Ultraflex 2 maldi tof mass spectrometer

All reactions were performed under an inert atmosphere of nitrogen or argon, unless otherwise noted. d-Galactose, d-glucosamine hydrochloride, and l-fucose were purchased from Carbosynth LLC (CA, USA). All other reagents were purchased from commercial sources and used directly. All solvents were dried and distilled following standard protocols. All glycosylation reactions were performed in an oven-dried round-bottom flask. Proton nuclear magnetic resonance (¹H NMR) and ¹³C NMR spectra were recorded with a Varian 400 MHz spectrometer and a Bruker 600 MHz spectrometer. High resolution mass spectra (HRMS) were acquired using an UltraFlex II MALDI/TOF mass spectrometer (Bruker Corporation, MA, USA) and Orbitrap Lumos mass spectrometer (Thermo Fisher Scientific, CA, USA). Thin layer chromatography (TLC) was performed on a silica gel matrix with a 254 nm fluorescent indicator, and flash column chromatography purification was performed on silica gel 60 (Sigma-Aldrich Corporation, WI, USA).

β-eliminated glycans were permethylated and analyzed at the Glycomics Core at Beth Israel Deaconess Medical Center as previously described (36 (link)). Mass spectrometry data were acquired on an UltraFlex II MALDI-TOF Mass Spectrometer (Bruker Daltonics). Reflective positive mode was used, and data were recorded between 500 m/z and 6000 m/z. The mass spectrometry O-glycan profile was acquired by aggregating at least 20,000 laser shots. Mass peaks were manually annotated and assigned to a particular O-glycan composition based on known core structures.

β-eliminated glycans were permethylated, lyophilized and then redissolved in 10 μL of 75% methanol, from which 1 μL was mixed with 1 μL 2,5-dihydroxybenzoic acid (DHB) (5 mg/mL in 50% acetonitrile with 0.1% trifluoroacetic acid) and spotted on a MALDI polished steel target plate (Bruker Daltonics). MS data from each spot was then acquired on a Bruker UltraFlex II MALDI-TOF Mass Spectrometer (Bruker Daltonics). Reflective positive mode was used, and data were recorded between m/z 300 and m/z 4000. For each MS O-glycan profile, an aggregation of 20,000 laser shots or more were collected for data extraction. Mass signals of a signal/noise ratio of at least 4 were considered. Once acquired, the data were manually analyzed, and possible O-glycan compositions were annotated based on previously reported insect core structures.