Abstract
Despite the recent advances in mass spectrometry (MS)-based methods for glycan structural analysis, characterization of glycomes remains a significant analytical challenge, in part due to the widespread presence of isomeric structures and the need to define the many structural variables for each glycan. Interpretation of the complex tandem mass spectra of glycans is often laborious and requires substantial expertise. Broad adoption of MS methods for glycomics, within and outside the glycoscience community, has been hindered by the shortage of bioinformatics tools for rapid and accurate glycan sequencing. Here, we developed an online porous graphitic carbon liquid chromatography (PGC-LC)-electronic excitation dissociation (EED) MS/MS method that takes advantage of the superior isomer resolving power of PGC and the structural details provided by EED MS/MS for characterization of glycan mixtures. We also made improvements to GlycoDeNovo, our de novo glycan sequencing algorithm, so that it can automatically and accurately identify glycan topologies from EED tandem mass spectra acquired online. The majority of linkages can also be determined de novo, although in some cases, biological insight may be needed to fully define the glycan structure. Application of this method to the analysis of N-glycans released from ribonuclease B not only revealed the presence of 18 high-mannose structures, including new isomers not previously reported, but also provided relative quantification for each isomeric structure. With fully automated data acquisition and topology analysis, the approach presented here holds great potential for automated and comprehensive glycan characterization.