Profiling of Permethylated Mucin O-glycans Using Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

We are studying the glycobiology of host microbe-interactions in the gastrointestinal tract with a focus on the role of the mucus layer covering the GI tract, and in particular, mucins and their glycans in health and disease. Mucin glycans are increasingly recognized as important factors in host-microbe interactions as they provide nutrients and binding sites for bacteria. An alteration in the glycosylation profile can lead to disturbance in the microbiotic composition and vice versa, phenotypes that are commonly observed in disease states.

However, mucins are notoriously challenging to study as they're heavily glycosylated. Innovations in sampling techniques and follow-up bioinformatic analysis can take the field forward as well as new sample processing approaches to increase the throughput and reduce the time from sample collection to results. Other analytical techniques allow more detailed structural characterization of glycans, but require long running and analysis time.

The technique described here using MALDI-TOF mass spectrometry has advantage of speed, which makes it high-throughput and suitable for screening and profiling. To begin, mix purified mucins with 250 microliters of the beta-elimination buffer in a four milliliter glass vial. Seal the vial tightly with a polytetrafluoroethylene-lined cap and incubate the reaction at 45 degrees Celsius for 16 hours.

On ice, add one milliliter of the 5%acetic acid solution to the reaction mix in a drop-wise manner. For desalting the sample, plug a glass pipette with a small amount of glass wool. Add 1.5 milliliters of the resin suspension to the glass pipette from the top, then allow it to settle and drain.

Now, wash the resin with two milliliters of 5%acetic acid and discard the flowthrough. Then, add the mucin sample to the desalting column and collect the flowthrough in a five milliliter tube. Wash the column two times with one milliliter of 5%acetic acid to collect the flowthrough.

Then using a centrifugal evaporator, remove the acetic acid and concentrate the sample at five millibar and 30 degrees Celsius for two hours. Dissolve the sample in 0.5 milliliters of methanolic acetic acid, and dry it under a stream of nitrogen. Using a glass syringe, add four milliliters of DMSO to the vial containing sodium hydroxide and methanol mixture.

After vortexing, centrifuge the solution at 2, 000 G for two minutes. Carefully decant the supernatant and remove the salts without disturbing the gel. After ensuring no more salts are formed, resuspend the gel in four milliliters of anhydrous DMSO to prepare the permethylation base suspension.

Next, to the dried mucin sample, add anhydrous DMSO and permethylation base immediately followed by iodomethane. Incubate the permethylation reaction for 30 minutes at room temperature with vigorous shaking. Then, add 0.5 milliliters of water to terminate the reaction.

After the sample turns cloudy, flush it with nitrogen until it turns clear. Load the sample onto a hydrophilic lipophilic balance 96-well extraction plate. Apply positive pressure to push the samples through the solid phase at a flow rate of approximately one milliliter per minute.

After discarding the flowthrough, wash the wells two times with one milliliter of water. Elute the glycans from the plate two times with one milliliter of methanol. Apply pressure only until methanol starts exiting the plate, then allow gravity flow to maintain the required rate.

Dry the sample using a centrifugal evaporator and dissolve it in 10 microliters of TA50. After mixing one microliter of sample and the matrix, load it onto a steel MALDI target plate and allow it to dry. For data analysis, load the exported ASCII file of the mass spectrum in GlycoWorkbench.

After performing baseline correction, compute the peak centroids and in the pop-up window, select the appropriate mass range, minimum signal-to-noise ratio, and minimum mass spectrometry peak intensity. Then, use the Glyco-Peakfinder tool to identify the structure compositions that match the peak list. Choose perMe as the derivitization, redEnd as the reducing end, and set the minimum and maximum number of expected residues.

For mucins, select Hexose, N-Acetyl-Hexosamine, Deoxy-Hexose, and N-Acetylneuraminic Acid. For sulfated glycans, use the Other residue option with a mass of 87.9. For MALDI-TOF data, set the maximum number of sodium ions and charges to one each, and set the accuracy to 0.5 Daltons.

Select all correct annotations with the Control key and click on each of them. Right-click on the selected annotations and choose Add to the annotated peak list. To generate a report comparing multiple annotated spectra, go to Tools followed by Reporting and create a report comparing different profiles.

Print the report as a PDF to save it. For fragmentation spectra analysis, load the spectra onto GlycoWorkbench and generate the peak list as demonstrated earlier. Draw putative glycan structures that correspond to the annotated glycan compositions.

To generate fragments for each structure, right-click to select Copy fragments into canvas and choose Compute fragments under the Fragments tool. For further fragmentation analysis, click on a peak of interest in the spectrum viewer. Right-click and select Find all structures matching the peaks to query the master charge ratio of the selected peak against online databases.

Select putative structures of interest and right-click to copy them to the canvas window with the appropriate option. To compute possible fragments from each glycan structure, select the structure in the canvas. Then, under tools and fragments, select compute fragments for the current structure.

If fragments for more than one glycan structure have been computed, go to the Fragments table view under the Summary tab to view the comparison table. Select the fragments matching the peak list for each potential glycan structure. Right-click and choose Copy fragments to canvas from the dropdown menu.

Finally, in the Tools tab, go to Profiler followed by Annotate peaks with all structures. Then select Tools, Reporting, and Create a report of the annotations. Desalting by ion exchange resulted in better recovery of larger glycans with these larger structures making up 31%of total glycans compared to 21%for PGC extraction.

Among the glycan structures identified, one glycan was identified only through in the ion exchange desalted sample. Furthermore, desalting by ion exchange and borate removal led to spectra with increased signal-to-noise ratios compared to those produced after desalting by solid phase extraction with PGC. Permethylation reaction times of 30 and 90 minutes led to the identification of 33 glycan peaks, while a five-minute reaction identified only 31 peaks.