Nature excels at breaking down glycans into their components,often in a systematic and predictable manner,typically via enzymatic acid-base catalysis to achieve selective cleavage of the glycosidic bond.Noting the importance of proton transfer in the active site of many of these enzymes,we developed a proton reagent for acid-catalyzed glycan sequencing (PRAGS) that derivatizes the reducing terminus of glycans with a pyridine moiety possessing moderate proton affinity.Gas-phase collisional activation of PRAGS-derivatized glycans predominately generated glycosidic bond cleavages retaining the charge on the reducing terminus (Y ions),rendering glycan composition and structure analysis.Glycans are also highly susceptible to dissociation by free radicals,which led us to develop a related free radical activated glycan sequencing (FRAGS) reagent,in which a free radical initiator is coupled to to the recuding terminus of the glycan.Collisional activation of FRAGS-derivatized glycans produced abundant cross-ring cleavages (X ions),glycosidic bond cleavages (Y and Z ions),and combinations of these types of cleavages (X+Y,Y+Y,Y+Z,and Z+Z).Branched sites were readily identified with the FRAGS reagent by the combination of Y+Z and Z+Z (Z+Z-H and Z+Z+H) ions that are observed only at these locations.Moreover,the presence of B ions upon CID of singlyprotonated FRAGS-derivatized glycans was utilized to identify the existence and location of an N-acetylated glycan unit,and the Z+Z-H ion can be employed to elucidate the site of reducing terminus.Furthermore,the fragmentation efficiency of this method is much higher than those of ECD,EDD,and ETD.The mechanism of dissociation as well as characteristic examples of the utility of these techniques for glycan sequencing are investigated and discussed.