Ionization Characteristics of Glycan Homologues in Various Modes of Electrospray

Fluorescence labeled glycan homologous mixtures were quantified using fluorescence and then used to evaluate ionization performances in electrospray ionization at micro, nano, and femto flow modes. nanoESI produced higher (2+ and 3+) charged ions adducted with sodium and calcium. In comparison, femtoESI was found to favor the generation of [M + H]⁺ ions against metal adducts, even with nonvolatile salts up to 1 mM for NaCl and 100 μM for CaCl₂. For labeled glucose homopolymer (GHP) glycans, nanoESI and femtoESI had 0.81 and 3 nM detection limits, respectively. With LC separation and a much higher flow rate, conventional microflow ESI detected all glycans with 10-fold lower concentrations. Overall, nanoESI had the optimum uniformity in the relative ionization efficiency (RIE). When summing up intensities of analyte ions formed with all charge carriers, the RIE of the midsized glycans (10 to 16 glucose units) appear to be uniform (RIE 95%-105%). For the smaller (1-5 glucose units) glycan components, femtoESI provided better uniformity than nanoESI and conventional ESI. For the labeled IgG N-glycans, the impact of chemical structure on the ionization efficiency was revealed by the strong correlation between their RIE trends in different ionization modes.