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New immunological and mass spectrometric methods for complex analysis of persistent oligosaccharides in corn stover pretreated with AFEX. Lignocellulosic biomass is a sustainable alternative to fossil fuels and is widely used to develop biotechnologies for the production of products such as food, feed, fuels and chemicals. The key to these technologies is the development of cost-competitive processes for converting complex carbohydrates present in plant cell walls into simple sugars such as glucose, xylose and arabinose. Because lignocellulosic biomass is very stubborn, it must be subjected to thermochemical treatments (e.g., ammonia fiber exfoliation (AFEX), dilute acids (DA), ionic liquids (IL)) and biological treatments (e.g., enzymatic hydrolysis and microbial fermentation) in combination to obtain the desired product. . However, when commercial fungal enzymes are used in the hydrolysis process, only 75-85% of the soluble sugars formed are monosaccharides, and the remaining 15-25% are soluble, intractable oligosaccharides, which are not always available to microorganisms. Previously, we have successfully isolated and purified soluble stubborn oligosaccharides using a combination of carbon and diatomaceous earth separation and size exclusion chromatography, and also investigated their enzyme inhibitory properties. We have found that oligosaccharides containing higher degree of polymerization (DP) methylated uronic acid substitutions are more difficult to process with commercial enzyme blends than low DP and neutral oligosaccharides. Here we report the use of several additional methods, including glycan profiling using monoclonal antibodies (mAbs) specific to plant biomass glycans to characterize glycan bonds in plant cell walls and enzymatic hydrolysates, matrix-assisted laser desorption ionization, time-of-flight mass- spectrometry. . MALDI-TOF-MS) uses structure-informative diagnostic peaks obtained by spectroscopy after secondary decay of negative ions, gas chromatography and mass spectrometry (GC-MS) to characterize oligosaccharide bonds with and without derivatization. Due to the small size of oligosaccharides (DP 4–20), these molecules are difficult to use for mAb binding and characterization. To overcome this problem, we applied a new biotin conjugation-based oligosaccharide immobilization method that successfully labeled the majority of low DP soluble oligosaccharides on the microplate surface, which was then used in a high throughput mAb system for specific ligation analysis. This new method will facilitate the development of more advanced high throughput glycome assays in the future that can be used to isolate and characterize oligosaccharides present in biomarkers for diagnostic purposes.
Lignocellulosic biomass, composed of agricultural, forestry, grass and woody materials, is a potential feedstock for the production of bio-based products, including food, feed, fuel and chemical precursors to produce higher value products1. Carbohydrates (such as cellulose and hemicellulose) present in plant cell walls are depolymerized into monosaccharides by chemical processing and biotransformation (such as enzymatic hydrolysis and microbial fermentation). Common pre-treatments include ammonia fiber expansion (AFEX), dilute acid (DA), ionic liquid (IL), and steam explosion (SE), which use a combination of chemicals and heat to reduce lignocellulose production by opening plant cell walls3,4. stubbornness of matter, 5. Enzymatic hydrolysis is carried out at a high solids load using commercial active carbohydrate-containing enzymes (CAZymes) and microbial fermentation using transgenic yeasts or bacteria to produce bio-based fuels and chemicals 6 .
CAZymes in commercial enzymes are composed of a complex mixture of enzymes that synergistically cleave complex carbohydrate-sugar bonds to form monosaccharides2,7. As we reported earlier, the complex network of aromatic polymers of lignin with carbohydrates makes them highly intractable, which leads to incomplete sugar conversion, accumulating 15-25% of sex oligosaccharides that are not produced during enzymatic hydrolysis of the pretreated biomass. This is a common problem with various biomass pretreatment methods. Some reasons for this bottleneck include enzyme inhibition during hydrolysis, or the absence or low levels of essential essential enzymes that are required to break sugar bonds in plant biomass. Understanding the composition and structural characteristics of sugars, such as the sugar bonds in oligosaccharides, will help us improve sugar conversion during hydrolysis, making biotechnological processes cost-competitive with petroleum-derived products.
Determining the structure of carbohydrates is challenging and requires a combination of methods such as liquid chromatography (LC)11,12, nuclear magnetic resonance spectroscopy (NMR)13, capillary electrophoresis (CE)14,15,16 and mass spectrometry (MS)17. ,eighteen. MS methods such as time-of-flight mass spectrometry with laser desorption and ionization using a matrix (MALDI-TOF-MS) are a versatile method for identifying carbohydrate structures. Recently, Collision-Induced Dissociation (CID) tandem MS of sodium ion adducts has been most widely used to identify fingerprints corresponding to oligosaccharide attachment positions, anomeric configurations, sequences, and branching positions 20, 21 .
Glycan analysis is an excellent tool for in-depth identification of carbohydrate bonds22. This method uses monoclonal antibodies (mAbs) directed to plant cell wall glycan as probes to understand complex carbohydrate linkages. More than 250 mAbs are available worldwide, designed against various linear and branched oligosaccharides using various saccharides24. Several mAbs have been widely used to characterize the structure, composition, and modifications of the plant cell wall, as there are significant differences depending on plant cell type, organ, age, developmental stage, and growth environment25,26. More recently, this method has been used to understand vesicle populations in plant and animal systems and their respective roles in glycan transport as determined by subcellular markers, developmental stages, or environmental stimuli, and to determine enzymatic activity. Some of the different structures of glycans and xylans that have been identified include pectin (P), xylan (X), mannan (M), xyloglucans (XylG), mixed bond glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG) , glucuronic acid-arabinoxylan (GArbX) and arabino-galactan (ArbG)29.
However, despite all these research efforts, only a few studies have focused on the nature of oligosaccharide accumulation during high solids load (HSL) hydrolysis, including oligosaccharide release, oligomeric chain length changes during hydrolysis, various low DP polymers, and their curves. distributions 30,31,32. Meanwhile, although glycan analysis has proven to be a useful tool for a comprehensive analysis of glycan structure, it is difficult to evaluate water-soluble low DP oligosaccharides using antibody methods. Smaller DP oligosaccharides with a molecular weight of less than 5-10 kDa do not bind to ELISA plates 33, 34 and are washed away before antibody addition.
Here, for the first time, we demonstrate an ELISA assay on avidin-coated plates using monoclonal antibodies, combining a one-step biotinylation procedure for soluble refractory oligosaccharides with glycome analysis. Our approach to glycome analysis was validated by MALDI-TOF-MS and GC-MS based analysis of complementary oligosaccharide linkages using trimethylsilyl (TMS) derivatization of hydrolyzed sugar compositions. This innovative approach can be developed as a high-throughput method in the future and find wider application in biomedical research35.
Post-translational modifications of enzymes and antibodies, such as glycosylation,36 affect their biological activity. For example, changes in glycosylation of serum proteins play an important role in inflammatory arthritis, and changes in glycosylation are used as diagnostic markers37. Various glycans have been reported in the literature to readily appear in a variety of diseases, including chronic inflammatory diseases of the gastrointestinal tract and liver, viral infections, ovarian, breast, and prostate cancers38,39,40. Understanding the structure of glycans using antibody-based glycan ELISA methods will provide additional confidence in disease diagnosis without the use of complex MS methods.
Our previous study showed that stubborn oligosaccharides remained unhydrolyzed after pretreatment and enzymatic hydrolysis (Figure 1). In our previously published work, we developed an activated charcoal solid-phase extraction method to isolate oligosaccharides from AFEX-pretreated corn stover hydrolyzate (ACSH)8. After initial extraction and separation, the oligosaccharides were further fractionated by size exclusion chromatography (SEC) and collected in order of molecular weight. Sugar monomers and oligomers released from various pretreatments were analyzed by sugar composition analysis. When comparing the content of sugar oligomers obtained by various pretreatment methods, the presence of stubborn oligosaccharides is a common problem in the conversion of biomass to monosaccharides and can lead to a reduction in sugar yield of at least 10-15% and even up to 18%. US. This method is used for further large-scale production of oligosaccharide fractions. The resulting ACH and its subsequent fractions with different molecular weights were used as experimental material for the characterization of oligosaccharides in this work.
After pretreatment and enzymatic hydrolysis, persistent oligosaccharides remained unhydrolysed. Here (A) is an oligosaccharide separation method in which oligosaccharides are isolated from AFEX-pretreated corn stover hydrolysate (ACSH) using a packed bed of activated carbon and diatomaceous earth; (B) Method for separation of oligosaccharides. The oligosaccharides were further separated by size exclusion chromatography (SEC); (C) Saccharide monomers and oligomers released from various pretreatments (diluted acid: DA, ionic liquid: IL and AFEX). Enzymatic hydrolysis conditions: high solids loading of 25% (w/w) (approximately 8% glucan loading), 96 hours hydrolysis, 20 mg/g commercial enzyme loading (Ctec2:Htec2:MP-2:1:1 ratio) and ( D) Sugar monomers and oligomers of glucose, xylose and arabinose released from AFEX pre-treated corn stover (ACS).
Glycan analysis has proven to be a useful tool for a comprehensive structural analysis of glycans in extracts isolated from solid biomass residues. However, water-soluble saccharides are underrepresented using this traditional method41 because low molecular weight oligosaccharides are difficult to immobilize on ELISA plates and are washed out before antibody addition. Therefore, for antibody binding and characterization, a one-step biotinylation method was used to coat soluble, non-compliant oligosaccharides on avidin-coated ELISA plates. This method was tested using our previously produced ACSH and a fraction based on its molecular weight (or degree of polymerization, DP). One-step biotinylation was used to increase oligosaccharide binding affinity by adding biotin-LC-hydrazide to the reducing end of the carbohydrate (Fig. 2). In solution, the hemiacetal group at the reducing end reacts with the hydrazide group of biotin-LC-hydrazide to form a hydrazone bond. In the presence of the reducing agent NaCNBH3, the hydrazone bond is reduced to a stable biotinylated end product. With the modification of the sugar reducing end, binding of low DP oligosaccharides to ELISA plates became possible, and in our study this was done on avidin-coated plates using glycan-targeted mAbs.
Screening of monoclonal antibodies based on ELISA for biotinylated oligosaccharides. Here (A) combined biotinylation of oligosaccharides and subsequent ELISA screening with glycan-targeted mAbs on NeutrAvidin coated plates and (B) shows a one-step procedure for biotinylation of reaction products.
Avidin-coated plates with oligosaccharide-conjugated antibodies were then added to primary and secondary antibodies and washed in a light- and time-sensitive medium. After antibody binding is complete, add TMB substrate to incubate the plate. The reaction was finally stopped with sulfuric acid. The incubated plates were analyzed using an ELISA reader to determine the binding strength of each antibody to detect antibody-specific cross-linking. For details and parameters of the experiment, see the corresponding section “Materials and Methods”.
We demonstrate the utility of this newly developed method for specific applications by characterizing the soluble oligosaccharides present in ACSH as well as in crude and purified oligosaccharide fractions isolated from lignocellulosic hydrolysates. As shown in Figure 3, the most common epitope-substituted xylans identified in ACSH using bioacylated glycome assay methods are usually uronic (U) or methyluronic (MeU) and pectic arabinogalactans. Most of them were also found in our previous study on the analysis of glycans of non-hydrolyzed solids (UHS)43.
Detection of recalcitrant oligosaccharide epitopes using a monoclonal antibody directed to the cell wall glycan. The “neutral” fraction is the ACN fraction and the “acidic” fraction is the FA fraction. Brighter reds on the heatmap indicate higher epitope content, and brighter blues indicate a blank background. Color values on the scale are based on raw OD values for formulations N=2. The main epitopes recognized by the antibodies are shown on the right.
These non-cellulose structures could not be cleaved by the most common cellulases and hemicellulases in the tested commercial enzyme mixture, which includes the most commonly used commercial enzymes. Therefore, new auxiliary enzymes are required for their hydrolysis. Without the necessary non-cellulose accessory enzymes, these non-cellulose bonds prevent complete conversion to monosaccharides, even if their parent sugar polymers are extensively hydrolyzed into shorter fragments and dissolved using commercial enzyme mixtures.
Further study of signal distribution and its binding strength showed that binding epitopes were lower in high DP sugar fractions (A, B, C, DP up to 20+) than in low DP fractions (D, E, F, DP) in dimers) (Fig. 1). Acid fragments are more common in non-cellulose epitopes than neutral fragments. These phenomena are consistent with the pattern observed in our previous study, where high DP and acid moieties were more resistant to enzymatic hydrolysis. Therefore, the presence of non-cellulose glycan epitopes and U and MeU substitutions can greatly contribute to the stability of the oligosaccharides. It should be noted that binding and detection efficiency can be problematic for low DP oligosaccharides, especially if the epitope is a dimeric or trimeric oligosaccharide. This can be tested using commercial oligosaccharides of different lengths, each containing only one epitope that binds to a specific mAb.
Thus, the use of structure-specific antibodies revealed certain types of recalcitrant bonds. Depending on the type of antibody used, the appropriate ligation pattern, and the strength of the signal it produces (most and least abundant), new enzymes can be identified and added semi-quantitatively to the enzyme mixture for more complete glycoconversion. Taking the analysis of ACSH oligosaccharides as an example, we can create a database of glycan bonds for each biomass material. It should be noted here that the different affinity of antibodies should be taken into account, and if their affinity is unknown, this will create certain difficulties when comparing the signals of different antibodies. In addition, comparison of glycan bonds may work best between samples for the same antibody. These stubborn bonds can then be linked to the CAZyme database, from which we can identify enzymes, select candidate enzymes and test for bond-breaking enzymes, or develop microbial systems to express these enzymes for use in biorefineries44.
To evaluate how immunological methods complement alternative methods for characterizing low molecular weight oligosaccharides present in lignocellulosic hydrolysates, we performed MALDI (Fig. 4, S1-S8) and analysis of TMS-derived saccharides based on GC-MS on the same panel (Fig. 5) oligosaccharide part. MALDI is used to compare whether the mass distribution of oligosaccharide molecules matches the intended structure. On fig. 4 shows MS of the neutral components ACN-A and ACN-B. ACN-A analysis confirmed a range of pentose sugars ranging from DP 4–8 (Fig. 4) to DP 22 (Fig. S1), whose weights correspond to MeU-xylan oligosaccharides. ACN-B analysis confirmed the pentose and glucoxylan series with DP 8-15. In supplementary material such as Figure S3, FA-C acidic moiety mass distribution maps show a range of (Me)U substituted pentose sugars with a DP of 8-15 that are consistent with the substituted xylans found in ELISA-based mAb screening. The epitopes are consistent.
MALDI-MS spectrum of soluble non-compliant oligosaccharides present in ACS. Here, (A) ACN-A low weight range fractions containing methylated uronic acid (DP 4-8) substituted glucuroxylan oligosaccharides and (B) ACN-B xylan and methylated uronic acid oligosaccharides substituted with glucuroxylan (DP 8-15).
Analysis of the composition of the glycan residue of refractory oligosaccharides. Here (A) TMS saccharide composition of various oligosaccharide fractions obtained using GC-MS analysis. (B) Structures of various TMS-derived sugars present in oligosaccharides. ACN – acetonitrile fraction containing neutral oligosaccharides and FA – ferulic acid fraction containing acid oligosaccharides.
Another interesting conclusion was drawn from the LC-MS analysis of the oligosaccharide fraction, as shown in Figure S9 (methods can be seen in the electronic supplementary material). Fragments of hexose and -OAc groups were repeatedly observed during ligation of the ACN-B fraction. This finding not only confirms the fragmentation observed in glycome and MALDI-TOF analysis, but also provides new information about potential carbohydrate derivatives in pretreated lignocellulosic biomass.
We also performed glycan composition analysis of oligosaccharide fractions using TMS glycan derivatization. Using GC-MS, we determined the composition of neural (non-derivative) and acidic sugars (GluA and GalA) in the oligosaccharide fraction (Fig. 5). Glucuronic acid is found in acidic components C and D, while galacturonic acid is found in acidic components A and B, both of which are high DP components of acidic sugars. These results not only confirm our ELISA and MALDI data, but are also consistent with our previous studies of oligosaccharide accumulation. Therefore, we believe that modern immunological methods using biotinylation of oligosaccharides and subsequent ELISA screening are sufficient to detect soluble recalcitrant oligosaccharides in various biological samples.
Since ELISA-based mAb screening methods have been validated by several different methods, we wanted to further explore the potential of this new quantitative method. Two commercial oligosaccharides, xylohexasaccharide oligosaccharide (XHE) and 23-α-L-arabinofuranosyl-xylotriose (A2XX), were purchased and tested using a new mAb approach targeting the cell wall glycan. Figure 6 shows a linear correlation between the biotinylated binding signal and the log concentration of oligosaccharide concentration, suggesting a possible Langmuir adsorption model. Among the mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, and CCRC-M151 correlated with XHE, and CCRC-M108, CCRC-M109, and LM11 correlated with A2XX over a range of 1 nm to 100 nano. Due to the limited availability of antibodies during the experiment, limited experiments were performed with each oligosaccharide concentration. It should be noted here that some antibodies react very differently to the same oligosaccharide as a substrate, presumably because they bind to slightly different epitopes and can have very different binding affinities. The mechanisms and implications of accurate epitope identification will be much more complex when the new mAb approach is applied to real samples.
Two commercial oligosaccharides were used to determine the detection range of various glycan-targeting mAbs. Here, linear correlations with log concentration of oligosaccharide concentration indicate Langmuir adsorption patterns for (A) XHE with mAb and (B) A2XX with mAb. The corresponding epitopes indicate the structures of the commercial oligosaccharides used as substrates in the assay.
The use of glycan-targeted monoclonal antibodies (glycocomic analysis or ELISA-based mAb screening) is a powerful tool for in-depth characterization of most of the major cell wall glycans that make up plant biomass. However, classical glycan analysis only characterizes larger cell wall glycans, as most oligosaccharides are not efficiently immobilized on ELISA plates. In this study, AFEX-pretreated corn stover was enzymatically hydrolyzed at a high solids content. Sugar analysis was used to determine the composition of recalcitrant cell wall carbohydrates in the hydrolyzate. However, mAb analysis of smaller oligosaccharides in hydrolysates is underestimated, and additional tools are needed to effectively immobilize oligosaccharides on ELISA plates.
We report here a novel and efficient oligosaccharide immobilization method for mAb screening by combining oligosaccharide biotinylation followed by ELISA screening on NeutrAvidin™ coated plates. The immobilized biotinylated oligosaccharides showed sufficient affinity for the antibody to enable rapid and efficient detection of recalcitrant oligosaccharides. Analysis of the composition of these stubborn oligosaccharides based on mass spectrometry confirmed the results of this new approach to immunoscreening. Thus, these studies demonstrate that the combination of oligosaccharide biotinylation and ELISA screening with glycan-targeted monoclonal antibodies can be used to detect crosslinks in oligosaccharides and can be widely applied in other biochemical studies characterizing the structure of oligosaccharides.
This biotin-based glycan profiling method is the first report capable of investigating the recalcitrant carbohydrate bonds of soluble oligosaccharides in plant biomass. This helps to understand why some parts of biomass are so stubborn when it comes to biofuel production. This method fills an important gap in glycome analysis methods and extends its application to a wider range of substrates beyond plant oligosaccharides. In the future, we may use robotics for biotinylation and use the method we have developed for high-throughput analysis of samples using ELISA.
Corn straw (CS) grown from Pioneer 33A14 hybrid seeds was harvested in 2010 from Kramer Farms in Ray, Colorado. With the permission of the landowner, this biomass can be used for research. The samples were stored dry < 6% moisture in zip-lock bags in room temperature. The samples were stored dry < 6% moisture in zip-lock bags in room temperature. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Samples were stored dry at <6% humidity in zippered bags at room temperature.样品在室温下以干燥< 6% 的水分储存在自封袋中。样品在室温下以干燥< 6% Образцы хранят в пакетах с застежкой-молнией при комнатной температуре с влажностью < 6%. Samples are stored in zipper bags at room temperature with humidity < 6%. The study complied with local and national guidelines. Compositional analysis was performed using the NREL protocol. The composition was found to contain 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protein and 13. 4% ash.
Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) is a complex mixture of cellulase, β-glucosidase and Cellic® HTec2 (157 mg protein/ml, lot VHN00001) from Novozymes (Franklinton, NC, USA)). Multifect Pectinase® (72 mg protein/mL), a complex blend of pectin degrading enzymes, was donated by DuPont Industrial Biosciences (Palo Alto, CA, USA). Enzyme protein concentrations were determined by estimating protein content (and subtracting the contribution of non-protein nitrogen) using Kjeldahl nitrogen analysis (AOAC method 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). Diatomaceous earth 545 was purchased from EMD Millipore (Billerica, MA). Activated carbon (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
AFEX pretreatment was performed at GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Pre-treatment was carried out at 140° C. for 15 minutes. 46 residence time at 1:1 ratio of anhydrous ammonia to biomass at 60% (w/w) loading in a stainless steel benchtop batch reactor (Parr Instruments Company). It took 30 minutes. The reactor was brought to 140°C and the ammonia was rapidly released, allowing the biomass to quickly return to room temperature. The composition of AFEX pre-treated corn stover (ACS) was similar to that of untreated corn stover (UT-CS).
High solids ACSH 25% (w/w) (approximately 8% dextran loading) was prepared as a starting material for large scale production of oligosaccharides. Enzymatic hydrolysis of ACS was performed using a commercial enzyme mixture including Cellic® Ctec2 10 mg protein/g glucan (in pretreated biomass), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, and Multifect Pectinase (Genencor Inc, USA). ). ), 5 mg protein/g dextran. Enzymatic hydrolysis was carried out in a 5-liter bioreactor with a working volume of 3 liters, pH 4.8, 50°C and 250 rpm. After hydrolysis for 96 hours, the hydrolyzate was collected by centrifugation at 6000 rpm for 30 minutes and then at 14000 rpm for 30 minutes to remove unhydrolyzed solids. The hydrolyzate was then subjected to sterile filtration through a 0.22 mm filter beaker. The filtered hydrolyzate was stored in sterile bottles at 4° C. and then fractionated on carbon.
Analysis of the composition of extract-based biomass samples according to NREL laboratory analysis procedures: preparation of samples for composition analysis (NREL/TP-510-42620) and determination of structural carbohydrates and lignin in biomass (NREL/TP-510 – 42618)47.
Oligosaccharide analysis of the hydrolyzate stream was performed on a 2 ml scale using an autoclave-based acid hydrolysis method. Mix the hydrolyzate sample with 69.7 µl of 72% sulfuric acid in a 10 ml screw cap culture tube and incubate for 1 h on a benchtop at 121 °C, cool on ice and filter into a high performance liquid chromatography (HPLC) vial . The concentration of oligosaccharides was determined by subtracting the concentration of monosaccharides in the non-hydrolyzed sample from the total sugar concentration in the acid-hydrolyzed sample.
Glucose, xylose, and arabinose concentrations in the acid hydrolysed biomass were analyzed using a Shimadzu HPLC system equipped with an autosampler, column heater, isocratic pump, and refractive index detector on a Bio-Rad Aminex HPX-87H column. The column was maintained at 50°C and eluted with 0.6 ml/min 5 mM H2SO4 in water. flow.
The hydrolyzate supernatant was diluted and analyzed for monomer and oligosaccharide content. Monomeric sugars obtained after enzymatic hydrolysis were analyzed by HPLC equipped with a Bio-Rad (Hercules, CA) Aminex HPX-87P column and an ash guard column. The column temperature was maintained at 80°C, water was used as the mobile phase with a flow rate of 0.6 ml/min. Oligosaccharides were determined by hydrolysis in dilute acid at 121°C according to the methods described in refs. 41, 48, 49.
Saccharide analysis was performed on raw, AFEX pre-treated and all non-hydrolysed biomass residues (including production of serial cell wall extracts and their mAb screening) using previously described procedures 27, 43, 50, 51 . For glycome analysis, alcohol-insoluble residues of plant cell wall material are prepared from biomass residues and subjected to serial extraction with increasingly aggressive reagents such as ammonium oxalate (50 mM), sodium carbonate (50 mM and 0.5% w/v), CON. (1M and 4M, both with 1% w/v sodium borohydride) and acid chlorite as previously described52,53. The extracts were then subjected to ELISA against a complex panel of mAb50s directed to the cell wall glycan, and the mAb binding reactions were presented as a heat map. mAbs targeting plant cell wall glycan were purchased from laboratory stocks (CCRC, JIM and MAC series).
One-step biotinylation of oligosaccharides. The conjugation of carbohydrates with biotin-LC-hydrazide was performed using the following procedure. Biotin-LC-hydrazide (4.6 mg/12 μmol) was dissolved in dimethyl sulfoxide (DMSO, 70 μl) by vigorous stirring and heating at 65° C. for 1 min. Glacial acetic acid (30 µl) was added and the mixture was poured onto sodium cyanoborohydride (6.4 mg/100 µmol) and completely dissolved after heating at 65° C. for about 1 minute. Then, from 5 to 8 μl of the reaction mixture was added to the dried oligosaccharide (1-100 nmol) to obtain a 10-fold or more molar excess of the label over the reducing end. The reaction was carried out at 65°C for 2 h, after which the samples were immediately purified. No sodium cyanoborohydride was used in the labeling experiments without reduction, and the samples were reacted at 65° C. for 2.5 hours.
ELISA coating and washing of samples of biotinylated oligosaccharides. 25 μl of biotinylated samples (100 μl of each concentrated sample diluted in 5 ml of 0.1 M Tris buffer solution (TBS)) were added to each well of the avidin-coated plate. Control wells were coated with 50 μl of biotin at a concentration of 10 μg/ml in 0.1 M TBS. Deionized water was used as a coating for blank measurements. The tablet was incubated for 2 hours at room temperature in the dark. Wash the plate 3 times with 0.1% skimmed milk in 0.1 M TBS using program no. 11 for Grenier flat 3A.
Addition and washing of primary antibodies. Add 40 µl of primary antibody to each well. Incubate the microplate for 1 hour at room temperature in the dark. The plates were then washed 3 times with 0.1% milk in 0.1M TBS using wash program #11 for Grenier Flat 3A.
Add secondary antibody and wash. Add 50 µl of mouse/rat secondary antibody (diluted 1:5000 in 0.1% milk in 0.1 M TBS) to each well. Incubate the microplate for 1 hour at room temperature in the dark. The microplates were then washed 5 times with 0.1% milk in 0.1 M TBS using Grenier Flat 5A plate wash program #12.
Adding a substrate. Add 50 µl of 3,3′,5,5′-tetramethylbenzidine (TMB) to the base substrate (by adding 2 drops of buffer, 3 drops of TMB, 2 drops of hydrogen peroxide to 15 ml of deionized water). Prepare the TMB substrate. and vortex before use). Incubate the microplate at room temperature for 30 minutes. In the dark.
Complete the step and read the tablet. Add 50 µl of 1 N sulfuric acid to each well and record the absorbance from 450 to 655 nm using an ELISA reader.
Prepare 1 mg/ml solutions of these analytes in deionized water: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine . (glcNAc), N-acetylgalactosamine (galNAc), inositol (internal standard). Two standards were prepared by adding the 1 mg/mL sugar solutions shown in Table 1. Samples are frozen and lyophilized at -80° C. until all water is removed (usually about 12-18 hours).
Add 100–500 µg of sample to screw cap tubes on an analytical balance. Record the amount added. It is best to dissolve the sample in a specific concentration of solvent and add it to the tube as a liquid aliquot. Use 20 µl of 1 mg/ml inositol as an internal standard for each sample tube. The amount of internal standard added to the sample must be the same as the amount of internal standard added to the standard tube.
Add 8 ml of anhydrous methanol to a screw cap vial. Then 4 ml of 3 N. methanolic HCl solution, capped and shaken. This process does not use water.
Add 500 µl of 1 M HCl methanol solution to the oligosaccharide samples and standard TMS tubes. Samples were incubated overnight (168 hours) at 80° C. in a thermal block. Dry the methanolysis product at room temperature using a drying manifold. Add 200 µl MeOH and dry again. This process is repeated twice. Add 200 µl of methanol, 100 µl of pyridine and 100 µl of acetic anhydride to the sample and mix well. Incubate samples at room temperature for 30 minutes. and dried. Add 200 µl of methanol and dry again.
Add 200 µl of Tri-Sil and heat capped tube for 20 minutes. 80°C, then cooled to room temperature. Use a drying manifold to further dry the sample to a volume of approximately 50 µl. It is important to note that we did not allow the samples to dry completely.
Add 2 ml of hexane and mix well by vortexing. Fill the tips of Pasteur pipettes (5-8 mm) with a piece of glass wool by inserting the glass wool on top of a 5-3/4 inch diameter pipette. Samples were centrifuged at 3000 g for 2 minutes. Any insoluble residues are precipitated. Dry the sample to 100-150 µl. A volume of approximately 1 μl was injected into the GC-MS at an initial temperature of 80 °C and an initial time of 2.0 minutes (Table 2).