Release of N-glycans and fluorescent labelling
N-glycans were released from human serum IgG and bovine fetuin (15 µg, both from Sigma-Aldrich, St Louis, MO, USA, cat. number I4506 and F3004). The glycoproteins were dried down and reconstituted in water (22.8 µL) and denatured using Rapigest™ SF (5% w/v, 6 µL) (Waters Corporation, Milford, MA, USA, cat. number 186001861) at 90 ºC for 10 min. After the denatured proteins have cooled to room temperature, PNGase F (600 U, 1.2 µL) (New England Biolabs, Ipswich, MA, USA, cat. number P0709L) was added with incubation at 55 ºC for 10 min to release the N-glycans. RFMS label (Waters Corporation, Milford, MA, USA, cat. number 186008091-1) was reconstituted in 131 µL anhydrous dimethyl formamide (DMF) and 12 µL of the label was added to the N-glycans followed by incubation at room temperature for 10 min.
For labelling with ProC (Sigma-Aldrich, St Louis, MO, USA, cat. number SML2088) and 2-AB (Acro Organics, Geel, Belgium, cat. number 104901000), the labelling protocol was as previously described16. In brief, the labelling mixture was prepared freshly by either dissolving ProC (38.3 mg/mL) or 2-AB (19.2 mg/mL) and 2-picoline borane (44.8 mg/mL) in dimethylsulfoxide (DMSO) and glacial acetic acid (70 : 30, v/v). The label (25 µL) was then added to the released N-glycans and incubated at 65 ºC for 2 h.
Purification of fluorescent derivatized N-glycans using hydrophilic 96-well micro SPE
After fluorescent derivatization, RFMS labelled N-glycans were diluted with 358 µL acetonitrile (ACN) and purified with Glycoworks HILIC µElution plate (Waters Corporation, Milford, MA, USA, cat. number 186002780) according to the manufacturer’s protocol (Waters Corporation). Each well was equilibrated with water (200 µL), followed by 85% (v/v) ACN (200 µL). The N-glycans were loaded to the well and the well was washed twice with 90% (v/v) ACN containing 1% formic acid (600 µL). Elution of the N-glycans was accomplished by addition of 3 × 30 µL of the GlycoWorks SPE elution buffer (Waters Corporation, Milford, MA, USA, cat. number 186007992). The eluted glycans were subsequently dried down in a vacuum centrifuge and reconstituted in a buffer consisting of water, dimethylformamide (DMF) and ACN (in ratio of 9 : 10 : 21, v/v) (40 µL) for analysis.
Purification of fluorescent derivatized N-glycans using PD MiniTrap G10 column
PD MiniTrap G10 column (Cytiva, Marlborough, MA, USA, cat. number GE28-9180-10) was equilibrated with 8 mL of water under gravity flow. RFMS labelled N-glycans were diluted with water (final volume of 300 µL) and loaded onto the column. The column was rinsed with 400 µL of water and N-glycans were eluted from the column with 600 µL of water. The eluted glycans were subsequently dried down and reconstituted as described in the HILIC-SPE procedure.
Purification of fluorescent derivatized N-glycans using cellulose magnetic beads (CMBs)
Fluorescent derivatized N-glycans were diluted with ACN to a final concentration of 90% (v/v) ACN. Storage buffer from the CMBs (200 µg) (Life Technologies Corporation, Carlsbad, CA, USA, cat. number, 4489112) was first removed and the beads were washed twice with water before being added to the labelled N-glycans, incubated at 24 °C with mixing on the Thermomixer® C (Eppendorf, Hamburg, Germany) at 1200 rpm for 5 min. After 5 min, the beads were immobilized onto a magnetic stand and the supernatant was removed. The beads were rinsed twice with 200 µL of 90% (v/v) ACN. N-glycans were eluted from the beads by addition of 200 µL of water with incubation at 24 °C and mixing at 1200 rpm for 5 min. The beads were immobilized and the purified N-glycans were collected and dried down using a vacuum centrifuge. 2-AB and ProC labelled N-glycans were reconstituted in 40 µL of 75% (v/v) ACN while RFMS labelled N-glycans were reconstituted as described in the HILIC-SPE procedure for injection into the LC-FLR-MS.
Automation of release of N-glycans, fluorescent labelling and purification procedure on liquid handler
The steps for release of N-glycans, RFMS labelling and their purification using CMBs were automated onto a robotic liquid handler (Tecan Freedom Evo 150, Tecan, Männedorf, Switzerland). The release of N-glycans from protein and RFMS labelling on the liquid handler was adapted from Waters Corporation47. In brief, the samples were dried down in a 96-well non-treated PCR plate. Water (10 µL) and Rapigest™ SF (3%w/v, 10 µL) were added to the samples in the plate and the plate was incubated at 90 ºC for 10 min for denaturation followed by addition of PNGase F (600 U, 10 µL) and incubation at 55 °C for another 10 min. After the plate is cooled for 5 min, RFMS label (10 µL), reconstituted in 110 µL anhydrous DMF, was then added and incubated at room temperature for at least 10 min.
CMBs (200 µg) were aliquoted into a 2 mL 96 deep-well non-treated plate. The storage buffer was removed and the beads washed twice and the RFMS labelled samples were transferred from the PCR plate to the deep-well plate containing the CMBs. ACN was added to the plate to a final concentration of 90% (v/v). The plate was then transferred manually to an external mixer for mixing on the Thermomixer® C at 1200 rpm for 5 min due to the speed limitation of the mixer on the workstation. After 5 min, the plate was transferred back to the liquid handler where subsequent removal of the supernatant and the washing steps were carried out. For elution, the mixing was similarly carried out on the external mixer before being transferred back to the workstation for collection of the eluate onto a new plate.
LC-FLR-MS analysis of N-glycans
Fluorescent derivatized N-glycans were analysed on a H-class Acquity UPLC system (Waters Corporation) consisting of a quaternary solvent manager, sampler manager and fluorescence detector coupled to a Xevo G2-S Q-TOF MS (Waters Corporation) system.
The labelled N-glycans were separated on a Waters Acquity UPLC glycan BEH amide column (2.1 mm x 150 mm, 1.7 μm) with mobile phase A comprising of 50 mM ammonium formate at pH 4.4 and ACN as mobile phase B. The separation gradient was 75 − 51% B at a flow rate of 0.4 mL/min for 40 min with the temperature of the column maintained at 60 °C. N-glycans were detected on the fluorescence detector at an excitation wavelength of 265 nm and emission wavelength of 425 nm for RF-MS labelled glycans, at 310 nm and 370 nm for ProC labelled glycans, and at 250 nm and 428 nm for 2-AB labelled glycans. An external standard of RF-MS, 2-AB (both from Waters Corporation, Milford, MA, USA, cat. number 186006841, 186007982) and ProC (Ludger, Oxfordshire, UK, cat. number CPROC-GHP-30) labelled dextran ladder was used respectively to establish a calibration curve for the different fluorescently labelled N-glycans from which the retention time of each chromatographic peak was converted into glucose units (GU).
Full MS scan data was acquired in positive ion mode in the range of 100 to 2,000 m/z using a capillary voltage of 2.75 kV, with the cone voltage set to 15 V, source temperature at 120 °C, desolvation gas flow at 800 L/H, desolvation temperature at 300 ˚C and scan time of 1 s. Glu-fibrinopeptide (m/z 785.8421) was used as a lock spray mass to calibrate the mass accuracy of the instrument during the analytical runs.
Data processing and structural assignment of N-glycans
Data acquisition and processing was performed using UNIFI scientific information system software (version 1.8.2.169, Waters Corporation). The retention time of each chromatographic peak was processed and converted into GU values and structural assignment of each peak was performed by matching their GU values against commercial library databases. For GU values that correspond to multiple structures, the MS data was used in addition to assign the correct structure based on mass confirmation (mass accuracy of 5 ppm). Relative abundances of each glycan was calculated and normalized by dividing the area of each glycan peak over the total area of all the glycans identified in the chromatograph.
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- Source: https://www.nature.com/articles/s41598-024-80649-y