Quantitative Glycomics of Cultured Cells Using Isotopic Detection of Aminosugars with Glutamine(IDAWG)

 Culture mES cells at 37°C under 10% CO₂ using mES cell culture media lacking Gln and supplemented with either amide-15N-Gln or corresponding normal-abundance 14N-Gln at 2 mM final concentration.

mESCell Culture Media

Dulbecco's modified Eagle's medium (DMEM) Gln-Free

10% fetal calf serum

2 mM L-glutamine

either ¹⁴N or amide-¹⁵N

0.1 mM 2-mercaptoethanol

1000U/mL recombinant murine leukemia inhibitory factor (LIF).

 Change the media daily for 3 days.

 Day 3 collect the ESCs (1 × 10⁷) with PBS (do not use trypsin) and cell scraper.

 Transfer cells to a 15 mL conical tube, and centrifuge at 1,000 × g.

Phosphate-buffered saline (PBS)

80.0 g NaCl

2.0 g KCl

14.4 g Na2HPO4

2.4 g KH2PO4

Milli-Q water (18.2 MΩ) to 1 liter

 Wash the cells with ice cold phosphate buffered saline (PBS), centrifuge at 1000 × g, decant the supernatant, and repeat 2 more times.

 After removal of supernatant the third time store the cell pellets at −80ºC until analysis.

 To each pellet, add water to 100 μL, and transfer the suspension to 1.5 mL microcentrifuge tubes (treating bothe Heavy and Light samples separate but equal).

 Add 500 μL ice-cold methanol to the tube and move the cells to a 10 mL Dounce homogenizer.

 Disrupt the cells well on ice by Dounce homogenization (6 to 8 strokes), then transfer the mixture to a tared 8 mL glass tube.

 Add 3.5 mL methanol, 1.5 mL water, and 2 mL chloroform to yield a final ratio of 4:8:3 chloroform:methanol:water.

 Incubate the mixture for 3 h at room temperature with end-over-end rotation to extract lipids.

 Centrifuge 30 min at 3,300 × g, 4°C.

 Remove the supernatant, add 4 mL methanol, 1.5 mL water, and 2 mL chloroform to the insoluble materials, and incubate for 2 more hour at room temperature to extract the lipids again.

 Centrifuge 30 min at 3,300 × g, 4°C, and remove the supernatant.

 Add 1 mL water to the insoluble materials in glass tube and vortex well.

 Add 6 mL ice-cold acetone to the glass tube and vortex well to precipitate the proteins.

 Incubate for 10 min on ice and then centrifuge 30 min at 3,300 × g, 4°C, and remove the supernatant.

 Repeat steps 9 to 11 two more times.

 Dry the insoluble protein powder under a stream of nitrogen at 40°C using the Pierce Reacti-Vap Evaporating Unit and Reacti-Therm Heating/Stirring Module.

 Weigh the protein powders in the tared 8 mL glass tubes.

 Take the same amount of protein powder (3 to 5 mg each) of normal and ¹⁵N-labeled cell populations and mix together. Prepare one sample of mixed protein powder to prepare N-linked glycans and another sample to prepare O-linked glycans.

 Resuspend the mixed protein powder in an 8-mL glass tube with 200 μL of 40 mM NH₄HCO₃, seal the tube, then place in a heating block with temperature set at 100°C for 5 min.

 After cooling to room temperature, centrifuge briefly to collect the solution at the bottom of the tube.

 Add 25 μL of 2 mg/mL trypsin solution in 40 mM NH₄HCO₃ and 25 μL of 2 mg/mL chymotrypsin in 40 mM NH4HCO3. Finally, add 250 μL of 2 M urea (in 40 mM NH₄HCO₃ to yield a final concentration of 1 M urea. Seal the tube.

 Incubate the solution for 18 h at 37°C to digest the proteins.

 After incubation, place the tube in the 100°C heating block for 5 min to deactivate the enzymes. Allow to cool to room temperature.

 While the solution is cooling, equilibrate a Sep-Pak C18 cartridge column by washing three times with 100% acetonitrile followed by three washes with 5% acetic acid.

 Add 500 μL of 10% acetic acid to the cooled solution, then load the solution onto the equilibrated Sep-Pak C18 cartridge column.

 Wash the column with 1 mL of 5% acetic acid three times.

 Put a clean glass collection tube under the column and elute the peptides stepwise, first with 1 mL of 20% isopropanol in 5% acetic acid, then with 1 mL of 40% isopropanol in 5% acetic acid, and finally with 1 mL of 100% isopropanol.

 Combine the elutions and dry down in a Speed-Vac evaporator to remove solvents.

(It is possible either to use stepwise elutions or simply to use 100% isopropanol for all three washes.)

 Resuspend the peptides with 45 μL of 100 mM sodium phosphate (pH 7.5) and then add 5 μL PNGase F stock to release the N-linked glycans from the peptides. Incubate for 20 h at 37°C.

 After PNGase F digestion, add 450 μL of 5% acetic acid to solution, then the load solution onto an equilibrated Sep-Pac C18 column (see step 19 for equilibration). Elute the N-linked glycans with three 1 mL aliquots of 5% acetic acid and collect them in a glass tube. Dry down in Speed-Vac evaporator for permethylation (proceed to step 28).

 In an 8 mL glass tube, add 1 mL of freshly prepared 1 M sodium borohydride to the mixed protein powder.

 Vortex, and sonicate the sample tube quickly in a Branson Ultrasonic Cleaner at room temperature using the default settings.

 Incubate for 18 h at 45°C in a heating block.

 After incubation, cool the sample to room temperature.

 Neutralize the sample by adding 10% acetic acid dropwise to the tube while vortexing, until bubbling stops.

 Pack a 1 mL bed volume of AG 50W–X8 resin stock into a Pasteur pipet to make the cation-exchange column and wash the column sequentially with 2 mL methanol, 1 mL of 1 M hydrochloric acid and 2 mL 5% acetic acid.

 Place a clean glass collection tube under the column.

 Load sample onto equilibrated column, elute the O-linked glycans with 4-5 mL of 5% acetic acid, in 1mL steps.

 Dry down in a Speed-Vac evaporator.

 Add 1.5 mL of a 9:1 methanol:glacial acetic acid mixture.

 Dry the sample under a stream of dry nitrogen using the Pierce Reacti-Vap Evaporating Unit and Reacti-Therm Heating/Stirring Module.

 Repeat this step two more times to remove the borate.

 Prepare the dry sodium hydroxide solution for permethylation:

a. In a glass tube, combine 100 μL of 50% w/w sodium hydroxide solution and 200 μL anhydrous methanol and vortex

briefly.

b. Add 4 mL anhydrous dimethylsulfoxide (DMSO) and vortex.

c. Centrifuge the tube quickly (30 sec) at 1,068 × g, room temperature, and pipet off dry DMSO, salts, and white

residue, leaving clean sodium hydroxide solution in the tube.

d. Repeat steps 2 and 3 four to five more times to remove all of the white residue in the tube.

e. Once the tube is clean, add 2 mL dry DMSO and pipet up and down gently.

 Add 200 μL dry DMSO to the released N- or O-linked glycan samples

 Purge the tube with dry N2 to remove air, sonicate 2 min in a Branson Ultrasonic Cleaner at room temperature using the default settings, and vortex quickly to dissolve sample.

 Add 250 μL of the prepared sodium hydroxide solution to sample tube, purge with dry N2, and sonicate quickly.

 Add 100 μL iodomethane with a 500 μL microsyringe to the sample, purge with dry N2, and vortex vigorously for 5 min.

 Add 2 mL water and bubble off iodomethane with dry N2 gently.

 After the solution becomes clear, add 2 mL dichloromethane and vortex.

 Centrifuge the tube quickly (30 sec) at 1,068 × g, room temperature.

 Remove and discard the top aqueous layer.

 Add 2 mL water, vortex, and centrifuge quickly (30 sec) at 1,068 × g, room temperature, and then remove the aqueous layer.

 Repeat steps 9 and 10 four more times.

 Dry the sample under a gentle stream of dry N2 using the Pierce Reacti-Vap Evaporating Unit and Reacti-Therm Heating/Stirring Module.

 Dissolve the permethylated N- or O-linked glycans in 10 μL of 100% methanol/1 mg of protein powder.

 Trasnfer 15 μL of the solution into a 1.5 mL microcentrifuge tube, then add 35 μL of 1 mMNaOH in 50% methanol to achieve a final volume of 50 μL.

 Directly nfuse the solution directly into the mass spectrometer using a nanospray ion source with a fused-silica emitter (360 × 75 × 30 μm, SilicaTip) at 2.0 kV capillary voltage, 240°C capillary temperature, and a syringe flow rate of 0.4 μL/min.

 Acquire the full FTMS (Fourier Transform Mass Spectrometry) spectra at 400 to 2000 m/z in positive ion and profile mode with 2–4 microscans and 1000 maximum injection time (msec).

* This step must be optimized for the particular instrument and can be combined with tandem mass spectrometry analysis of the analytes.

 Calculate the ratios of the same glycan structures in each sample (normal and 15N-labeled).