Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis

The main experimental stages of the MAPP method are summarized in Figure 1.

1. Preparation of samples

NOTE: Here, the method is applied to plant tissues for illustrative purposes. The plants selected were Coffea arabica, Allium sativum var. ophioscorodon, and several Thai mango varieties (Aokrong, Kam, Rad, Chokanan, Mamkamdang, Talabnak, Mahachanok, and Nga). The plants were selected for their commercial importance. Their processing for human consumption generates currently underutilized agro-industrial waste, which may provide a source of value-added products, including pure glycans. Thus, MAPP was applied to characterize the glycan composition of the waste plant biomass for bioprospecting purposes.

1. Separate the plant material into different tissues (e.g., root, stem, and leaves).
2. Dry the plant tissues in a hot air oven at 40 °C for 12-24 h (decrease/increase the time in accordance with the sample). Alternatively, snap freeze the samples in liquid nitrogen and subsequently lyophilize for ~4 days.
3. Homogenise the samples to a fine powder using a pestle and mortar or a mechanical tissue lyser (see Table of Materials) with a ball bearing in each tube.
   ​NOTE: For fresh plant tissue, drying or lyophilizing prior to homogenization is recommended. Generally, homogenization with a pestle and mortar is sufficient for most dry samples. For particularly resilient samples, such as grains, legumes, and processed food items like pasta, snap freezing in liquid nitrogen can increase the speed and efficiency of sample homogenization. We have found that mechanical homogenization is compatible with nearly all sample types, is considerably faster and less labor-intensive, and effectively minimizes the risk of sample cross-contamination from repeated use of the same equipment (i.e., pestle and mortar).

2. Preparation of alcohol insoluble residue (AIR)

1. Add 1.5 mL of 70% (v/v) ethanol to 50-100 mg of air-dried and homogenized sample material.
2. Vortex thoroughly to mix and then centrifuge at 10,000 x g for 10 min at room temperature. Discard the resulting supernatant using a pipette and retain the pellet.
3. To the remaining pellet, add 1.5 mL of methanol and chloroform (1:1 [v/v]). Vortex, centrifuge, and discard the supernatant, as per step 2.2.
4. To the remaining pellet, add 1.5 mL of 100% acetone. Vortex, centrifuge, and discard the supernatant, as per step 2.2.
5. Place the resultant pellet either overnight in a fume hood to allow the residual acetone to evaporate or in a vacuum centrifuge until dry.
   NOTE: AIR material can be stored at ambient temperature until required.

3. Glycan extraction

NOTE: If possible, perform all extraction steps in a tissue lyser with a ball bearing in each tube to aid resuspension. If a tissue lyser is unavailable, extractions can be performed instead with continuous stirring or shaking. It may be necessary to extend the extraction time if this is not possible.

1. To 10 mg of AIR material, add 30 µL/mg of 50 mM cyclohexanediaminetetraacetic acid (CDTA; see Table of Materials), pH 7.5.
2. Shake at 27 Hz for 2 min, followed by 10 Hz for 2 h.
3. Centrifuge at 10,000 x g for 10 min at 4 °C. Retain the resultant supernatant, add it to a sterile microcentrifuge tube, and store it at 4 °C on a rotary shaker.
4. To the residual pellet, add 30 µL/mg 4 M NaOH + 0.1 % (w/v) NaBH₄.
   CAUTION: NaBH₄ is toxic if swallowed. Use personal protective equipment (PPE). Handle under a fume hood. Avoid dust formation. Avoid breathing dust. Do not allow the product to come into contact with water.
5. Repeat steps 3.2 to 3.3. Wash the residual pellet two or three times with dH₂O to remove the residual NaOH.
6. Add 30 µL/mg cellulase (preferably GH5 endo-1,4-β-glucanase, in appropriate enzyme buffer-as recommended by the manufacturer; see Table of Materials) to the pellet and incubate at the enzyme optimal temperature for 16 h.
7. Centrifuge the samples at 10,000 x g for 10 min at 4 °C . Retain the supernatant, transfer to clean microcentrifuge tubes, and store at 4 °C on a rotary shaker. Once extracted, the samples must be printed as soon as possible.
8. Centrifuge all the stored extracts again at 10,000 x g for 10 min at 4 °C.
   ​NOTE: The samples must be free of particulates and debris. Pass through a 0.2 µm spin filter prior to microarray printing if necessary. Particularly viscous samples are incompatible with microarray analysis as the microarrayer capillaries and the print head can become easily clogged. As a general rule, users should be able to pipette all the samples intended for printing with a standard low-volume pipette.

4. Preparation of standards

1. Prepare 1 mg/mL solutions of defined glycan standards (Table 1) in sterile dH₂O. If using pachyman as a standard, dissolve in 4 M NaOH instead and neutralize with glacial acetic acid following solubilization.
2. Store the prepared standards overnight at 4 °C on a rotary shaker to allow complete solubilization.
3. Centrifuge all the standards for 10 min at 10,000 x g at 4 °C to pellet any debris. The resultant supernatant is used for subsequent printing.

5. Microarray printing

1. Prepare a 1:20 (v/v) dilution of black Indian ink/drawing ink in glycerol system buffer (GSB; combine 47% glycerol, 52.9% dH₂O, 0.06% Triton X-100, and 0.04% biocide [0.15%-0.17% cupric nitrate and 1.4%-2.0% magnesium nitrate in water] and filter sterilize) (see Table of Materials) and centrifuge for 10 min at 15,000 x g (room temperature).
   NOTE: Ink solution is necessary to create a top and bottom border around the printed samples, so that the printed microarrays can be visually detected on the membrane. However, the ink solution is likely to contain sediment. All solutions must be free of particulates for printing, so avoid disturbing the sediment when pipetting. Discard and prepare fresh when this is no longer possible. The solution cannot easily be filtered to remove particulates.
2. Add 40 µL of ink solution and GSB to the first section of the first 384-well plate (Figure 2).
3. Add 25 µL of GSB to all dilution 1 (D1) wells. Add 40 µL of GSB to all dilution 2, 3, and 4 (D2-D4) wells.
4. Dilute the extracted glycan samples and defined glycan substrates 1:1 (v/v) with GSB by adding 25 µL of extracted glycan sample to the D1 wells in order.
5. Serially dilute each sample four times by taking 10 µL of the sample from the D1 well and adding to the D2 well. Aspirate gently with a pipette to mix.
6. Repeat the process by taking 10 µL of sample from the D2 well and adding it to the D3 well.
7. Repeat for the D4 well. After mixing, discard 10 µL from the D4 well so that each well contains a final volume of 40 µL.
8. Add 40 µL of ink solution and GSB to the final block of the final plate.
9. Cover the plates with an adhesive plate cover and centrifuge for 10 min at 3,000 x g (room temperature). Ensure no bubbles remain following centrifugation and repeat if necessary.
10. Using a non-contact piezoelectric microarray printing robot, print the samples onto the nitrocellulose membrane (see Table of Materials) following the steps below.
    NOTE: Below are actions recommended for optimum print quality; however, the specific parameters required will ultimately depend on the instrument used. We advise that users contact the instrument manufacturer to discuss the appropriate customizations and print settings required for their device.
    1. Prior to printing, empty the waste buffer reservoir and fill the clean buffer reservoir with clean GSB, as necessary. Switch on the instrument and allow it to stabilize for >10 min if it has an integrated humidity and temperature control system. Switch on the microarrayer and initialize the system.
       NOTE: Running a test to determine the internal pressure of the instrument is recommended. If the pressure is too low, then it may be necessary to perform a high-pressure purge. Again, contact the instrument manufacturer to discuss the specific operation of the specific instrument.
    2. Purge the print head and capillaries several times with GSB to remove debris and potential contaminants. Perform a test print run by either loading a plate of GSB alone or setting the instrument up to print from the clean buffer reservoir directly, bypassing sample aspiration from a loaded plate.
       NOTE: It is not necessary to perform the test print using a nitrocellulose membrane; clean microscope slides are sufficient and offer the advantage that the spot size, shape, and quality can be visually assessed prior to sample printing.
    3. When printing extracted glycan samples, program the system to flush with clean GSB between each sample.
       NOTE: Typically, the sample volume per printed spot is 100 pL to 10 nL, and the spot size ranges from 20 µm to 100 µm, depending on the sample volume selected. Printing 100 microarrays from one source 384-well plate takes approximately 40 min, including system flushing. The resultant microarrays will be approximately 1 cm² in size. Additional plates will increase the length of the array by approximately 1 cm per plate. A schematic of the printed microarray design is shown in Figure 3. Once printed, the microarrays are ready to use immediately and can be stored for several years. Should the print job need to be repeated for any reason, due to the potential evaporation of samples during printing, it is recommended that a new sample plate be loaded and the original plate discarded.
    4. Once a week, thoroughly clean the print head and capillaries. Do this by loading a 384-well containing a 1:20 dilution of concentrated NaOH in GSB and perform a 40 min to 1 h print run onto microscope slides. Users should ensure this cleaning solution is compatible with their instrument before proceeding.
11. Save the unique grid file (.gal file) produced for the printed microarray ready for downstream analysis.

6. Microarray probing

1. Cut out individual, identical printed microarrays from the nitrocellulose membrane and place them into an appropriately sized vessel for probing (e.g., a 12- or 24-well microtiter plate) (see Table of Materials). The array should lie flat on the base of the vessel. One microarray is required per probe and represents one technical replicate.
2. To reduce non-specific binding, incubate the microarrays for 1 h in MP-TBST blocking buffer (1x Tris-buffered saline, pH 7.5, + 0.1% [v/v] TWEEN 20 [TBST] and supplemented with 5% [w/v] skimmed-milk powder; see Table of Materials) on a rotating/rocking shaker. Ensure that the volume is sufficient to submerge the entire array.
3. Following incubation, remove the MP-TBST and replace it with a fresh volume of MP-TBST.
4. Incubate the arrays with monoclonal antibodies (mAbs) or His-tagged CBMs, or other GRMPs (e.g., lectins), diluted 1:10-1:1,000 (as specified by the manufacturer; see Table of Materials) in MP-TBST for 2 h on a rotating/rocking shaker.
5. Following incubation, remove the molecular probe solution and cover the arrays in clean TBST, ensuring the arrays are fully submerged. To remove residual probe solution, immediately remove the TBST and replace with a fresh volume. Place the arrays on a rotating/rocking shaker for 5 min. After 5 min, remove the TBST, replace with a fresh volume, and place on a rotating/rocking shaker for 5 min.
   NOTE: This process should be repeated three times, not including the initial addition and immediate removal of TBST.
6. Incubate the arrays with alkaline-phosphatase conjugated secondary antibodies (anti-mouse, anti-rat, anti-rabbit, anti-His, as appropriate; see Table of Materials) diluted 1:1,000 in MP-TBST for 2 h on a rocking/rotating shaker.
   NOTE: Horseradish peroxidase-conjugated secondary antibodies are also suitable, used in conjunction with tetramethyl benzidine (TMB)/hydrogen peroxide substrate for color development.
7. Following incubation, repeat the wash procedure, as per step 6.5, to remove non-specifically bound secondary antibodies.
8. Cover the arrays in nitro-blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) color development solution (see Table of Materials) for the chromogenic detection of antibody binding. Leave until purple precipitate spots develop at the antigen binding sites (typically 5-30 min, however the arrays should be monitored closely to avoid oversaturation, as the reaction can occur rapidly).
   CAUTION: BCIP is harmful when in contact with the skin and may cause respiratory irritation. Use PPE. Avoid dust formation. Avoid breathing dust.
9. To terminate the reaction, submerge the arrays in clean tap water and wash extensively.
10. Place the arrays between blotting paper overnight at ambient temperature to dry.
11. Discard the arrays with obvious defects and repeat the probing protocol in such instances (Figure 4).

7. Analysis and quantification

1. Scan the developed arrays at a 2,400 dots per inch (dpi) resolution using a desktop scanner. Convert the images to TIFF files and then to negatives.
2. Using microarray analysis software (see Table of Materials), overlay the unique .gal grid file onto each microarray image to calculate the color intensity of the spot produced at each antigen binding site and subtract the local background.
3. Export the grid data as a .txt file. These can then be manually imported into an Excel sheet for analysis.
4. Generate a mean spot signal intensity value for each sample by averaging the spot intensity firstly across each sample dilution and then across any biological replicates included.
5. Assign a value of 100 to the highest mean spot signal intensity and normalize the remaining data accordingly.
   NOTE: Normalized mean spot signal intensities can then be presented as a heatmap of relative glycan epitope abundance using the conditional formatting function in Excel³³, or using the geom_tile function in R ggplot2 package^40,41.