DETERMINING THE SEQUENCE OF OLIGOSACCHARIDES BY NSI-MS

University Of Georgia, Athens GA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Preparation of the per-O-methylated glycans Fifty micrograms of HF2-2P1t20A17 was dissolved in dimethylsulfoxide and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992). The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were dried under a stream of nitrogen. Nanospray Ionization-Linear Ion Trap Mass Spectrometry Mass analysis by NSI-MSn was performed on a LTQ Orbitrap Discoverer mass spectrometer (Thermo Scientific) equipped with a nanospray ion source. Briefly, permethylated glycans were dissolved in 1 mM NaOH in 50% methanol and infused directly into a linear ion trap mass spectrometer using a nanospray source at a syringe flow rate of 0.5 [unreadable]l/min. The capillary temperature was set to 210 [unreadable]C, and MS analysis was performed in positive ion mode. Fragmentation by CID in MS/MS and MSn modes 25% collision energy was applied. The nomenclature of Domon and Costello was used to guide the depiction of fragmentation derived from MSn spectra.