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Human serum glycan profiling with mass spectrometry (MS) has been employed to study several disease conditions and is demonstrating promise in, for example, clinical biomarker discovery. However, the low glycan ionization efficiency and the large dynamic range of glycan concentrations in human sera can hinder comprehensive profiling. In particular, large glycans are problematic because they are present at low concentrations and are prone to fragmentation. Here we show that, following liquid chromatographic separation on graphite columns, subambient pressure ionization with nanoelectrospray (SPIN)-MS can expand the serum glycome profile in comparison with the conventional atmospheric pressure electrospray ionization (ESI)-MS with a heated capillary inlet. Notably, the ions generated by the SPIN interface were observed at higher charge states for approximately half of the annotated glycans. Out of a total of 130 detected glycans, 34 were only detected with the SPIN-MS, resulting in improved coverage of glycan families as well as of glycans with larger numbers of labile monosaccharides.

Improving N-Glycan Coverage using HPLC-MS with Electrospray Ionization at Subambient Pressure