Name: automated modular high throughput exopolysaccharide screening platform coupled with highly sensitive carbohydrate fingerprint analysis
Uploaded: 2016-04-11T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis at JoVE.com

We sincerely thank Thomas Howe and J&#246;rg Carsten for the programming and technical support with the liquid handling systems.

<ol>
	<li>Milani, J., Maleki, G., Valdez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2,+Hydrocolloids+in+Food+Industry.">Ch. 2, Hydrocolloids in Food Industry.</a> Food Industrial Processes - Methods and Equipment. 2, (2012).</li><li>Thibodeau, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+the+skin+from+environmental+stresses+with+an+exopolysaccharide+formulation.">Protecting the skin from environmental stresses with an exopolysaccharide formulation.</a> Cosmet Toiletries. 120 (12), 81-90 (2005).</li><li>Prajapati, V. D., Jani, G. K., Zala, B. S., Khutliwala, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insight+into+the+emerging+exopolysaccharide+gellan+gum+as+a+novel+polymer.">An insight into the emerging exopolysaccharide gellan gum as a novel polymer.</a> Carbohydr Polym. 93 (2), 670-678 (2013).</li><li>Schmidt, W., Brouwers, H. J. H., Kuehne, H. C., Meng, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+working+mechanism+of+starch+and+diutan+gum+in+cementitious+and+limestone+dispersions+in+presence+of+polycarboxylate+ether+superplasticizers.">The working mechanism of starch and diutan gum in cementitious and limestone dispersions in presence of polycarboxylate ether superplasticizers.</a> Appl. Rheol. 23 (5), 52903 (2013).</li><li>Srinivasan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+Polysaccharides+as+Treatment+Agents+for+Wastewater.">Natural Polysaccharides as Treatment Agents for Wastewater.</a> Green Materials for Sustainable Water Remediation and Treatment. , 51-81 (2013).</li><li>R&#252;hmann, B., Schmid, J., Sieber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+exopolysaccharide+screening+platform:+From+strain+cultivation+to+monosaccharide+composition+and+carbohydrate+fingerprinting+in+one+day.">High throughput exopolysaccharide screening platform: From strain cultivation to monosaccharide composition and carbohydrate fingerprinting in one day.</a> Carbohydr Polym. 122, 212-220 (2015).</li><li>Ortega-Morales, B. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+extracellular+polymers+synthesized+by+tropical+intertidal+biofilm+bacteria.">Characterization of extracellular polymers synthesized by tropical intertidal biofilm bacteria.</a> J Appl Microbiol. 102 (1), 254-264 (2007).</li><li>Xu, R., Ma, S., Wang, Y., Liu, L., Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+identification+and+statistic+optimization+of+a+novel+exopolysaccharide+producing+Lactobacillus+paracasei+HCT.">Screening identification and statistic optimization of a novel exopolysaccharide producing Lactobacillus paracasei HCT.</a> Afr. J. Microbiol. Res. 4 (9), 783-795 (2010).</li><li>Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., Smith, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+method+for+determination+of+sugars+and+related+substances.">Colorimetric method for determination of sugars and related substances.</a> Anal. Chem. 28 (3), 350-356 (1956).</li><li>Masuko, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+analysis+by+a+phenol-sulfuric+acid+method+in+microplate+format.">Carbohydrate analysis by a phenol-sulfuric acid method in microplate format.</a> Anal Biochem. 339 (1), 69-72 (2005).</li><li>Geater, C. W., Fehr, W. R., Wilson, L. A., Robyt, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+more+rapid+method+of+total+sugar+analysis+for+soybean+seed.">A more rapid method of total sugar analysis for soybean seed.</a> Crop Sci. 41 (1), 250-252 (2001).</li><li>Sutherland, I. W., Dumitriu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+16,+Microbial+Exopolysaccharides.">Ch 16, Microbial Exopolysaccharides.</a> Polysaccharides Structural Diversity and Functional Versatility. , 431-457 (2005).</li><li>Wingender, J., Neu, T. R., Flemming, H. C., Wingender, J., Neu, T. R., Flemming, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+Bacterial+Extracellular+Polymeric+Substances?.">What are Bacterial Extracellular Polymeric Substances?.</a> Microbial extracellular polymeric substances: characterization, structure and function. , 258 (1999).</li><li>R&#252;hmann, B., Schmid, J., Sieber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+carbohydrate+analysis+via+liquid+chromatography+coupled+with+ultra+violet+and+electrospray+ionization+ion+trap+detection+in+96-well+format.">Fast carbohydrate analysis via liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection in 96-well format.</a> J. Chromatogr. A. 1350, 44-50 (2014).</li></ol>

We have nothing to disclose.

Polysaccharide detection with the phenol-sulfuric-acid method: Different monosaccharides show different absorption maxima and molar extinction coefficients by use of this method9. This results in different absorption maxima of polymers, which contain several sugars in different amounts. The different wavelengths of absorption maxima for 16 different commercially available polymers are given in Table 5. The polymers were dissolved (1 g/L) in ddH2O, stirred (150 rpm) overnight and measured with the phenol-sulfuric-acid-method. Diutan gum showed the lowest absorption maxima at 470 nm and scleroglucan and hyaluronic acid the highest at 484 nm. Based on these results 480 nm was chosen for this screening platform. The relative absorbance of the polymers was calculated based on the absorbance obtained with 1 g/L glucose (set as 100%). The lowest results were obtained with scleroglucan and succinoglycan, both with 92%. This was expected because scleroglucan only contains glucose and succinoglycan contains glucose and galactose in a ratio of 7:1. Both commercial polymers have different losses of drying and different ash contents, this is the reason why the theoretical value of ~110% was not reached. Xylan showed the highest relative absorbance with 311%. The reason for this is the high molar extinction coefficient achieved from xylose due to the more dominant furanose form. At a level of 0.1 g/L glucose the quantification limit was reached, as well as the detection limit at a concentration &#60;0.05 g/L. However, the detection limit for positive strains in the screening is higher than 0.7 g/L and therefore, the assay showed a good performance. In order to get reliable results, the remaining glucose after gel-filtration was determined with a glucose-assay and this value was subtracted from the value from the phenol-sulfuric-acid method.
Automated glucose-assay dilution: The performance of the glucose determination after cultivation (dilution 1:100) was investigated. For this, 10 &#181;l of supernatant were transferred to 990 &#181;l of ddH2O in a deep well plate and mixed via ten times aspirating and dispensing 180 &#181;l out of this dilution. The second critical step was the correct pipetting of only 5 &#181;l aliquot for the 1:10 dilution from the glucose-assay after gel-filtration. In order to generate the dilution 25 &#181;l of ddH2O were transferred with a 50 &#181;l-tip first, afterwards 20 &#181;l of ddH2O and 5 &#181;l gel-filtrate were aspirated together. This ensures a better removal of the 5 &#181;l aliquot out of the tip. Both dilution steps were verified with various glucose standards via a glucose-assay. The results for two exemplary concentrations are given in Table 6. The 1:100 dilution for the determination of the glucose content after cultivation showed high precision for both standards with a CV &#60;1.2%. At the same time, the accuracy for the higher standard was up to 11.6 (% error). However, this is negligible as the glucose determination represents only the remaining glucose content after cultivation and therefore, is not important for the polymer detection. The 1:10 dilution for the remaining glucose after gel-filtration showed very reliable results with a CV &#60;1.4% and an accuracy &#60;2.1% error.
Consideration of evaporation: The screening requires 3.5 hr from the first step to the first glucose-assay. In order to find out, whether this time frame has an influence on uncapped MTP storage, 50 &#181;l of glucose-assay calibration standards were stored with and without cover for 3.5 hr in the robot carousel. In the calibration range (45 to 4.5 mg/L) the sample concentration hardly increased. An increase &#8212; caused by evaporation &#8212; was below 2.1% and only for the two lowest concentrations (1.8 and 0.9 mg/L) it reached up to 12.4% (Table 7).
Gel-filtration: High amounts of non-metabolized glucose disturb the quantitative detection of glucose from the hydrolyzed polymer. Therefore, a gel-filtration step was required to remove the remaining glucose after cultivation. Additionally, the gel-filtration purifies the polymer containing supernatant from salts and monomeric carbohydrate compounds, others than glucose, to minimize the analytical background in the monomer analysis. At the gel-filtration step 35 &#181;l of filtrate were placed in the center of the well. For validation of the robustness of gel-filtration in the automated system, eight calibration standards from 0.045 up to 9 g/L glucose were filtrated (n = 8). The glucose of every concentration was always reduced by more than 95% of the initial value (Table 8). In doing so, the gel-filtration showed very good results for various concentrations of glucose. Additionally, the remaining glucose after gel-filtration was also determined with a glucose-assay and subtracted from the phenol-sulfuric-acid determination to receive the correct amount of glucose equivalent for the hydrolyzed polymer.
Pyruvate-assay: First of all, it was investigated whether the neutralized and diluted (1:10) TFA-matrix from the hydrolysis step interferes with the enzymatic reaction. Therefore, the complete assay was performed twice, one time with and one time without matrix and showed reliable results. Finally, the pyruvate content of 16 commercially available polymers was successfully measured and is depicted in Figure 3. It is generally known that out of those 16 polymers only succinoglycan and xanthan naturally contain pyruvate. With our pyruvate-assay both of these polymers were correctly identified. In scleroglucan, welan gum and xylan pyruvate was also detected in significant amounts. Overall, the capability of the approach was validated and the pyruvate-assay showed a high performance. It proved to be able to detect pyruvate in different polymers after hydrolysis.
Carbohydrate fingerprint: After performing all analytical modules in the automated screening, potential EPS producers were selected for the carbohydrate fingerprint analysis. For this, several criteria were applied: 1) Positive observation of viscosity after centrifugation and/or after filtration. 2) Precipitation before and after filtration. Observed fibers and flakes were evaluated as positive. 3) Glucose equivalent value from the phenol-sulfuric-acid method. Values &#62;700 mg/L were rated as positive and values between 300 and 700 mg/L were rated as putative EPS producers. When two or three criteria were evaluated as positive, the strains were selected for further carbohydrate fingerprint analysis. The criteria can be customized towards the individual purpose of the EPS screening (e.g. low viscosity EPS). Our approach aimed at finding efficient EPS producers. When searching for strains that only produce small amounts of EPS the evaluation limit of the glucose equivalent should be reduced.
Technical benefit and future applications: One interesting feature of this protocol is the modular character of the steps and the different analytical modules. They can be combined in different ways, adjusted to individual requirements and novel modules can easily be implemented. Furthermore, the analytical modules can be used separately, e.g. the hydrolysis module in combination with the HT-PMP-derivatization module is able to perform a monomeric composition analysis from different polymer solutions (1 g/L) in 96-well format. For laboratories without having access to a liquid handling system the complete screening can be handled manually without any changes in the pipetting scheme. However, using a liquid handling system increases the throughput to up to 768 strains (instead of 192 strains if screened manually) per day. The protocol that is described here is capable of a screening for different genera and therefore, for the screening of large strain collections to identify novel EPS producers and analyze their carbohydrate fingerprint in one approach (Figure 4). Furthermore, a targeted screening for polysaccharides containing rare sugars like fucose, uronic acids or even unknown sugars can be performed via the detailed monosaccharide analysis. Also, different sugar combinations in defined ratios can be detected. This enables the simple identification of structurally related variants of already known EPS or novel EPS.

Microbial exopolysaccharides (EPS) are a structurally highly diverse group of polymers that fulfill various biological functions. They usually are built of complex repeat units, which are distinguished by different types of monomers (sugar, sugar derivatives, sugar acids), the bonds between these monomers and their substitutions. The structural diversity of microbial polysaccharides confers rather different characteristics to the members of this molecule class, which allows their application in different fields like food1, cosmetics2,3, construction chemistry4 or water treatment5. To further extend the field of application of these bio-based and such sustainable polymers the identification of novel natural microbial polysaccharides as well as the engineering of structural variants represents promising approaches. Either way, a fast screening method is required to quickly scan a vast number of microbial strains for their polysaccharide formation, and to analyze their products. Therefore, we have recently developed a 96-well HT-screening platform for the analysis of microbial polysaccharide production from natural isolates or engineered strain variants that includes the identification of the monomeric composition6.
Applying this platform for a first screening round of ~500 natural isolates allowed us to identify only about 10 to 20% of the isolated strains as being able to produce EPS (data not shown). This means that 80-90% of the analyzed strains did not produce EPS under the conditions applied, and therefore, a further analysis of the detailed carbohydrate fingerprint was not necessary. As this highly sophisticated identification of the monomeric composition is a time consuming process, especially for data analysis, a fast pre-screening method to identify the strains positive in EPS production, would drastically increase the efficiency. Furthermore, reagents, consumables and measurement time at the UHPLC-UV-ESI-MS would be reduced. Additionally, the different analytical modules, while on one hand make the method highly reliable, are on the other hand complicating the manual handling of more than two 96-well plates in parallel, and as such restrict the full potential of the method. For these reasons, we decided to develop an automated screening platform. Therefore, we combined the modular format of the existing carbohydrate fingerprint technique with a fully automated fast detection method of the total sugar content, based on absorbance measurement.
The phenol-sulfuric-acid method still represents the method of choice for the fast determination of total carbohydrate content of bacterial and plant polysaccharides7,8. This method was first described by Dubois et al.9 and adapted for different applications and sample sizes, even for small scale in 96-well plates10,11. The phenol-sulfuric-acid method measures the total carbohydrate content by one value, summating all monomeric, oligomeric and polymeric carbohydrates of the samples.
Taking these aspects into account, the choice of a suitable cultivation medium is essential to apply this method. Complex media containing oligomeric or polymeric carbohydrate compounds like yeast extract might lead to an altered polymer content and therefore, should strictly be avoided. Furthermore, high amounts of sugars are used as C-source for the cultivation of the strains. High levels of remaining carbohydrates from the cultivation process might negatively interfere with the measurement of the EPS content.
Therefore, the use of defined and pure sugars is advised. In our experiments we used glucose for the cultivation of the cells. The remaining glucose after cultivation was reduced via a gel-filtration and determined via a glucose-assay. Finally, the glucose equivalents of the polysaccharides were calculated by subtracting the remaining glucose after the gel-filtration from the total carbohydrate content that had been detected with the phenol-sulfuric-acid method. Gel-filtration and glucose-assay in combination with the phenol-sulfuric-acid method ensure reliable results and are capable of being our first, completely automated detection system.
Two new analytical modules were included in the automated screening platform to increase the amount of information from the EPS-detection system: the precipitation and the observation of an increase in viscosity.
Many different EPS &#8212; e.g. succinoglycan, xanthan and colonic acid12&#160;&#8212; are reported to be modified with a non-carbohydrate pyruvate ketal on sugar positions C4 and C6. Those pyruvate ketals (just as succinyl half esters and uronic acids) contribute to the polyanionic nature and therefore, to the physical properties of the polymer by interacting via divalent cation bridges13. In order to identify those particular polymers the determination of pyruvate was established as another additional analytical module. This increases the information about the polysaccharide substituents and their potential macroscopic properties.
Combining all the modules enables the identification of different EPS as well as a fast and efficient determination of EPS producers. By that approach the screening platform could be divided into two major parts (Figure 1). Within the automated screening (part I) the workflow occurs fully automated (Table 1) to quickly preselect the EPS producing strains. The carbohydrate fingerprint analysis (part II) quantitatively determines the monomer composition of the EPS produced by the preselected strains. Thereby, the data analysis was minimized in order to optimize the screening of large strain collections. This offers the possibility to analyze 384 strains in one single automated screening run and with two runs that are possible per day of 768 strains per day. Additionally, the carbohydrate fingerprint analysis gives an even more detailed overview of all the identified EPS. This enables the directed analysis and identification of only slightly differing EPS variants or completely new ones compared to the already described chemical structures of EPS.

<table><tbody><tr><td>96 well deep well plate 2.0 ml (DWP)</td><td>Greiner Bio-One</td><td>780271</td><td>Main-culture (CP 1-1 to 1-4), equilibration plates for gel-filtration (CP 1-6, 1-7, 2-6 and 2-7), 1:100 dilution for the glucose assay (CP 4-1 to 4-4) containing 990&amp;nbsp;&amp;micro;l of ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Breathable Sealing Film</td><td>Axygen</td><td>BF-400-S</td><td>Incubation film for pre- and main-culture DWP</td></tr><tr><td>Aluminum Sealing Film</td><td>Axygen</td><td>PCR-AS-200</td><td>-80 &amp;deg;C storage film for glycerol-stock plates</td></tr><tr><td>MCA96 Nested Disposable Tips 200 &amp;micro;l</td><td>TECAN</td><td>30038619</td><td>Worktable position (WTP 2-1 to 2-4)</td></tr><tr><td>A/B glass-filter-plate 1&amp;nbsp;&amp;micro;m</td><td>Pall Corporation</td><td>PN 8031</td><td>Stored on collector plate (CP 2-1 to 2-4)</td></tr><tr><td>96-well micro titer plate V-Bottom</td><td>Greiner Bio-One</td><td>651201</td><td>Collector plate for filtration plate (CP 2-1 to 2-4)</td></tr><tr><td>96-well SpinColumn G-25</td><td>Harvard Apparatus</td><td>74-5612</td><td>Stored on washing DWP (CP 1-6, 1-7, 2-6 and 2-7)</td></tr><tr><td>96-well micro titer plate V-Bottom</td><td>Nunc</td><td>249944</td><td>Collector plate for gel-filtration plate (CP 3-1 to 3-4)</td></tr><tr><td>Nested Disposable Tips SBS 50 &amp;micro;l tips</td><td>TECAN</td><td>30038609</td><td>Carousel position (CP 4-6, 4-7 and 5-1 to 5-6)</td></tr><tr><td>Trough 250 ml</td><td>Axygen</td><td>Res-SW96-HP</td><td>Water WTP 1-1, Glucose assay reagent-mix WPT 1-2, ammonium-acetate buffer pH 5.6 (CP 5-7)</td></tr><tr><td>96-well micro titer plate F-Bottom (MTP)</td><td>Greiner Bio-One</td><td>655101</td><td>precipitation 1 (CP 6-1 to 6-4), pH-value (CP 7-1 to 7-4), precipitation 2 (CP 8-1 to 8-4), phenol-sulfuric-acid method (CP 8-5 to 8-8), glucose-assay (CP 9-1 to 9-9), dummy plates (WTP 3-1, 3-2)</td></tr><tr><td>96-well silicon cap mat&amp;nbsp;</td><td>Whatmann</td><td>7704-0105</td><td>Cover mat for MTP</td></tr><tr><td>200 &amp;micro;l pipette tips</td><td>Sarstedt</td><td>70.760.002</td><td>For manually handling</td></tr><tr><td>1,000 &amp;micro;l pipette tips</td><td>Sarstedt</td><td>70.762</td><td>For manually handling</td></tr><tr><td>96-well-PCR micro titer plate&amp;nbsp;</td><td>Brand</td><td>781350</td><td>Hydrolysis, PMP-derivatisation</td></tr><tr><td>TPE (thermoplastic elastomer) cap mat&amp;nbsp;</td><td>Brand</td><td>781405</td><td>Hydrolysis, PMP-derivatisation</td></tr><tr><td>Filter plate 0.2&amp;nbsp;&amp;micro;m Supor</td><td>Pall Corporation</td><td>PN 8019</td><td>Filtration of samples for UHPLC-ESI-MS analysis with a MTP collector plate</td></tr><tr><td>Pipette Tips LHS 5-300&amp;nbsp;&amp;micro;l</td><td>Brand</td><td>732150</td><td>Brand LHS system</td></tr><tr><td>Glucose oxidase&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G2133</td><td>Glucose-assay</td></tr><tr><td>Horseradish peroxidase&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P6782</td><td>Glucose-assay, pyruvat-assay</td></tr><tr><td>DA-64 (&lt;em&gt;N&lt;/em&gt;-(Carboxymethylaminocarbonyl)-4.4&#039;-bis(dimethylamino)-diphenylamine sodium salt)</td><td>Wako Chemicals GmbH</td><td>043-22351</td><td>Pyruvat-assay</td></tr><tr><td>Pyruvate oxidase&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P4591</td><td>Pyruvat-assay</td></tr><tr><td>Potassium phosphate dibasic</td><td>Carl-Roth</td><td>P749.3</td><td>Pyruvat- and glucose-assay</td></tr><tr><td>Potassium phosphate monobasic</td><td>Carl-Roth</td><td>3904.3</td><td>Pyruvat- and glucose-assay</td></tr><tr><td>Thiamine pyrophosphate</td><td>Sigma-Aldrich</td><td>C8754</td><td>Pyruvat-assay</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma-Aldrich</td><td>31413</td><td>Pyruvat-assay</td></tr><tr><td>2-Propanol</td><td>VWR</td><td>20922.466</td><td>Precipitation</td></tr><tr><td>Phenol</td><td>VWR</td><td>20599.231</td><td>Phenol-sulfuric-acid method</td></tr><tr><td>Sulfuric acid</td><td>Carl-Roth</td><td>4623.4</td><td>Phenol-sulfuric-acid method</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T6508</td><td>Hydrolysis</td></tr><tr><td>Ammonium solution</td><td>Carl-Roth</td><td>P093.1</td><td>Hydrolysis, PMP-derivatization</td></tr><tr><td>Ethanol absolut</td><td>VWR</td><td>20821.321</td><td>Hydrolysis, PMP-derivatization</td></tr><tr><td>Phenol red&amp;nbsp;</td><td>Alfa Aesar</td><td>B21710</td><td>Hydrolysis, PMP-derivatization</td></tr><tr><td>1-Phenyl-3-methyl-5-pyrazolone</td><td>Sigma-Aldrich</td><td>M70800</td><td>PMP-derivatization</td></tr><tr><td>Methanol LC-MS</td><td>VWR</td><td>83638.320</td><td>PMP-derivatization</td></tr><tr><td>Acetonitril LC-MS</td><td>VWR</td><td>83040.320</td><td>PMP-derivatization</td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>338826</td><td>PMP-derivatization</td></tr><tr><td>Ethanol absolut</td><td>VWR</td><td>20821.321</td><td>PMP-derivatization</td></tr><tr><td>Methyl red</td><td>Alfa Aesar</td><td>36667</td><td>pH-value</td></tr><tr><td>Robotic liquid handling system&amp;nbsp;</td><td>Tecan</td><td>Freedom EVO</td><td>Worktable setup in &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr><tr><td>Liquid handling station LHS</td><td>Brand</td><td>709400</td><td>Worktable setup in &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr><tr><td>Tip-Adapter</td><td>Brand</td><td>709434</td><td>Worktable setup in &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr><tr><td>Liquid Ends MC 20-300&amp;nbsp;&amp;micro;l</td><td>Brand</td><td>709416</td><td>Worktable setup in &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr><tr><td>Adapter 60 mm</td><td>Brand</td><td>709430</td><td>Worktable setup in &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr></tbody></table>

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.

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

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.

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

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11.4K Views.  Technische Universität München. 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.

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

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Biochemistry

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

Environment

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Bioengineering

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Chemistry

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Automated Lectin-Based Glycan Profiling Using Bead Array in a Single Tip (BIST)

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of &lt;10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

Author Spotlight: Advancing Protein Glycosylation Research Using a Fully Automated System

A system for the automated and rapid analysis of glycans on proteins was previously developed. This article presents the protocol for "glyco-qualitative analysis," optimized for a broad spectrum of users, such as those engaged in analyzing glycan structures in biopharmaceuticals and other glycoconjugate materials.

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

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 ...

Elucidating Glycan Structure and Occurrence Using Microarray Polymer Profiling

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.

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) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans ...

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

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