This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Introduction: Sialylation of oligosaccharides affects the fragmentation and the energetics of dissociation. Using dissociation energy profiles of asialo- and sialylated oligosaccharides, the mechanism of glycosidic bond cleavage was observed to depend upon the number of sialic acid residues. Analysis by negative ion nanospray-MS/MS on a quadrupole-orthogonal time-of-flight instrument revealed that sialylated ions require substantially more energy to undergo fragmentation than asialo-forms. In asialo forms, deprotonation of a hydroxyl oxygen destabilized the glycosidic bond, but with sialylated forms, the charge resided on the sialic acid, and more energy was required to fragment the glycosidic bond. The energetic nature of deprotonated asialo-glycan ions facilitates glycosidic bond fragmentation during CID, while deprotonated sialylated glycan ions are lower in energy and resist glycosidic fragmentation. Methods: Lewis A, Lewis X, Sialyl Lewis A, Sialyl Lewis X, LNT, LNnT, DSLNT, LST-a, LST-d, and LST-c were analyzed using negative ion nanospray on an ABI Pulsar i QStar QoTOF MS. Solutions were sprayed from 30% MeOH in water with 0.1% ammonium hydroxide at concentrations between 1 pM and 10 pM. Tandem mass spectra of either singly or doubly charged precursor ions were acquired at varying collision energies, -2.5V to -47.5V. Spectra were collected for one minute and averaged for analysis. Breakdown curves for ions of interest were plotted as the percentage of the total ion intensity versus the collision energy. The threshold for peak selection was held to 5% of the intensity of the base peak. Results: Sialylation of milk oligosaccharides significantly changes the dissociation energetics of this class of compounds. In negative ion nanospray, the energy required to deplete the precursor intensity by 50% of neutral LNT and LNnT is roughly -12V; however the sialylated forms, LST-a and LST-d respectively, are much more stable and require roughly -37V to fragment to the same extent. LNT and LNnT produce abundant Y2 and Y3 ions at low energies, indicating the destabilization of the glycosidic bond, while sialylated structures produce abundant B1 ions showing the loss of a sialic acid residue as well as higher energy C ions. When comparing the energetics of asialo- and sialylated forms of Lewis antigens, large differences were also seen in the collision energy required to fragment the precursor ion to 50% of its initial intensity. Lewis X and Lewis A required -6V, while the sialylated forms required four times the amount of energy, -27-29V. Charge is produced in neutral sugars by deprotonation of a hydroxyl oxygen during the ionization process. This anion is evidently energetic enough to undergo glycosidic bond cleavage at relatively low collision energies. As the ions have gained energy during the ionization process, the energy for complete fragmentation during CID is minimal. The sialylated sugars have the negative charge residing on the terminal sialic acid, remote from the glycosidic oxygen; therefore, to cause glycosidic bond fragmentation energy needs to be added to the system in order to induce homolytic bond cleavage or deprotonate one of the hydroxyl oxygens. Sialylated sugars thus require more energy during the CID process to induce glycosidic cleavages.