Ultraflex time of flight mass spectrometer 3

The N-glycans purified by glycoblotting were diluted with 5 µl of 1% AcOH in 5% ACN/water. A total of 1 µl of sample was mixed with 1 µl of 2,5-dihydroxy benzoic acid (FUJIFILM Wako Pure Chemical Corporation) as ionic liquid matrices, and spotted onto a MALDI target plate. The analytes were then subjected to MALDI-TOF MS analysis using an Ultraflex time-of-flight mass spectrometer III (Bruker Daltonics, Inc.) in a reflector, using the positive-ion mode and typically accumulating 2,000 shots. The N-glycan peaks in the MALDI-TOF MS spectra were selected using FlexAnalysis v. 3 (Bruker Daltonics, Inc.). The glycan structures were estimated using the GlycoMod Tool (http://br.expasy.org/tools/glycomod/).

N-Linked oligosaccharides were analyzed using a glycoblotting-based method, as described previously (Nishimura et al., 2004) . Purified hemocyanin (0.1 mg) was reduced and alkylated in the presence of a detergent and then digested by trypsin. N-Glycans of glycopeptides were released from trypsindigested samples by incubation with N-glycosidase F (New England Biolabs) or N-glycosidase A (Roche Applied Science) and then specifically captured on BlotGlyco H beads via hydrazone bonds. The glycans blotted on beads were purified using the Mass PREP HILIC mElution Plate (Waters) and subjected to MALDI-TOF mass spectrometry (MS) analysis using an Ultraflex time-of-flight mass spectrometer III (Bruker Daltonics) in reflector positive ion mode with 2,5-dihydroxylbenzoic acid as a matrix. The detected N-glycan peaks in MALDI-TOF MS spectra were chosen using FlexAnalysis v. 3 software (Bruker Daltonics). The intensity of the isotopic peaks of each glycan was normalized to a 10-pmol internal standard (A2 amide glycan). Glycan structures were determined using the GlycoMod Tool (http://web.expasy.org/glycomod/) and the GlycoSuite website (http://unicarbkb.org/).