Before starting this protocol, users need to bring the following reagents and equipment (taking 1 IgG sample as an example):
(1) 100uL of 0.85% sodium chloride solution; Acetonitrile (LCMS grade); Ultrapure water;
(2) 15/85 = ultrapure water/acetonitrile solution (v/v), 2 mL; 1/9/90 = formic acid/ultrapure water/acetonitrile (v/v), 20 mL; 50 mM ammonium formate, pH = 4.4, 500 mL;
(3) SPE vacuum pump; A 96-well plate negative pressure device or positive pressure device; Heating module or metal bath (90 °C and 50 °C); Vortex instrument; A pipette gun; Centrifuge; Reaction tube.

Step 1: Rapid Glycosyl Release
(1) The IgG standard and the surfactant in the kit were taken out, and the powder adhered to the tube wall was centrifuged to the bottom of the tube using a centrifuge at 5000 rpm for 15 s.  
(2) Preparation standard: IgG standard (1.2 mg/5 mg) was dissolved in (600 uL/2500 uL) 0.85% sodium chloride solution so that the final concentration of IgG standard was 2 mg/mL.Note:Use 0.85% sodium chloride solution or ultrapure water to dissolve the IgG standard.
(3) Preparation of glycosyl release buffer: Dissolve one bottle of surfactant (1.2 mg/3. 6mg) in (24uL/72uL) PNGase F buffer, mix well and place at room temperature. (The Glycosyl Release Buffer Solution is Prepared for Ready Use)
(4) Add 20uL of the pre-prepared sample to be tested (2mg/mL) and IgG standard (2mg/mL) to the reaction tube, add 3uL of glycosyl release buffer, pipette and mix well for 5 times, then add 3.3 uL of ultrapure water, pipette and mix well for 5 times to mix well.Note:The concentration of the sample to be tested needs to be diluted to 2mg/mL first, and loading 20uL means the load volume is 40ug. A 200uL reaction tube can be used when heating with a PCR instrument, and a 0.6 mL/1. 5mL reaction tube can be used according to the instrument specifications when using other heating instruments.
(5) The mixture was incubated at 90 °C for 3 minutes to denature, and after the reaction was completed, the mixture was taken out and allowed to cool at room temperature for 3 minutes.Note:This step requires room temperature cooling for 3 minutes to avoid the excessive temperature of the reaction system affecting the activity of glycosidase in the next step, and the extension of room temperature cooling time will not affect the experiment.
(6) Add another 1.2 uL of PNGase F, pipette and mix well 5 times to mix well, and incubate at 50 °C for 5 minutes.Note:Glycosidase (PNGase F) was removed from-20 ° when added to the system. It should not be removed in advance and placed at room temperature to prevent the enzyme activity from decreasing.
(7) After the reaction is completed, take it out and let it cool at room temperature for 3 minutes.

Step 2: Quickly label glycosyl groups
(1) Labeling reagent: Dissolve 1 bottle of labeling substance (8.2 mg/24. 6 mg) in (60.13 uL/180. 4 uL) anhydrous DMF. The labeling reagent is ready for use, and the solution is aspirated and dispersed 5-10 times to completely dissolve. (This mark is currently used and prepared)Note:Because the labeling reagent is placed in a brown bottle, the brown bottle is inconvenient to centrifuge. After adding anhydrous DMF, try to fully dissolve the powder on the upper part of the brown bottle when sucking and mixing.
(2) Add 6uL of labeling reagent solution to the reaction tube, pipette and mix well for 5 times, and react at room temperature for 5 minutes.
(3) Add 179uL of acetonitrile to the reaction tube, pipette and mix well for 5 times, and then use for purification.

Step 3: Purification
(1) Install negative pressure or positive pressure device and 96-well extraction plate.Note:When using the vacuum device, the pressure should not be too large, and the appropriate pressure can be set according to different instrument models.
(2) Activation: Add 200uL of ultrapure water to activate the hole to be used, vacuum suction the liquid and collect the waste liquid tray.
(3) Equilibrium: Add 200uL 15/85 water/acetonitrile solution to the equilibrium hole, vacuum suction the liquid and collect the waste liquid tray.
(4) Loading: Load the sample diluted with acetonitrile (212.5 uL), and collect the waste liquid tray after vacuum suction.
(5) Add 600uL 1/9/90 formic acid/ultrapure water/acetonitrile to clean the hole, and collect the waste liquid tray after vacuum suction.
(6) Add 600uL 1/9/90 formic acid/ultrapure water/acetonitrile to clean the hole, and collect the waste liquid tray after vacuum suction. (i.e., washing twice)
(7) Negative pressure air suction once to suck the residual liquid at the filter element cleanly.
(8) Remove the waste liquid tray and replace it with a collection plate. 50 uL of elution buffer was added to the wells, and the glycosyl groups were eluted by vacuum aspiration to the collection plate, and 50 uL of elution buffer was added to the wells, and the glycosyl groups were eluted by vacuum aspiration to the collection plate. (i.e. final elution volume 100uL)Note:The liquid added to the wells is added to the middle of the wells as much as possible, so as to completely cover the packing and improve the purification efficiency and elution efficiency.
(9) Add 200uL of sample diluent, suck 5 times and mix well, and wait for testing on the machine.

Step 4: HILIC-FLR detection and analysis
(1) Column ACQUITY UPLC ® Glycan BEH Amide, 130 Å, 1.7 um, 2.1 x 150 mm (waters part number 186004742).
(2) Column temperature: 60 ° C.
(3) Mobile phase A: 50 mM ammonium formate (LC-MS grade recommended) solution, pH = 4.4.
(4) Mobile phase B: 100% acetonitrile (LC-MS grade is recommended).
(5) Flow rate: 0.4 mL/min.
(6) Gradient:

(7) FLR wavelength: EX 265/EM 425 nm.
(8) FLR sampling rate: 2 Hz.
(9) Injection volume: 10uL.
Remarks: The above parameters are based on the LCMS equipped with "ACQUITY ® RDa" detector, and the user adjusts the appropriate parameters according to the equipment.

Absin provides information on the routine maintenance and use of N-sugar analysis kits for rapid enzymatic release and rapid labeling of N-sugars. This protocol has been validated using monoclonal antibodies and has been tested for use with a variety of other N-linked glycoproteins. We recommend that users confirm the enzyme release of their own specific samples.

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