The overall goal of this procedure is to break down all glycans in a biological sample while conserving monosaccharide composition and linkage information. Facilitating distillation of interesting glycan features such as sick cyalalation and bisecting N-Acetylglucosamine into single analytical signals. This method can help to directly answer key questions in the field of glycomites such as which unique glycan features are altered in particular disease states.
The main advantage of this technique is that glycan features that tend to be altered in disease such as, beta 1-6 branching and corfulcosulation are distilled into single analytical signals rather than being spread across multiple intact glycans. Generally, individuals need to do this method really struggle with poor yeilds of N-Acetylcysteines due to improper processing of freshly permethylated glycans. Prep the samples, by first transferring nine micro-liters of each biological analyte into separate 1.5 milliliter test tubes.
To each sample, add one micro-liter of deionized water and 270 micro-liters of high purity of DMSO. And vigorously mix by vortexing them. After sample preparation, assemble the sodium hydroxide loaded spin columns as described in the text protocol.
Using and analytical syringe, add 105 micro-liters of iodomethane to each analyte and immediately cap the tubes. Thoroughly vortex the samples. Unplug the previous prepared spin columns and centrifuge them for 15 seconds at 2, 400 Gs.After discarding the reservoir tubes and affluent, re-plug the columns and place them inside of a fresh set of tubes, labeled for each analyte.
Use a 1000 micro-liter pipe head to transfer each analyte solution onto the corresponding spin column. Once the analyte solution is added, crimp about two milliliters from the end of a 200 micro-liter pipe head tip and leave the tip in the column to use as a stirrer. Use the pipe head tips to gently stir the columns once ever two to three minutes.
After 11 minutes, unplug the columns and centrifuge them for 15 seconds at 2, 400 Gs.Immediately remove the spin columns and place them in new reservoir tubes, saving the flow-through solution but discarding the plugs. To the columns, add 300 micro-liters of acetonenitrile and centrifuge them 30 seconds at 9, 600 Gs.Now transfer the first permethylated flow-through solutions into silanized glass test tubes containing 3.5 milliliters of sodium phosphate buffer. Be sure that the transferred supernaned is clear and that no solid material is transferred.
A small white pellet will have formed at the bottom of the plastic tubes. Avoid transferring any obvious solid material. Failure to do so will result poor yields of N-Acetylcysteine-glucosamine glycan notes.
Immediately vortex every sample in the batch once all samples have been transferred. Then combine the acetonenitrile flow-through solutions with the rest of the liquid in the respective test tubes. Again, avoid transferring any solid, white residue during this step.
Cap, shake and vortex the combined solutions immediately before moving to the next sample. After permethylation, add 1.2 milliliters of chloroform to each test tube inside a fume hood. Then cap the tubes and mix the contents vigorously.
To separate the layers, briefly centrifuge the solutions at 3, 000 Gs.Use a pasture pipe head to extract and discard most of the top aqueous layer leaving about three milliliters of the layer behind. Add 3.5 milliliters of the 0.2 molar sodium phosphate buffer and then cap, mix and centrifuge the test tube. Execute this liquid/liquid extraction step a total of three times.
Leaving about one milliliter of the aqueous layer in the final extraction. Now use a clean silanized pasture pipe head to carefully transfer the chloroform layer to a clean and labeled silanized glass test tube as described in the text protocol. In a pre-heated evaporation manifold, set to 75 degrees celsius, dry the samples under a gentle stream of nitrogen gas.
Make sure that the gas flow disturbs the liquid surface but without splattering it. Once the samples are dry, they are ready for hydrolysis. Add 325 micro-liters of 2 Molar trifluoro acetic acid or TFA to each sample and mix well.
Tightly cap each sample tube immediately after addition to all the samples to prevent TFA evaporation and sample loss during the subsequent heating step. Incubate the samples at 121 degrees celsius for two hours to hydrolize the glycans. While samples are incubating, again, prepare the evaporation manifold at 75 degrees celsius.
After incubation, dry the samples under a steady stream of nitrogen in the evaporation manifold. Frequently check samples to ensure they are dried using the minimum amount of drying time possible. To each sample, add 475 micro-liters of 10 grams per liter sodium borohydride.
And then mix samples thoroughly to dissolve any residue. Cap the test tubes and allow the reduction reaction to continue for one hour. Next, add 63 micro-liters of methanol to each sample.
Mix and then dry the samples at 75 degrees celsius under nitrogen. Add 125 micro-liters of methanol to acetic acid solution to each sample. Mix and then dry the samples again at 75 degrees celsius under nitrogen.
Ensure samples are completely dry by placing under a vacuum for 20 minutes. After drying, add 18 micro-liters of deionized water to each sample. Then centrifuge the samples briefly and mix them well to dissolve an precipitate.
Next, add 250 micro-liters of acetic anhydride to each tube. Cap the tubes, thoroughly vortex and sonicate the samples in a water bath for two minutes to ensure complete dissolution of any residue. Then incubate the samples at 50 degrees celsius for 10 minutes.
Now add 230 micro-liters of concentrated TFA to each sample. After capping and mixing the samples, incubate them for 10 minutes an internal temperature of 50 degrees celsius. Add 1.8 milliliters of dichloromethane to each sample and mix well.
Next, add two milliliters of dionized water to the tube and perform liquid/liquid extraction twice. Recover the dichloromethane layer with a silanized pasture pipe head. Transfer to a auto-sampler vial and then dry the sample under nitrogen at 74 degrees celsius.
Prior to GC-MS, reconstitute the samples each with 120 micro-liters of acetone, capping and mixing them thoroughly. In split mode, use an auto-sampler to inject one micro-liter of each sample into the GC split-mode liner. For time of flight mass spectrometers, a split ration of forty and helium gas in constant flow mode at 0.8 milliliters per minute is recommended.
Analyze the sample using a two-step gradient that reaches 325 degrees celsius. Subject the eluding sample components to electron ionization. For a time of flight mass analyzer, analyze fragments M over Z 40 to 800 with a 0.1 second pulse summation rate.
To demonstrate the sensitivity of this protocol, a biological sample was processed carefully. The resulting chromatogram gives adequate intensity between the desired product peaks for ease of comparison and analysis. The same starting biological sample was then processed with the same procedure.
But the white residue in the permethylation solution that was spun through the NAOH column was carried into the subsequent mixture for liquid/liquid extraction. The chromatogram gives a different ratio of peak intensity of the same key sample products. When compared directly, the difference is stark.
The clear depression of the 4-position linked N-acetylglucosamine peak in second chromatogram is a recognizable trait of an unsuccessful permethylation and extraction. To appreciate the utility of this approach toward detecting differences in glycan composition in disease states, extracted ion chromatograms from a normal blood plasma sample and a cancer patient blood plasma sample, are normalized for 2-linked manos and overlaid. Quantitative differences in unique glycan features such as beta 1-6 branching as well as sick cyalalation are readily apparent.
After watching this video, you should have a good understanding of how to carry out bottom up glycomics glycan node anaylsis. Facilitating distillation of interesting glycan features such as corfulcosulation, sick cyalalation and bisecting glycanite into single analytical signals.
