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

The overall goal of this procedure is to harvest Olink Glycans. This is accomplished by first resolving glycoproteins by SDS page. The second step is to wash gel pieces containing the glycoprotein of interest.

Next, the olink glycans are released by reductive beta elimination. The final step is glycan per methylation with phase partition. Ultimately perm methylated glycans are analyzed by tandem mass spectrometry to detect structures and quantify abundances.

The main advantage of this technique over existing methods like high performance liquid chromatography, is that our method separates neutral and charged per methylated glycans in a rapid phase partition. This method can help answer key questions in the glyco science field by allowing comprehensive characterization of or linked glycans released from resolved glycoproteins. Prior to starting this procedure, resolve glycoproteins using gel electrophoresis.

After electrophoresis, place the gel on a glass plate and excise the region of interest using a clean scalpel to increase the yield of OG glycans. Transfer the gel band of interest onto another glass plate and cut piece into approximately two millimeter cubes. Transfer the small gel pieces into a 13 by 100 millimeter screw top glass tube with a stainless steel micros spatula.

Next, add one milliliter of 25 millimolar ammonium bicarbonate or ambi to the sample tube. After capping the tube with a Teflon line screw top cap, gently mix the contents by flicking the tube before letting it stand for 10 minutes. Then carefully remove ambit from the glass tube.

Using a pasture or glass pipette, add one milliliter of aceto nitrile to the glass tube to completely cover the gel pieces. After capping the tube, gently mix the contents by flicking the tube before letting it stand for 10 minutes. When finished, remove the aceto nitrile from the glass tube.

After repeating the previous steps until the bright blue color is eliminated, add two milliliters of ethyl acetate and recap the tube. Place the tube at four degrees Celsius overnight with end over end agitation. Once the ethyl acetate wash has been removed, perform three washes with two milliliters of deionized water.

Once the gel pieces have been dried, add 500 microliters of a 100 millimolar sodium hydroxide solution. And let's stand for three to five minutes on ice to equilibrate the gel to the basic conditions that enhance glycan recovery. Following this, add 500 microliters of two molar sodium boro hydride in 100 millimolar sodium hydroxide, resulting in final concentrations of one molar sodium boro hydride in 100 millimolar sodium hydroxide.

After gently mixing the tube, incubate the sample at 45 degrees Celsius for 18 hours. During the first hour of incubation, gently mix the tube every 15 minutes. After the beta elimination reaction is complete and the tube has been placed on ice, slowly add 10%acetic acid dropwise.

To neutralize the base, gently vortex the sample tube between additions of acetic acid, centrifuge the tube to eliminate bubbles and prevent spillover if necessary. To make a small glass column scratch and break the tip of a pasture or glass pipette using a ceramic cutter so that the taper of the pipette tip is approximately one centimeter long. After teasing apart a plug of glass wool, push it towards the tip, forming a support for the resin bed.

Secure the pipette column with a clothes pin and place over a glass test tube. Next, wash the empty pipette containing glass wool with one milliliter of methanol and three milliliters of 5%acetic acid. After swirling Dow X hydrogen cation exchange resin slurry in 5%Acetic acid.

Transfer enough of it to produce a one milliliter bed volume in the pasture pipette column, rinse the column using five volumes of 5%acetic acid checking flow through for the appearance of resin particles. After placing the column over a new 16 by 125 millimeters screw top glass tube, load the sample onto the column. Once the flow through has been collected, elute glycans with at least three volumes of 5%acetic acid into the same tube.

Following lyophilization, add 300 microliters of 10%acetic acid in methanol to the dried sample tube. After vortexing, remove the resulting trimethyl bate by evaporation under a nitrogen stream at 37 degrees Celsius. After repeating the resuspension and drying steps at least four times, reconstitute the sample in 5%acetic acid.

Once the sample has been loaded onto an equilibrated C 18 column, collect the flow through into a 13 by 100 millimeter glass screw cap tube. Elute the OOG glycans with three milliliters of 5%acetic acid into the same tube. Then lyophilize the sample to prepare the base reagent for perm methylation.

Add 400 microliters of 50%sodium hydroxide to a clean 13 by 100 millimeter glass screw top tube. Then add 800 microliters of anhydrous methanol to the same tube. Add four milliliters of anhydrous dimethyl sulf oxide and vortex to generate a white precipitate.

Centrifuge the sample at 600 times G for one minute to pellet the precipitate. After discarding the supernatant and adding four milliliters of anhydrous dimethyl sulf oxide to the pellet vortex the reagent Repeat four more times until the base slurry becomes translucent. Dissolve the pelleted base in three milliliters of anhydrous dimethyl sulf oxide, and gently mix by pipetting up and down with a clean pasture pipette.

Next, add 200 microliters of anhydrous dimethyl sulf oxide to the dried sample and vortex. To resus suspend it, add 300 microliters of the resuspended base slurry to the sample, and immediately add 100 microliters of ITOM methane. Seal the tube with a Teflon lined cap and vigorously mix for five minutes by vortex.

To stop the perm methylation reaction, place the tube on ice and add two milliliters of 5%acetic acid. After vortexing, pipette the solution up and down five times to reduce the remaining volume of ITOM methane by evaporation. At this point, add two milliliters of di chloro methane to the sample following vortexing and centrifugation.

Transfer the top aqueous layer into a new 13 by 100 millimeter glass tube. After repeating the aqueous wash of the di chloro methane layer, combine the second aqueous phase with the first aqueous layer. Once the water wash of the di chloro methane phase has been repeated two to three times, transfer the di chloro methane phase to a clean glass tube with a clean pasture pipette.

Then dry the organic phase under a nitrogen stream at 42 degrees Celsius. After e equilibrating a C 18 column and loading the aqueous layer onto it. Wash the C 18 column with water and then elute the perm methylated sulfated o glycans into a new glass tube with two milliliters of 50%Aceto nitrile analyze perm methylated OG glycans by direct infusion into an appropriate mass spectrometer using a nano electro spray source at a syringe flow rate of 0.4 to 0.6 microliters per minute at 210 degrees Celsius.

Capillary temperature for fragmentation by collision induced dissociation in MSM S and multidimensional MS of an ion trap instrument apply 30 to 40%collision energy representative mass spectra of perm methylated olink glycan samples released from bovine mucin using ingel reductive beta elimination are shown here in the top panel. The spectra is dominated by a poly disperse contaminant derived from the poly acrylamide gel. The bottom panel demonstrates that the ethyl acetate wash of the gel pieces before beta elimination eliminates this problem.

Oinked glycans were released from bovine sub maxillary mucin by Ingel reductive beta elimination using gel pieces that were either sliced small or large. The recovery of oinked glycans from small gel pieces was almost tenfold greater than from large gel slices. Neutral perm methylated glycans are recovered in the organic di chloro methane phase.

While onic perm methylated glycans are quantitatively partitioned into the water phase. The efficiency and simplicity of the phase partition method greatly facilitates sample throughput and subsequent analytic approaches. Nons sulfated perm methylated O glycans were recovered from the organic phase, and all of the perm methylated sul glycans were recovered from the aqueous phase, which facilitates the identification and characterization of nearly Isobar sulfated and nons sulfated glycans.

These glycans differ by only 0.1 mass units and would be difficult to resolve without physically separating the two species by phase partition After its development. This technique has allowed thorough exploration of glycan diversity in tissue samples, cultured cells, model systems, and patient samples. After watching this video, you should have a good understanding of how to successfully palmate and partition all linked glycans released from the gly proteins.

Resolved by SDS page.