and untreated sialic acid-linkage isomers are resolved in a single CZE-MS analysis.