Deglycosylation of Ribonuclease B by PNGase F and Reductive Amination of N-linked Glycans with 2-AB. The dried sample containing 15 μg (1 nmol) of ribonuclease B (Sigma) was incubated in 40 μL of water containing 10 μL denaturing buffer at 100 °C for 10 min. The sample was added with 10 μL G7 buffer, NP40 and 60 μL water. This solution was added 1 μL PNGase F (NEB, 500 unit/μL) and incubated for 24 h at 37 °C. The released glycans were purified by porous graphitized carbon (PGC) column (Alltech) and eluted by 25% acetonitrile. Eluted glycan mixture was completely dried in speed vac. This glycan mixture and standard sample were reductively aminated by 2-aminobenzamide labeling kit (Prozyme). These derivatives were heated at 65 °C for 3 h. The labeled glycans were washed using Whatman paper (3017 915), dried and then stored at −20 °C.
Normal-phase Capillary LC-MALDI Tandem Mass Spectrometry. The normal-phase column was made by packing amide-80 resin (5 μm, TOSOH) in the fused silica capillary tubing (ODxIDxL, 360 μm × 250 μm × 17 cm) using He gas and bomb loader (Proxeon). Normal-phase capillary LC was carried out using a LC-Packings Ultimate System (LC Packings, Amsterdam). Solvent A was 10 mM ammonium formate adjusted to pH 4.4 with formic acid. Solvent B was 20% solvent A in acetonitrile.8 The labeled glycans dissolved in 80% acetonitrile were loaded onto an amide-80 column and eluted with increasing aqueous concentration. The column was equilibrated at 5% A and the gradient was initiated 10 min at flow rate of 1.5 μL/min. after injection and increased to 52% A over 60 min, 95% A over 90 min and 5% A over 100 min. The elution fractions were directly spotted onto a 192 disposible MALDI plate at 30 sec interval using Probot spotter system (LC Packings). The sample spots were overlaid with 0.6 μL of 2,5-dihydroxybenzoic acid (DHB, LaserBio Lab) added with NaI (5 μg) and rapidly dried in vacuum. All mass spectra were obtained with 4700 Proteomic Analyzer (Applied Biosystems, Framingham, MA) and each well on the plate was irradiated with 355 nm UV light of an Nd:YAG laser with a repetition rate of 200 Hz. 1500 to 2000 laser shots were averaged to normal mass spectra and 4800 to 5500 laser shots to MS/MS spectra. The samples were analyzed at 25 kV of source acceleration voltage with two-stage reflection in MS mode. In the MS/MS experiments, collision energy, defined by the potential difference between the source acceleration voltage (8 kV) and the floating collision cell (7 kV), was set to 1 kV.
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