Resin- and Magnetic Nanoparticle-Based Free Radical Probes for Glycan Capture, Isolation, and Structural Characterization
By combining the merits of solid supports and free radical activated glycan sequencing (FRAGS) reagents, we develop a multifunctional solid-supported free radical probe (SS-FRAGS) that enables glycan enrichment and characterization. SS-FRAGS comprises a solid support, free radical precursor, disulfide bond, pyridyl, and hydrazine moieties. Thio-activated resin and magnetic nanoparticles (MNPs) are chosen as the solid support to selectively capture free glycans via the hydrazine moiety, allowing for their enrichment and isolation. The disulfide bond acts as a temporary covalent linkage between the solid support and the captured glycan, allowing the release of glycans via the cleavage of the disulfide bond by dithiothreitol. The basic pyridyl functional group provides a site for the formation of a fixed charge, enabling detection by mass spectrometry and avoiding glycan rearrangement during collisional activation. The free radical precursor generates a nascent free radical upon collisional activation and thus simultaneously induces systematic and predictable fragmentation for glycan structure elucidation. A radical-driven glycan deconstruction diagram (R-DECON) is developed to visually summarize the MS² results and thus allow for the assembly of the glycan skeleton, making the differentiation of isobaric glycan isomers unambiguous. For application to a real-world sample, we demonstrate the efficacy of the SS-FRAGS by analyzing glycan structures enzymatically cleaved from RNase-B.
© 2019 American Chemical Society. Received: March 13, 2019; Accepted: November 13, 2019; Published: November 13, 2019. This work is supported by the National Institutes of Health through Grant 1R15GM121986-01A1, National Science Foundation through Grant CHEM1709272, Beckman Institute at Caltech, and National Science Foundation through Grant CHEM1508825.
Supplemental Material - ac9b01303_si_001.pdf
Accepted Version - nihms-1633117.pdf