Name: microarray polymer profiling (mapp) for high-throughput glycan analysis
Uploaded: 2023-09-29T13:00:00.000+00:00
Duration: 7 min 12 s
Description: Watch this Scientific Journal Video about MAPP Protocol – Advancing Glycan Analysis at JoVE.com

The authors would like to acknowledge ArrayJet for their expert advice regarding microarray robotics. SS and JS would like to acknowledge the support from Fundamental Fund 2022 (FF65/004), Chiang Mai University.

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.
<ol>
	<li>Separate the plant material into different tissues (e.g., root, stem, and leaves).</li>
	<li>Dry the plant tissues in a hot air oven at 40 &#176;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.</li>
	<li>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. 
	&#8203;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).</li>
</ol>
2. Preparation of alcohol insoluble residue (AIR)
<ol>
	<li>Add 1.5 mL of 70% (v/v) ethanol to 50-100 mg of air-dried and homogenized sample material.</li>
	<li>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.</li>
	<li>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.</li>
	<li>To the remaining pellet, add 1.5 mL of 100% acetone. Vortex, centrifuge, and discard the supernatant, as per step 2.2.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>To 10 mg of AIR material, add 30 &#181;L/mg of 50 mM cyclohexanediaminetetraacetic acid (CDTA; see Table of Materials), pH 7.5.</li>
	<li>Shake at 27 Hz&#160;for 2 min, followed by 10 Hz&#160;for 2 h.</li>
	<li>Centrifuge at 10,000 x g for 10 min at 4 &#176;C. Retain the resultant supernatant, add it to a sterile microcentrifuge tube, and store it at 4 &#176;C on a rotary shaker.</li>
	<li>To the residual pellet, add 30 &#181;L/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.</li>
	<li>Repeat steps 3.2 to 3.3. Wash the residual pellet two or three times with dH2O to remove the residual NaOH.</li>
	<li>Add 30 &#181;L/mg cellulase (preferably GH5 endo-1,4-&#946;-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.</li>
	<li>Centrifuge the samples at 10,000 x g for 10 min at 4&#160;&#176;C . Retain the supernatant, transfer to clean microcentrifuge tubes, and store at 4 &#176;C on a rotary shaker. Once extracted, the samples must be printed as soon as possible.</li>
	<li>Centrifuge all the stored extracts again at 10,000 x g for 10 min at 4&#160;&#176;C. 
	&#8203;NOTE: The samples must be free of particulates and debris. Pass through a 0.2 &#181;m 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.</li>
</ol>
4. Preparation of standards
<ol>
	<li>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.</li>
	<li>Store the prepared standards overnight at 4 &#176;C on a rotary shaker to allow complete solubilization.</li>
	<li>Centrifuge all the standards for 10 min at 10,000 x g at 4&#160;&#176;C to pellet any debris. The resultant supernatant is used for subsequent printing.</li>
</ol>
5. Microarray printing
<ol>
	<li>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.</li>
	<li>Add 40 &#181;L of ink solution and GSB to the first section of the first 384-well plate (Figure 2).</li>
	<li>Add 25 &#181;L of GSB to all dilution 1 (D1) wells. Add 40 &#181;L of GSB to all dilution 2, 3, and 4 (D2-D4) wells.</li>
	<li>Dilute the extracted glycan samples and defined glycan substrates 1:1 (v/v) with GSB by adding 25 &#181;L of extracted glycan sample to the D1 wells in order.</li>
	<li>Serially dilute each sample four times by taking 10 &#181;L of the sample from the D1 well and adding to the D2 well. Aspirate gently with a pipette to mix.</li>
	<li>Repeat the process by taking 10 &#181;L of sample from the D2 well and adding it to the D3 well.</li>
	<li>Repeat for the D4 well. After mixing, discard 10 &#181;L from the D4 well so that each well contains a final volume of 40 &#181;L.</li>
	<li>Add 40 &#181;L of ink solution and GSB to the final block of the final plate.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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 &#62;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.</li>
		<li>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.</li>
		<li>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 &#181;m to 100 &#181;m, 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.</li>
		<li>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&#160;a 40 min to 1 h print run onto microscope slides. Users should ensure this cleaning solution is compatible with their instrument before proceeding.</li>
	</ol>
	</li>
	<li>Save the unique grid file (.gal file) produced for the printed microarray ready for downstream analysis.</li>
</ol>
6. Microarray probing
<ol>
	<li>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&#160;flat on the base of the vessel. One microarray is required per probe and represents one technical replicate.</li>
	<li>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.</li>
	<li>Following incubation, remove the MP-TBST and replace it with a fresh volume of MP-TBST.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Following incubation, repeat the wash procedure, as per step 6.5, to remove non-specifically bound secondary antibodies.</li>
	<li>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.</li>
	<li>To terminate the reaction, submerge the arrays in clean tap water and wash extensively.</li>
	<li>Place the arrays between blotting paper overnight at ambient temperature to dry.</li>
	<li>Discard the arrays with obvious defects and repeat the probing protocol in such instances (Figure 4).</li>
</ol>
7. Analysis and quantification
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Export the grid data as a .txt file. These can then be manually imported into an Excel sheet for analysis.</li>
	<li>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.</li>
	<li>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 package40,41.</li>
</ol>

Elucidating Glycan Structure and Occurrence Using Microarray Polymer Profiling

The authors declare no conflict of interest.

The MAPP technique described here is now a well-established method for glycan analysis. The basic principles were first described in 200711, but the technique has undergone continuous development in order to capitalize on the latest innovations in microarray technology, molecular probe development, and advances in our understanding of glycan biochemistry. In general, glycans, especially polysaccharides, are more challenging to analyze than proteins and nucleotides due to their structural complexity and heterogeneity45, as well as the fact that they cannot readily be sequenced or synthesized1. In many cases, no single technique can decipher glycan complexity conclusively; thus, MAPP is often used with other methods. This is one of the reasons why AIR preparation is usually chosen as a starting point for MAPP, since AIR is compatible with most other glycan analysis methods34, facilitating the subsequent comparison of datasets.
Due to homogenization of the sample prior to AIR preparation, some spatial information is invariably lost. However, as polysaccharides are sequentially released from samples, the presence of epitopes in the fractions obtained provides information about the molecular architecture and composition of that sample17. Thus, selecting an appropriate extraction regime is critical to the success of the method. Multiple parameters determine the suitability of the extraction method: cellular structure, time, temperature, pH, pressure, ionic strength of the solvent, and fineness of the solid particulate sample49. It is recommended that a range of increasingly more aggressive solvents is used to maximize the likelihood of successfully extracting constituent glycans and building a representative compositional picture of the sample. For most samples, CDTA, NaOH, and cellulase are sufficient to remove plant-derived storage and cell wall polysaccharides33,50,51,52. For some tissue samples, a hybrid extraction regime that also includes CaCl2, HCl, and Na2CO3 has been shown to be successful53, while marine microalgal samples may require the addition of ethylenediaminetetraacetic acid (EDTA)10.
Microarrays should include a range of pure, defined glycan standards to be used as positive controls5. Included standards should be modified according to the nature of the sample. Once printed, appropriate GRMPs need to be selected. The generation of hybridoma mAbs to polysaccharide structures is challenging54; glycan-binding antibodies are difficult to raise and can have low affinity55. Fortunately, gene sequence information for CBMs can be obtained with relative ease for recombinant expression4 and engineering their binding specificities56,57. Whilst an impressive catalog of GRMPs has been developed, with most now available from commercial sources, relative to the diversity of glycan structures existing in nature, only a small proportion have been produced and successfully characterised58. This can limit the ability to detect and discriminate between certain structures. It is advisable to perform an initial probing experiment using one or two probes representative of each major glycan structure anticipated to be present, for which the binding specificity is well characterized. In subsequent probing experiments, the probe list can be expanded to cover a broader range of glycans and delve deeper into fine structures.
Although mundane, ensuring that microarrays are washed thoroughly after each incubation step is fundamental to the success of the probing procedure. The ineffective removal of non-specifically bound probes is likely to obscure the result by causing a high background signal following color development. In this case, it is necessary to repeat the probing procedure, starting with a new microarray. Furthermore, arrays should be touched sparingly and only by holding the edges with forceps; the nitrocellulose membrane is brittle and easily damaged. The color development solution collects in cracks and creases, causing oversaturation, which impedes array analysis.
MAPP is quick, adaptable, and convenient. This method is compatible with animal, microbial, or plant glycans derived from any biological or industrial system, so long as they can be extracted and immobilized onto nitrocellulose, and for which one has appropriate molecular probes. The data generated provide detailed, semi-quantitative, compositional insight, which cannot readily be obtained via other glycan analysis methods.

Glycans are ubiquitous in all domains of life and exhibit unparalleled diversity in structure and function compared to other macromolecules1. However, due to their complexity, variability in biosynthesis and glycosidic linkages, and the paucity of appropriate methods for dissecting glycan structures, our understanding of this diversity in structures and functions is relatively limited2.
Many glycan analysis techniques are destructive and necessitate the breakdown of glycans into their constituent monosaccharides, which can obscure relevant three-dimensional and biological contexts3. Conversely, monoclonal antibodies (mAbs), carbohydrate binding modules (CBMs), lectins, viral agglutinins, and microbial adhesins, known collectively as glycan-recognizing molecular probes (GRMPs)4, recognize and bind to specific epitopes and can be used as tools to detect and discriminate between glycans within complex multi-glycan matrices5,6.
Here, we present microarray polymer profiling (MAPP), a rapid, versatile, and nondestructive method for glycan analysis that is applicable to a broad spectrum of biological samples. The method aims to provide a robust and high-throughput technology for analyzing glycans from diverse biological and industrial/commercial systems. MAPP unites the recognition specificity of glycan-directed molecular probes with reproducible, high-performance microarray screening technology to allow thousands of molecular interactions to be profiled in parallel. The output of this approach is diagnostic insight into the composition and relative abundance of glycans within a sample or tissue of interest.
MAPP can be used as an independent, stand-alone method, or in conjunction with other biochemical techniques, such as immunofluorescence microscopy7,8,9 and monosaccharide or linkage analysis10,11. The technique can also be used to map epitope specificities of novel GRMPs, using arrays printed with pure and structurally well-defined oligosaccharide standards12. A major advantage of MAPP over other methods, such as enzyme-linked immunosorbent assay (ELISA), is its compatibility with small sample volumes13,14. Moreover, MAPP offers significantly higher-throughput analysis15 and provides an effective form of sample preservation, as printed samples are dry and stable when immobilized onto nitrocellulose16.
The binding of GRMPs is generally dependent on the presence of a number of contiguous sugar residues that collectively form a binding site (epitope) that is unique to a particular polysaccharide class (xylan, mannan, xyloglucan, etc.)17. In contrast, the individual sugar residues (xylose, mannose, glucose) that are quantified using most biochemical techniques, for example monosaccharide composition or methylation analysis, can be components of multiple polysaccharide classes and thus difficult to assign18.
MAPP has been developed in response to a technology gap, namely the ability to rapidly analyze multiple glycans from a variety of sources using small amounts of material. MAPP capitalizes on the extensive repertoire of GRMPs that have been developed and characterized over the last three decades12,19,20,21,22,23,24,25,26,27,28,29,30,31,32. The development of MAPP has been an iterative process, with the technique being steadily refined and optimized. There is now a substantial body of literature describing the application of MAPP to various natural and industrial systems where glycans play central roles 5,6,9,10,21,33,34,35,36,37,38,39. Here, we describe the current state of the art for MAPP.

<table><tbody><tr><td>1,3:1,4-&amp;beta;-D-Glucan, Lichenan (icelandic moss)</td><td>Megazyme</td><td>P-LICHN</td><td></td></tr><tr><td>1,4-&amp;beta;-D-Mannan</td><td>Megazyme</td><td>P-MANCB</td><td></td></tr><tr><td>384-well microtiter plate</td><td>Greiner Bio-One</td><td>M1686</td><td></td></tr><tr><td>5-bromo-4-chloro-3-indolyl-phosphate (BCIP)</td><td>Melford</td><td>B74100-1.0</td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>270725</td><td></td></tr><tr><td>Alkaline Phosphatase AffiniPure Goat Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>115-055-003</td><td></td></tr><tr><td>Alkaline Phosphatase AffiniPure Goat Anti-Rat IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>112-055-003</td><td></td></tr><tr><td>Alkaline Phosphatase AffiniPure Rabbit Anti-His Tag</td><td>Jackson ImmunoResearch</td><td>300-055-240</td><td></td></tr><tr><td>Arabinoxylan (wheat)</td><td>Megazyme</td><td>P-WAXYL</td><td></td></tr><tr><td>Array-Pro Analyzer Software</td><td>Media Cybernetics</td><td>Version 6.3</td><td></td></tr><tr><td>&lt;em&gt;Bacillus &lt;/em&gt;sp. Cellulase 5A (BCel5A)</td><td>NZYTech</td><td>CZ0564</td><td></td></tr><tr><td>BAM antibodies</td><td>SeaProbes</td><td>Various</td><td></td></tr><tr><td>Black drawing ink (indian ink)</td><td>Winsor &amp;amp; Newton</td><td>GWD030</td><td></td></tr><tr><td>Carbohydrate binding modules</td><td>NZYTech</td><td>Various</td><td></td></tr><tr><td>CCRC antibodies</td><td>CarboSource</td><td>Various</td><td></td></tr><tr><td>CDTA</td><td>Sigma</td><td>319945</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>PHR1552</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>1.11727</td><td></td></tr><tr><td>Galactan (potato)</td><td>Megazyme</td><td>P-GALPOT</td><td></td></tr><tr><td>Galactomannan (carob)</td><td>Megazyme</td><td>P-GALML</td><td></td></tr><tr><td>Glycerol solution</td><td>Sigma</td><td>49781-5L</td><td></td></tr><tr><td>Gum tragacanth (legumes)</td><td>Sigma-Aldrich</td><td>G1128</td><td></td></tr><tr><td>INCh antibodies</td><td>INRA</td><td>Various</td><td></td></tr><tr><td>LM and JIM antibodies</td><td>PlantProbes</td><td>Various</td><td></td></tr><tr><td>Marathon Argus Microarray Printer</td><td>ArrayJet</td><td></td><td></td></tr><tr><td>Methanol</td><td>Sigma&amp;nbsp;</td><td>34860</td><td></td></tr><tr><td>Monoclonal antibodies</td><td>Biosupplies Australia</td><td>Various</td><td></td></tr><tr><td>NaBH&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>452882</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>S5881</td><td></td></tr><tr><td>Nitro-blue tetrazolium (NBT)</td><td>Melford</td><td>N66000-1.0</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Thermo Fisher Scientific</td><td>88018</td><td></td></tr><tr><td>Pectin (degree of methyl esterification 46%)</td><td>Danisco</td><td>NA</td><td></td></tr><tr><td>ProClin 200</td><td>Sigma</td><td>48171-U</td><td></td></tr><tr><td>Rhamnogalacturonan (soybean pectic fibre)</td><td>Megazyme</td><td>P-RHAGN</td><td></td></tr><tr><td>Rotating mixer</td><td>Fisher Scientific</td><td>88-861-050</td><td></td></tr><tr><td>Rotating/rocking Shaker</td><td>Cole-Parmer</td><td></td><td></td></tr><tr><td>Skimmed milk powder</td><td>Marvel</td><td></td><td></td></tr><tr><td>Spin filter</td><td>Costar Spin-X</td><td>8160</td><td></td></tr><tr><td>Stainless steel beads</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>TissueLyser II</td><td>Qiagen</td><td>85300</td><td></td></tr><tr><td>Tris</td><td>Sigma</td><td>93362</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-250ML</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416-100ML</td><td></td></tr><tr><td>Xylan (beechwood)</td><td>Megazyme</td><td>P-XYLNBE</td><td></td></tr><tr><td>Xyloglucan (tamarind)</td><td>Megazyme</td><td>P-XYGLN</td><td></td></tr><tr><td>&amp;beta;-Glucan (oat)</td><td>Megazyme</td><td>P-BGOM</td><td></td></tr></tbody></table>

microarray polymer profiling (mapp) for high-throughput glycan analysis

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans and glycoconjugates within a variety of biological samples, including plant and algal tissues, food materials, and human, animal, and microbial samples. Microarray technology underpins the efficacy of this method by providing a miniaturized, high-throughput screening platform, allowing thousands of interactions between glycans and highly specific glycan-directed molecular probes to be characterized concomitantly, using only small amounts of analytes. Constituent glycans are chemically and enzymatically fractionated, before being sequentially extracted from the sample and directly immobilized onto nitrocellulose membranes. The glycan composition is determined by the attachment of specific glycan-recognizing molecular probes to the extorted and printed molecules. MAPP is complementary to conventional glycan analysis techniques, such as monosaccharide and linkage analysis and mass spectrometry. However, glycan-recognizing molecular probes provide insight into the structural configurations of glycans, which can aid in elucidating biological interactions and functional roles.

MAPP was applied to determine the glycan composition of agricultural biomass waste, comprising mango peels from several northern Thai varieties, Coffea arabica cherry pulp and coffee bean processing waste, and root, stem, and leaf tissue from Thai black garlic, Allium sativum var. ophioscorodon. Several plant-derived polysaccharides are used in the food industry as functional ingredients42,43. Thus, the aim of this experiment was to deduce whether these abundant and currently underutilized agro-industrial waste materials may provide a source of value-added pure polysaccharides.
AIR material is routinely employed to prepare samples intended for glycan analysis44. There are several advantages to using AIR; treatment with solvents effectively removes endogenous CAZymes, metabolites, small saccharides, lipids, and pigments, resulting in samples enriched with polysaccharides and structural proteins34. Furthermore, producing AIR is a fast and effective way to increase sample longevity, as it is thermostable and can be stored for several years.
Three mixed fractions of constituent glycans were sequentially extracted from plant AIR material using CDTA, NaOH, and cellulase. CDTA chelates Ca2+ ions, which allow the removal of Ca2+ crosslinked de-esterified pectins from plant cell walls45. Alkaline conditions allow predominantly hemicelluloses, such as mannan, xylan, and &#946;-glucan, to be released due to the disruption of hydrogen bonding and saponification of ester linkages between cellulose microfibrils and hemicellulose, and lignin and hemicellulose, respectively46. A recombinant endo-1,4-&#946;-glucanase from Bacillus spp. was used to degrade amorphous regions of the structural cellulose microfibrils, releasing residual glycans bound to cellulose within the cell walls47. Although this method effectively separates glycans into these three broad groups, it should be noted that the samples are not pure; by the very nature of the extraction method, hemicellulose, if present in the sample, will inevitably be extracted and subsequently detected to varying degrees in the CDTA and cellulase fractions. Likewise, some pectin will be detected in the NaOH extraction if present in the sample.
A non-contact, piezoelectric microarray printing robot was used to immobilize extracted glycan fractions onto nitrocellulose via non-covalent attachment11, forming 300 identical microarrays. Defined glycan standards (Table 1) were also included in the printed microarrays as positive controls (Figure 5). The MAPP binding profile obtained for the selected glycan standards corresponds to previously reported epitope specificities. For example, LM21 exhibited strong binding to multiple mannan polysaccharides (galactomannan and glucomannan), while LM22 exhibited only weak binding to galactomannan25. Similarly, LM19 preferentially bound to de-esterified homogalacturonan48&#160;and LM15 bound to tamarind seed xyloglucan23.
The relative abundance of 16 epitopes, diagnostic of non-cellulosic plant cell wall polysaccharides, were detected by&#160;the attachment of&#160;glycan-directed monoclonal antibodies (Table 2) to printed extracts (Figure 6). The majority of extracted glycans were detected within the alkaline NaOH fraction. Strong binding signals were recorded for mAbs LM10 and LM11, representing xylan/arabinoxylan, within the peels of all the mango varieties tested. Within the garlic samples, LM10 and LM11 bound preferentially to root tissue extract (Garlic R) and exhibited only weak binding to the leaf tissue extract (Garlic L). LM19, representing partially methyl-esterified or un-esterified homogalacturonan, bound strongly to some mango variety extracts (Aokrong and Talabnak), but bound only weakly, or its binding was undetectable, in other varieties (Chokanan, Mamkamdang, Mahachanok, and Nga). In addition, LM19 bound only to the coffee pulp fractions and did not bind to the coffee bean processing waste material, previously thought to be composed of semi-purified coffee pectin (unpublished data).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65443/65443fig01.jpg" /> 
Figure 1: Major experimental steps in the MAPP method. (A) Samples are homogenized to form fine powders. (B) The homogenized samples are processed to isolate their AIRs. (C) The constituent glycans are sequentially extracted using a tailored extraction regime. (D) The extracted glycan fractions, ink, and GSB are transferred into 384-well plates, according to the plate layout, for printing onto nitrocellulose. (E) The printed microarrays are probed with selected GRMPs. (F) GRMP binding to the printed glycan fractions is quantified and analyzed before data are presented as a heatmap. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65443/65443fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65443/65443fig02.jpg" /> 
Figure 2: Example of a 384-well plate layout for sample, ink, and GSB loading with four dilutions per extracted glycan sample/standard. Different colors denote samples arising from different extraction reagents, while different shades represent serial dilutions. The first number in the code represents the sample number, while the end number represents the dilution number (D1 denotes dilution one, D2 denotes dilution two, and so on). For example, a well labeled &#39;12D3&#39; represents glycan sample 12, dilution three. Well plates should be divided into eight identical sections comprising six columns and eight rows. The first section of the first plate should contain only ink and buffer and resemble the example plate layout. Extracted glycan samples can then be loaded into subsequent plate sections according to the plate layout. Different extraction reagents should not be loaded into the same plate section. If there are insufficient samples to fill an entire section, fill all remaining wells in that section with buffer; do not leave any wells empty. If multiple plates are required, the next section after all samples have been loaded should contain three alternating columns of ink and GSB-this may not be section eight, depending on the number of samples being printed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65443/65443fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65443/65443fig03.jpg" /> 
Figure 3: Schematic representation of printed microarray design. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65443/65443fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65443/65443fig04.jpg" /> 
Figure 4: Representative microarrays. (A) No binding. (B) The binding signal is obscured by high background signal. (C) Generalized blue/purple staining due to oversaturation with NBT/BCIP. (D) Defective probing due to high substrate concentration. (E) Defective printing due to the unclean print head. (F) Strong binding to few samples. (G) Strong binding to many samples. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65443/65443fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65443/65443fig05.jpg" /> 
Figure 5: Monoclonal antibody binding to defined glycan standards, included to validate the printing and probing process. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65443/65443fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65443/65443fig06.jpg" /> 
Figure 6: MAPP of glycans extracted from agricultural biomass waste. The samples include coffee pulp waste (coffee pulp and coffee pectin), mango peels from several Thai varieties (AO, Aokrong; KO, Kam; RD, Rad; CH, Chokanan; MA, Mamkamdang; TL, Talabnak; MH, Mahachanok; NG, Nga) and black garlic leaves (Garlic L), stem (Garlic S), bulb (Garlic BG), and roots (Garlic R), using CDTA, NaOH, and cellulase (Bacillus spp. cellulase 5A). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65443/65443fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Defined commercial polysaccharide standards used in MAPP analysis as positive controls. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65443/65443_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Glycan-directed monoclonal antibodies selected for the interrogation of extracted plant glycan microarrays. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65443/65443_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about MAPP Protocol – Advancing Glycan Analysis at JoVE.com

Author Spotlight: MAPP Protocol &#8211; Advancing Glycan Analysis 

Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples.

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

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans ...

microarray-polymer-profiling-mapp-for-high-throughput-glycan-analysis

Nanyang Technological University

Research

JoVE Journal

Biology

1.6K Views.  Newcastle University. Ons onderzoek richt zich op het voorkomen van functies en structuren van complexe glycanen binnen diverse huishoudelijke, industriële en commerciële systemen. Verdere verfijningen van contactloze microarray-technologie en ons vermogen om snel toegang te krijgen tot sequentie-informatie voor koolhydraatbindende modules vergroten onze capaciteit om glycaanmicroarrays zowel te printen als te ondervragen. Glycanen zijn moeilijk te bestuderen vanwege hun structurele heterogeniteit, complexiteit ...

Video: Auteur in de schijnwerpers: MAPP-protocol - Geavanceerde glycaananalyse 

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Bioengineering

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

Chemistry

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

Behavior

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Biochemistry

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Cancer Research

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals.

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

Immunology and Infection

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays 

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

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

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