DOI: 10.5281/zenodo.4705546
title: Released N-glycans from IgG and Fetuin on Agilent QTOF and Orbitrap

description: <strong>Acknowledgements: </strong>This data was kindly prepared by and donated by the Biologics MS group at Charles River Labs, Inc. <strong>2-Aminobenzamide labeled glycans </strong> Released N-glycans were prepared from IgG (human serum, Sigma) and Fetuin (fetal bovine serum, Sigma) by using 2-Aminobenzamide (2-AB) labeling reagent. In summary the samples were buffer exchanged to 1X GlycoBuffer 2 (New England Biolabs) using Amicon 10K MWCO ultrafiltration device. N-glycans were released by adding PNGase F enzyme (New England Biolabs), using 2 µl of enzyme per 100 µg of protein, and incubating the sample at 37 °C for 18 hours. Released N-glycans were then purified by adding 90% ethanol and keeping the samples at -80 °C for 2 hours to precipitate the proteins. The supernatant was separated form the deglycosylated proteins by using centrifuge at 14000 rpm for 10 minutes and dried in speed-vacuum. 2-AB labeling was done by adding 2-AB-sodium cyanoborohydride derivatization solution, and vertexing the samples at 65 °C for 2.5 hours. Unreacted 2-AB was removed by acetone precipitation. For LC-MS analysis the samples were resuspended in 70% ACN:H2O, 0.05% Trifluoroacetic Acid (TFA), 10 µg of protein was used for the injection. <strong>Protein digestion </strong> Fetuin was used as a model glycoprotein for glycopeptide analysis. 100 µg of fetuin was dissolved in 90 µl of 6 M Guanidine hydrochloride in 100 mM of Tric/HCl, pH 8.5, then incubated at RT for 5 minutes to denature the protein. 10 µl of 100 mM Dithiothreitol (DDT, final concentration 10mM) was added for reduction, sample was incubated at 56 °C for 45 minutes. 6 µl of 500 mM Iodoacetamide (IAA, final concentration 30 mM) was added for reduction, and incubated at room temperature for 1 hour. The sample was then diluted with 100 mM of Tric/HCl, pH 8.5, for 7 times to decrease the Guanidine hydrochloride concentration to 0.8 M. The protein was then digested for overnight by adding 5 µl of 2 µg/µl trypsin (Promega) solution. The sample was purified using Top Tip HILIC SPE (PolyLC Inc., Columbia, MD), and dried in speed-vacuum. The sample was dissolved to 2% ACN:H2O, 0.1% formic acid (FA) solution, and 500 ng was used for the LC-MS analysis. <strong>Analysis by QTOF LC/MS/(MS)</strong> Released 2-AB labeled N-glycans were analyzed using Agilent QTOF LC/MS/(MS). The mass spectrometer was calibrated in 2GHz extended range (3000 m/z) positive-ion mode prior to analysis using Agilent calibration tuning mix. Separation was performed on Acquity BEH Amide column (1.7 μm, 2.1 x 150 mm, Waters). The column temperature was 45 °C, with fluorescence detector. Solvent A was 0.05% TFA in water and solvent B was 0.05% TFA in acetonitrile, with the flow rate at 500 µl/min. To achieve separation the following flow gradient was used: 72% solvent B for 5 minute,72-60% solvent B for 80 minutes, 60-10% solvent B for 4 minutes, stay at 10% solvent B for 10 minutes. Back to 72% B in 1 minute and keep at 72% B for the last 15 minutes. MS/(MS) data were obtained in positive ion mode with MS and MS/MS scan range: 300 to 3200 m/z and 100 to 3000 m/z, respectively. Fragmentor voltage was 250 V, octopole RF voltage was 750 V, nozzle voltage was 3500 V, gas temperature was 225 °C, drying gas flow rate was 18 L/min, nebulizer gas pressure was 35 psig, and sheath gas temperature was 275 °C. <strong>Analysis by Orbitrap nLC/MS/(MS)</strong> Glycopeptide analysis by LC-MS/MS was performed on a Dionex 3000 Ultimate nano-LC system (Dionex, Sunnyvale, CA) interfaced to Orbitrap FusionTM LumosTM mass spectrometer (Thermo Fisher Scientific) equipped with a nano-ESI source. Separation was performed using a PicoFrit C18 capillary column (75 μm id × 15 cm, New Objective). The flow rate was set at 450 nL/min and solvent A was 2% acetonitrile containing 0.1% formic acid, and solvent B was 98% acetonitrile with 0.1% formic acid. To achieve separation the following flow gradient was used: 5% solvent B for 0–5 minute, ramping of 5–35% solvent B for 10–60 minutes, ramping of 35–95% solvent B for 60–70 minutes and keep for 10 minutes, decreasing 95–5% solvent B from 85–86 minutes, and maintaining 5% solvent B from 86–100 minutes. Glycopeptides were electrosprayed at 2250V with NSI source, and the ion transfer tube was set at 350 °C. The mass spectrometer was operated in data-dependent and positive ion mode. The precursor ion (MS1) was analyzed in orbitrap detector, with 120,000 resolution, over a scan range from 300-2000 m/z, using 30% RF lens, with 60ms maximum injection time, and scanned every 3 seconds. Precursor ions that have charge states +2 to +6 were isolated for MS2. The CID MS2 was analyzed in ion trap, the isolation window was set at 1.6 m/z, using 30% CID energy and activation time was set at 10ms. HCD MS2 was analyzed in orbitrap with 30000 resolution, using 40% HCD energy, with the isolation window of 1.6 m/z. The dynamic exclusion was for the ions with a repeat count of 2. The repeat duration was set to 10 seconds, and the dynamic exclusion of an ion was maintained for 40 seconds.