Two-dimensional HPLC separation with reverse-phase-nano-LC-MS/MS for the characterization of glycan pools after labeling with 2-aminobenzamide.

N-Glycans, O-glycans, glycolipid glycan chains, and other oligosaccharide pools released from various natural sources often represent very complex mixtures. Various tandem mass spectrometric techniques may be applied to glycan pools, resulting in valuable structural information. For a very detailed analysis of these pools, however, the following approach may be preferable: In a first step, reducing-end oligosaccharides are labeled with the aromatic tag 2-aminobenzamide (2-AB). The 2-AB glycans are separated by analytical-scale, normal-phase (NP)-HPLC (first dimension) and peak-fractionated using fluorescence detection. Peak fractions are analyzed by nano-LC-ESI-IT-MS/MS using a conventional reverse-phase (RP) nanocolumn (second dimension). Chromatography may be monitored by measuring the UV absorbance of the AB tag. Tandem mass spectrometry may be performed on deprotonated species (negative-ion mode), on proton adducts, as well as on sodium adducts (positive-ion mode). This approach has the following particular advantages: (1) the combination of the two HPLC dimensions usually separates isobaric species from each other, thereby allowing the tandem mass spectrometric characterization of individual glycan structures; (2) the AB-mass tag helps with the unambiguous assignment of fragment ions; (3) the second-dimension RP-nano-LC-MS/MS analyses can be performed in many mass spectrometric laboratories, as only standard equipment is needed. Alternatively, for a less in-depth characterization of complex glycan pools, the RP-nano-LC-MS/MS technique may be used as a stand-alone technique. In conclusion, the presented methods allow the detailed mass spectrometric characterization of complex N-glycan pools released from various biological sources.