Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Novel Fluorescence and MS-Active Labeling Reagent

Conventional approaches to the preparation of N-glycans for HILICFLR-MS are either laborious, time-consuming, or require compromises in sensitivity. In the case of one of the most frequently employed 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 yet presented a solution that combines the desired attributes of simplicity, high MS sensitivity, and high throughput. One example is rapid tagging procedures that yield labeled glycans in a matter of minutes. Cook and co-workers have presented the use of a rapid tagging analog of aminobenzamide (AB). In a rapid reaction, the precursor glycosylamines of reducing, aldehyde terminated glycans are modified via a urea linked aminobenzamide. Although this rapid tagging reagent accelerates the labeling procedure, it does not provide the enhanced ionization efficiencies needed in modern N-glycan MS analyses. To address the above shortcomings, we have developed a sample preparation solution that enables unprecedented FLR and MS sensitivity for released N-glycan detection while also improving the throughput of Nglycan sample preparation. A novel labeling reagent has been synthesized that rapidly reacts with glycosylamines following their release from glycoproteins. Within a 5 minute reaction, N-glycans are labeled with RapiFluor-MS, a reagent comprised of an N-hydroxysuccinimide (NHS) carbamate rapid tagging group, an efficient quinoline fluorophore, and a tertiary amine for enhancing ionization. To further accelerate the preparation of N-glycans, rapid tagging has been directly integrated with a Rapid PNGase F deglycosylation procedure involving RapiGest SF surfactant and a HILIC μElution SPE clean-up step that provides highly quantitative recovery of the released and labeled glycans with the added benefit of not requiring a solvent dry-down step prior to the LC-FLR-MS analysis of samples. Syunya Sasaki, Matthew A. Lauber, Darryl W. Brousmiche, Zhengmao Hua, Stephan M. Koza, Ellen Guthrie, Paula Magnelli, Christopher H. Taron, Kenneth J. Fountain Nihon Waters, Tokyo, Japan Waters Corporation, Milford, MA New England BioLabs, Ipswich, MA