Traditional sample preparation techniques for the analysis of N-glycans have either been time-consuming and labor intensive, or require compromises in sensitivity.1 These restrictions result in throughput issues, or limit the level of detail that can be obtained regarding the glycosylation of a sample. In the case of one of the most frequently used labeling compounds, 2-aminobenzamide (2-AB), the resulting glycans can be readily detected by fluorescence but are difficult to detect by electrospray ionization mass spectrometry (ESI-MS). Variations of conventional approaches for N-glycan sample preparation have been explored, but have not presented solutions that combine the desired attributes of simplicity, high MS sensitivity, and high throughput. For example, rapid tagging procedures that yield labeled glycans in minutes have been developed2, however, they did not provide the enhanced ionization efficiencies needed in modern N-glycan MS analyses.
As part of our R&D work into improving biopharmaceutical workflows, we have developed a novel labeling reagent, RapiFluor-MS®, which reacts rapidly with released N-glycans and provides unrivalled sensitivity for their detection through a highly fluorescent chromophore and a tertiary amine for enhancing MS detection.3,4 By incorporating this reagent into an optimized sample preparation workflow for deglycosylation and HILIC-based sample cleanup, we have been able to achieve highly quantitative recovery of the tagged glycans, that are ready for analysis, in under an hour. This poster will provide a detailed overview of this reagent and workflow, along with comparisons to other commercially available tagging solutions.