Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

NOTE: Lab Safety Concerns

In keeping with standard laboratory best practices, observe the following. Store all organic solvents in appropriate locations. Keep all waste materials in chemical waste containers with clear labeling of chemical compositions. As several reagents used in these protocols are potential carcinogens or generate volatile combustible gases, handle all reagents in a fume hood with ventilation. Wear personal protective equipment such as gloves, lab coat, and eye protection when working with organic solvents.

Figure 1. Procedure for in-gel O-glycomics. (A) Proteins of interest are resolved by SDS-PAGE, detected by appropriate staining procedures (Coomassie or silver), and excised. Excised gel pieces are sliced into small cubes, destained, and washed with ethyl acetate to reduce contaminants that interfere with subsequent MS analysis. A portion of the gel slice can be reserved for in-gel tryptic digestion and subsequent proteomic characterization by LC-MS/MS. (B) O-linked glycans are released from resolved glycoproteins by in-gel reductive β-elimination. Essential steps include desalting and borate removal by azeotrope with methanol. (C) O-linked glycans released by reductive β-elimination are permethylated and subsequently partitioned into aqueous and organic phases. Sulfoglycans are quantitatively recovered in the upper (aqueous) phase while neutral and sialylated glycans partition into the lower (DCM) layer.

Figure 2. Detail flow chart for in-gel glycomics. Each experimental step shown in Figure 1 is individually illustrated. (A) gel destaining and EtOAc extraction, (B) direct in-gel reductive β-elimination for O-linked glycan release, and (C) permethylation and phase partition. Please click here to view a larger version of the figure.

1. Gel Excision and Removal of Gel-derived Contaminants

1. Before starting the procedure, prepare chemicals and reagents listed in Materials List.
2. Resolve protein on SDS-PAGE gel using standard Tris-Glycine buffer systems. Stain resolved proteins with either Coomassie Brilliant Blue or Silver Stain. In general, recovery of O-linked glycans from silver stained gels is approximately 80–90% of the recovery achieved from Coomassie stained gels.
3. After electrophoresis, place the gel on a glass plate and excise the region of interest using a clean scalpel or razor blade.
4. To increase yield of O-glycans, transfer the gel band of interest onto another glass plate and cut the excised gel piece into approximately 2 mm cubes. Avoid making gel pieces smaller than 1 mm cubes because glycan recovery is reduced. Transfer the small gel pieces into a screw top glass tube (13 x 100 mm) with a stainless steel microspatula.
5. Add 1 ml of 25 mM ammonium bicarbonate (AMBIC, NH₄HCO₃) to the sample tube, cap the glass tube with a Teflon-lined screw top cap, and gently mix by flicking the tube before letting stand for 10 min. Do not employ vigorous agitation in order to avoid ripping the gel pieces.
6. Carefully remove AMBIC from the glass tube using either a Pasteur glass pipette or a pipette equipped with an extra-long plastic tip. Avoid smashing and transferring the small gel pieces while removing the AMBIC solution.
7. Add 1 ml of acetonitrile (ACN) to the glass tube, cap, and gently mix by flicking tube before letting stand for 10 min. Ensure that the gel pieces are completely covered with solvent. If necessary, push gel pieces off of tube wall and into solvent with pipette tip.
8. Pipette off ACN from the glass tube and repeat steps 1.5–1.7 until the bright blue color is eliminated. Repeat a minimum of five sequential AMBIC/ACN washes if necessary. For silver stained gels, use a destain kit. Proceed to the next step when the gel piece turns uniformly white while in ACN.
9. Pipette off any liquid from the glass tube, recap the tube, add 1 ml of AMBIC and let stand for 5 min.
10. Remove AMBIC and add 2 ml of ethyl acetate (EtOAc) to the glass tube. Recap tube with Teflon-lined cap and place at 4 °C overnight with end-over-end agitation or alternatively perform three EtOAc washes at room temperature for 30 min each. The longer the EtOAc wash, the more efficient the removal of contaminants.
11. Remove the final EtOAc wash and perform three washes each with 2 ml of H₂O. Complete removal of EtOAc will ensure a robust reductive β-elimination reaction.
12. Pipette off H₂O and dehydrate the gel by washing once with 1 ml of ACN.
    NOTE: After drying, the dehydrated gels are ready for in-gel β-elimination. Dehydrated gel pieces can be stored at -20 °C until use.
13. Stop the washing process at any step and store the gel pieces at 4 °C overnight, regardless of which buffer or solution is applied to the gel (AMBIC, ACN, H₂O). Restart sample preparation anytime within the next 2 days.
14. Use a portion of the destained and dehydrated gel pieces for protein ID by standard proteomic approaches.

2. In-gel O-glycan Release by Reductive β-Elimination

1. Prepare a stock solution of 1 M sodium hydroxide (NaOH) using 50% NaOH or solid NaOH and store at 4 °C until use.
   1. Take 5.2 ml of 50% NaOH (or 4 g of solid NaOH) and adjust volume to 100 ml to make 1 M NaOH solution. Dilute the 1 M NaOH stock solution to 100 mM NaOH, which can be stored at 4 °C up to 6 months without loss of efficiency in reductive β-elimination reactions.
2. Make 2 M sodium borohydride (NaBH₄) in 100 mM NaOH. Dissolve 38 mg of NaBH₄ in 0.5 ml of 100 mM NaOH. Generally, use 0.5–1.0 ml of the borohydride solution for each sample. Vary the volume of the reaction solution on the amount of dried gel pieces prepared in step 1. Expect to use 0.5 ml of borohydride solution for 1–2 excised gel pieces (step 1.3).
3. Add 500 µl of 100 mM NaOH to the dehydrated gel pieces and let stand for 3–5 min on ice in order to equilibrate the gel to the basic conditions which enhance glycan recovery.
4. Add 500 µl of 2 M NaBH₄ in 100 mM NaOH, resulting in final concentrations of 1 M NaBH₄ in 100 mM NaOH. Gently mix the tube and incubate at 45 °C for 18 hr.
   1. During the first hour of incubation, gently mix the tube every 15 min. Avoid strong agitation, because it may fragment the gel pieces. Perform the reaction in a well-equilibrated incubator/oven or in a heating block. Ensure that the gel pieces are covered with reaction solution.
5. Stop the reaction by removing the sample tube from the incubator or heating block and placing it on ice.
   NOTE: After allowing the β-elimination reaction to proceed for 18 hr, the desalting process must be performed within the same day.

3. Desalting on Cation-exchange Chromatography

1. While maintaining the sample tube on ice to prevent excessive heat formation, slowly add 10% acetic acid (AcOH) dropwise to neutralize the base. Gently vortex the sample tube between additions of AcOH. Be careful to add acid slowly and drop wise as the addition of acid will produce a “volcano” of bubbles. Centrifuge the tube to eliminate bubbles and prevent spillover, if necessary.
   1. In order to remove the large amount of sodium in the reaction mixture, pass the reaction mixture through a small cation-exchange column (Dowex or AG-50W-X8 resin, H⁺ form).
   2. Wash the cation exchange resin with 1 M NaOH and 1 M HCl prior to use in order to remove contaminants that interfere with MS analysis, even if the resin is of analytical grade.
   3. Soak the resin in 1 M NaOH and remove solution by decantation. Add de-ionized water, remove water, then add 1 M HCl.
2. Repeat these steps until the solution above the resin is colorless. Wash the cleaned resin with deionized water and store in 5% AcOH at 4 °C.
3. To make a small glass column, scratch and break the tip of a Pasteur glass pipette using a ceramic cutter so that the taper of the pipette tip is approximately 1 cm long. Tease apart a plug of glass wool and push towards the tip forming a support for the resin bed. Secure the pipette column with a clamp or clothespin and place over a glass test tube.
4. Wash the empty pipette with 1 ml of methanol (MeOH) and 3 ml of 5% AcOH. Swirl H⁺ Dowex or AG50 cation exchange resin slurry stored in 5% AcOH and transfer enough of the slurry to produce a 1 ml bed volume in the Pasteur pipette column.
5. Rinse the column using five volumes of 5% AcOH. Check flow through for the appearance of resin particles and do not use the column if resin is detected.
6. Place column over a new screw top glass tube (16 x 125 mm) and load sample. Collect the flow through and elute glycans with at least 3 volumes of 5% AcOH into the same tube.
7. Cover the tube with Parafilm and make tiny holes with a needle. Place the sample tube at -80 °C or on dry ice. Once frozen, dry the sample by lyophilization. Store the dried material at -20 °C until use.

4. Borate Removal

1. Prepare 10% AcOH in MeOH. Take 10 ml of glacial acetic acid and adjust volume to 100 ml with methanol. Store this reagent in a glass bottle with Teflon-lined cap for up to 6 months.
2. Add 300 µl of 10% AcOH in MeOH to the dried, lyophilized sample tube and vortex. Remove the resulting trimethyl borate by evaporation under a N₂ stream.
   NOTE: Mixing borate salts with methanol under acidic conditions produces trimethyl borate which is a volatile compound.
3. Dry under N₂ stream at 37 °C. After drying, add another aliquot of AcOH in MeOH as described in step 4.2. Dry the liquid for 5 min under nitrogen (N₂) stream at 37 °C.
   1. Repeat resuspension and drying at least 3 times. Store the dried material at -20 °C until use.

5. C18 Clean-up

1. Equilibrate a C18 cartridge column (size 1 ml, 100 mg resin) using three volumes of ACN and five volumes of 5% AcOH. Accelerate passage of the equilibrating solutions by application of mild positive air pressure.
2. Add 500 µl of 5% AcOH to the desalted and borate-free sample tube and dissolve by vortex.
3. Load resuspended sample onto the equilibrated C18 column and collect the flow through into a glass screw cap tube (13 x 100 mm). Elute O-glycans with 3 ml of 5% AcOH into the same tube.
4. Parafilm the tube, make small holes with a small needle or use loosely closed PTFE-lined cap to cover the tube, and freeze the sample at -80 °C or on dry ice. Remove solvent by lyophilization. Store the dried sample at -20 °C until use.

6. Base Preparation for Permethylation

NOTE: In order to achieve robust permethylation, prepare NaOH slurry fresh. All glassware used for permethylation reactions should be extensively cleaned.

1. To prepare the base reagent for permethylation, add 400 µl of 50% NaOH to a clean glass screw-top tube (13 x 100 mm). Add 800 µl of anhydrous MeOH and vortex.
2. Add 4 ml of anhydrous dimethyl sulfoxide (DMSO) and vortex to generate a white precipitate.
3. Centrifuge at 600 x g for 1 min to pellet the precipitate. Pipette off supernatant and discard. Add an additional 4 ml of anhydrous DMSO to the pellet. Vortex.
4. Repeat step 6.2 and 6.3 a minimum of three more times or until no white precipitate forms.
5. Dissolve the pelleted base in 3 ml anhydrous DMSO and gently mix by pipetting up-and-down with a clean Pasteur pipette.
6. Use the base immediately for the following permethylation reaction as it cannot be stored.

7. Permethylation of Released Glycans

1. Add 200 µl of anhydrous DMSO to the C18-purified sample and vortex or sonicate to resuspend.
2. Add 300 µl of the resuspended base slurry to the sample and immediately add 100 µl of iodomethane (MeI). Seal tube with Teflon-lined cap and vigorously mix for 5 min by vortex.
3. To stop the permethylation reaction, add 2 ml of 5% AcOH on ice and vortex. Pipette solution up and down 5 times to reduce the remaining volume of MeI by evaporation.
   NOTE: The AcOH neutralizes the solution and removal of MeI increases the extraction efficiency of sulfated glycans into the water phase during the phase-partition.
4. Add 2 ml of dichloromethane (DCM) and vortex. Centrifuge at 600 x g for 1 min at room temperature to separate the aqueous and organic phases.
5. Transfer the top layer (aqueous phase that contains the majority of permethylated sulfated glycans) into a new glass tube (13 x 100 mm).
6. Add 2 ml of H₂O to the organic phase and vortex. Centrifuge at 600 x g for 1 min to separate the aqueous and organic phases and combine this second aqueous phase with the first aqueous phase (step 7.5).
7. Repeat step 7.6 and 7.5 three more times, but there is no need to save the aqueous phases resulting from the subsequent partitions.
8. Remove the final top layer (aqueous phase) as much as possible from the bottom layer (organic phase) and transfer the bottom layer (organic phase) into a separate new glass tube using clean Pasteur pipette.
9. Dry the organic phase under N₂ stream at 42 °C.
10. Cover the tube with Parafilm and store at -20 °C until use.

8. C18 Clean-up of Permethylated Sulfated O-glycans from the Aqueous Phase

1. Equilibrate a C18 cartridge column with three volumes of ACN and five volumes of 5% AcOH.
2. Load the aqueous phases collected and combined from steps 7.5 and 7.6 onto the column.
3. Wash the column with 10 ml of H₂O for desalting.
4. Elute the permethylated sulfated O-glycans into a new glass tube with 2 ml of 50% ACN. Use an additional elution with 85% ACN to enhance recovery of permethylated sulfated O-glycans on larger oligosaccharides.
5. Dry eluates under nitrogen stream at 42 °C.
6. Store the dried material at -20 °C up to 6 months prior to MS analysis.

9. Mass Spectrometry of Permethylated O-glycans

1. Analyze permethylated O-glycans by direct infusion into an appropriate mass spectrometer using a nanoelectrospray source at a syringe flow rate of 0.40–0.60 µl/min at 210 °C capillary temperature. For fragmentation by CID in MS/MS and MSⁿ of an ion trap instrument, apply 30–40% collision energy.
2. For negative ion mode, reconstitute permethylated sulfated O-glycans in 50 μl of methanol/2-propanol/1-propanol/13 mM aqueous ammonium acetate (16:3:3:2 by volume). For positive ion mode, reconstitute permethylated neutral/sialylated O-glycans in 50 µl of 1 mM sodium hydroxide in methanol/water (1:1).
3. Use the total ion mapping (TIM) and neutral loss scan (NL scan) functionality of the Xcalibur software package version 2.0 for MSn analysis to characterize individual O-glycan structures ^13,19.