| Usage | Before starting this protocol, users need to bring the following reagents and equipment (taking an 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 solid phase extraction 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 (0.25 mg/1. 2 mg) was dissolved in (125 uL/600 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 (1.5 mg/1. 2mg) in (30uL/24uL) 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) Configuration of labeling reagent: Dissolve 1 bottle of labeling substance (4.77 mg/8. 2mg) in (35.10 uL/60. 13uL) anhydrous DMF, pipping and mixing 5-10 times for complete dissolution. (The labeling reagent is now prepared and used)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 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) Take out the solid phase extraction cartridge in the kit and place it on the fixed frame.
(2) Activation: Add 200μL of ultrapure water to activate the hole to be used, blow the liquid with a pipette gun or ear cleaning ball, and collect the waste liquid tray.Note:When using a pipette gun or ear washing ball, you need to pay attention: first press the interface between the pipette gun/ear washing ball and the extraction cartridge tightly, and then blow the liquid vigorously; After blowing, first loosen the interface between the pipette/ear washing ball and the extraction cartridge, and then loosen the pipette/ear washing ball. Avoid the collapse of the packing due to the air retraction of the pipette gun/ear cleaning ball, which will affect the purification of the sample.
(3) Equilibrium: Add 200uL 15/85 water/acetonitrile solution to the equilibrium hole, blow the liquid with a pipette gun or ear cleaning ball and collect the waste liquid tray.
(4) Loading: Load the sample diluted with acetonitrile (212.5 uL), blow the liquid with a pipette gun or ear cleaning ball and collect it in the waste tray.
(5) Add 600uL 1/9/90 formic acid/ultrapure water/acetonitrile to clean the hole, blow the liquid with a pipette gun or ear cleaning ball and collect the waste liquid tray.
(6) Add 600uL 1/9/90 formic acid/ultrapure water/acetonitrile to clean the hole, blow the liquid with a pipette gun or ear cleaning ball and collect the waste liquid tray. (i.e., washing twice)
(7) Blow the pipette gun or ear cleaning ball once to clean the residual liquid at the filter element.
(8) Remove the waste liquid tray and replace it with a 1.5 mL reaction tube, add 50uL of elution buffer into the hole, collect the liquid after pipetting the liquid with a pipette gun or ear washing ball, and then add 50uL of elution buffer into the hole, collect the liquid after pipetting the liquid with a pipette gun or ear washing ball. (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 100uL 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:
| Time (min) | Flow Rate (mL/min) | % A | % B | Curve | | 0 | 0.4 | 25 | 75 | 6 | | 35 | 0.4 | 46 | 54 | 6 | | 36.5 | 0.2 | 100 | 0 | 6 | | 39.5 | 0.2 | 100 | 0 | 6 | | 43.1 | 0.2 | 25 | 75 | 6 | | 47.6 | 0.4 | 25 | 75 | 6 | | 55 | 0.4 | 25 | 75 | 6 |
(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 LCMS equipped with "ACQUITY ® RDa" detector, and the user adjusts the appropriate parameters according to the equipment. |
| Storage Temp. | | Name | Preservation conditions | | IgG | 2-8 °C, 12 months | | PNGase F | -25 °C--15 °C, 12 months, avoid repeated freezing and thawing | | PNGase F Buffer | 2-8 °C, 12 months | | Surfactant | Ambient temperature, 12 months | | Marker | 2-8 °C, 12 months, protected from light | | Anhydrous DMF | Ambient temperature, 12 months | | Elution Buffer | Ambient temperature, 12 months | | Sample dilution | Ambient temperature, 12 months | | Solid phase extraction cartridge | At room temperature, the unused cartridge is sealed and stored | | 200uL 8-row reaction tube, 8-row cover, 96-well collection plate, waste liquid tray | Normal temperature to the end of validity |
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